( G , H ) Western blotting revealed levels of POLH ( G ) and REV1 ( H ) in two independent clones upon selection following transfection with indicated shRNA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G, H) Western blotting revealed levels of POLH (G) and REV1 (H) in two independent clones upon selection following transfection with indicated shRNA.


( I ) Colony formation assay was conducted to reveal proliferation of WT , POLH KD , REV1 KD , HMCES KO , HMCES KO + POLH KD and HMCES KO + REV1 KD cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Colony formation assay was conducted to reveal proliferation of WT, POLH KD, REV1 KD, HMCES KO, HMCES KO + POLH KD and HMCES KO +REV1 KD cells.


( J ) Representative images showing proliferation of respective shRNA resistant reconstitution of POLH in 293A and HMCES KO cells stably expressing POLH shRNA . Experiment was repeated two independent times in duplicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Representative images showing proliferation of respective shRNA resistant reconstitution of POLH in 293A and HMCES KO cells stably expressing POLH shRNA. Experiment was repeated two independent times in duplicates.


( K ) Mutagenesis frequency analysis was conducted in 293T scrambled and 293T HMCES shRNA cells using sup F shuttle vector - based assay system . Experiments were repeated three independent times and data are presented as mean ± s . e . m . Student ' s t - test was used to calculate statistical significance ( * * , p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Mutagenesis frequency analysis was conducted in 293T scrambled and 293T HMCES shRNA cells using sup F shuttle vector-based assay system. Experiments were repeated three independent times and data are presented as mean±s.e.m. Student's t-test was used to calculate statistical significance (**, p<0.01).


( B ) Volcano plot showing DDR CRISPR - CAS9 screen for HMCES KO cells . - log10 ( p - value ) is plotted against log2 ( HMCES KO / WT ) fold change . Genes showing significant positive and negative enrichment are colored in red and top enriched candidates are also labeled .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Volcano plot showing DDR CRISPR-CAS9 screen for HMCES KO cells. -log10(p-value) is plotted against log2(HMCES KO/ WT) fold change. Genes showing significant positive and negative enrichment are colored in red and top enriched candidates are also labeled.


( C ) Plot showing number of sgRNAs that are negatively or positively enriched for each gene in DDR library .

O O O O O O O O O O O O O O O O O O O O O

(C) Plot showing number of sgRNAs that are negatively or positively enriched for each gene in DDR library.


Western blots showing levels of CtIP ( D ) upon shRNA mediated knockdown in 293A cells .

Western blots showing levels of CtIP (D) upon shRNA mediated knockdown in 293A cells.


Western blots showing levels of BRCA2 ( E ) upon shRNA mediated knockdown in 293A cells .

Western blots showing levels of BRCA2 (E) upon shRNA mediated knockdown in 293A cells.


Cell proliferation measured using CellTiter - Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding CtIP ( F ) shRNAs . Each experiment was repeated three independent times done in triplicates each time . Data was represented as mean ± s . e . m . Student ' s t - test was used to calculate statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell proliferation measured using CellTiter-Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding CtIP (F) shRNAs. Each experiment was repeated three independent times done in triplicates each time. Data was represented as mean±s.e.m. Student's t-test was used to calculate statistical significance (***, p<0.001; ****, p<0.0001).


Cell proliferation measured using CellTiter - Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding BRCA2 ( G ) shRNAs . Each experiment was repeated three independent times done in triplicates each time . Data was represented as mean ± s . e . m . Student ' s t - test was used to calculate statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) .

Cell proliferation measured using CellTiter-Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding BRCA2 (G) shRNAs. Each experiment was repeated three independent times done in triplicates each time. Data was represented as mean±s.e.m. Student's t-test was used to calculate statistical significance (***, p<0.001; ****, p<0.0001).


A , Hierarchical clustering analysis of RNA - sequencing data from cells treated with DMSO , 20 Gy IR , 250nM AZD6738 , or AZD6738 + IR ( n = 2 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O

A, Hierarchical clustering analysis of RNA-sequencing data from cells treated with DMSO, 20 Gy IR, 250nM AZD6738, or AZD6738 + IR (n=2 biological replicates).


C , Heat map of 33 representative inflammatory genes regulated by AZD6738 + IR .

O O O O O O O O O O O B-CONTROLLED_VAR O O O

C, Heat map of 33 representative inflammatory genes regulated by AZD6738 + IR.


D , MCF10A WT cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , cells were harvested at indicated time points and immunoblotting was performed with indicated antibodies . h , hours ; pSTAT1 , pSTAT1 ( Y701 ) ; pIRF3 , pIRF3 ( S396 ) ; pTBK1 , pTBK1 ( S172 ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, cells were harvested at indicated time points and immunoblotting was performed with indicated antibodies. h, hours; pSTAT1, pSTAT1(Y701); pIRF3, pIRF3(S396); pTBK1, pTBK1(S172).


E , MCF10A WT cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with indicated dose . 72 hours later , cells were harvested for immunoblotting with indicated antibodies .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, MCF10A WT cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with indicated dose. 72 hours later, cells were harvested for immunoblotting with indicated antibodies.


F , Same condition as E except the irradiation dose with 20 Gy , mRNA levels of inflammatory genes as indicated were assessed by real - time quantitative PCR , presented as mean + standard error of the mean of three biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, Same condition as E except the irradiation dose with 20 Gy, mRNA levels of inflammatory genes as indicated were assessed by real-time quantitative PCR, presented as mean + standard error of the mean of three biological replicates.


A , MCF10A WT cells were pretreated with AZD6738 ( 250 nM ) and RO - 3306 ( 9 μM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies .

O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, MCF10A WT cells were pretreated with AZD6738(250 nM) and RO-3306 (9 μM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.


B , Same condition except that RO - 3306 is replaced by PD - 0332991 ( 1µM ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

B, Same condition except that RO-3306 is replaced by PD-0332991 (1µM).


C , MCF10A WT cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 24 hours later , cell cycle distribution was measured by flow cytometry . EdU ( 10 μM ) was added into medium 2 hours before harvesting for flow cytometry .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, 24 hours later, cell cycle distribution was measured by flow cytometry. EdU (10 μM) was added into medium 2 hours before harvesting for flow cytometry.


D , MCF10A WT cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , and cells were harvested at indicated time points for immunoblotting .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

D, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, and cells were harvested at indicated time points for immunoblotting.


E , MCF10A control cells and CDC25C - WT / constitutively active mutant CDC25C - S216A over - expressed cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 24 hours later , cells were harvested for immunoblotting with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, MCF10A control cells and CDC25C-WT/constitutively active mutant CDC25C-S216A over-expressed cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, 24 hours later, cells were harvested for immunoblotting with indicated antibodies.


F , Same condition except that cells were harvested 72 hours later .

F, Same condition except that cells were harvested 72 hours later.


A , MCF10A Wild - type ( WT ) , cGAS knockout ( KO ) , and STING KO cells were treated with cGAMP at indicated concentrations for 4 hours , and cells were harvested for immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

A, MCF10A Wild-type (WT), cGAS knockout (KO), and STING KO cells were treated with cGAMP at indicated concentrations for 4 hours, and cells were harvested for immunoblotting.


B , MCF10A WT , cGAS KO , and STING KO cells were transfected with herring testis DNA ( HT - DNA , 2 µg / ml ) for 6 hours with lipofectamine 3000 . Immunoblotting was performed with indicated antibodies .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

B, MCF10A WT, cGAS KO, and STING KO cells were transfected with herring testis DNA (HT-DNA, 2 µg/ml) for 6 hours with lipofectamine 3000. Immunoblotting was performed with indicated antibodies.


MCF10A WT or two independent clones for cGAS KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies . Data are presented as mean + standard error of the mean of three biological replicates .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MCF10A WT or two independent clones for cGAS KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.


MCF10A WT or two independent clones for STING KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies . Data are presented as mean + standard error of the mean of three biological replicates .

MCF10A WT or two independent clones for STING KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.


MCF10A WT or two independent clones for cGAS , STING , and IRF3 KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , except that cells were harvested for real - time quantitative PCR . IFNB1 mRNA level was measured and statistical analyzed by two - way ANOVA ( * * * means p value < 0 . 001 ) . Data are presented as mean + standard error of the mean of three biological replicates .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MCF10A WT or two independent clones for cGAS, STING, and IRF3 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, except that cells were harvested for real-time quantitative PCR. IFNB1 mRNA level was measured and statistical analyzed by two-way ANOVA (*** means p value < 0.001). Data are presented as mean + standard error of the mean of three biological replicates.


MCF10A WT or two independent clones for IRF3 KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies . Data are presented as mean + standard error of the mean of three biological replicates .

MCF10A WT or two independent clones for IRF3 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.


( A MCF10A WT or two independent clones for MAVS KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A MCF10A WT or two independent clones for MAVS KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.


MCF10A WT or two independent clones for MAVS , RIG - I KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , ( B cells were harvested for real - time quantitative PCR instead of immunoblotting . IFNB1 mRNA level was measured by real - time quantitative PCR . Data are presented as mean + standard error of the mean of three biological replicates .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MCF10A WT or two independent clones for MAVS, RIG-I KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, (B cells were harvested for real-time quantitative PCR instead of immunoblotting. IFNB1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.


C , MCF10A WT or two independent clones for RIG - I KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, MCF10A WT or two independent clones for RIG-I KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.


D , MCF10A WT or two independent clones for MDA5 , KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, MCF10A WT or two independent clones for MDA5, KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.


MCF10A WT or two independent clones for MDA5 KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later E ) cells were harvested for real - time quantitative PCR instead of immunoblotting . IFNB1 mRNA level was measured by real - time quantitative PCR . Data are presented as mean + standard error of the mean of three biological replicates .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MCF10A WT or two independent clones for MDA5 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later E) cells were harvested for real-time quantitative PCR instead of immunoblotting. IFNB1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.


F ) MCF10A WT or two independent clones for IRF7 KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies .

F) MCF10A WT or two independent clones for IRF7 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.


A , 4T1 cells were pretreated with AZD6738 at indicated concentrations for 2 hours , and then irradiated with 10 Gy . 4 days later , Ifnb1 mRNA level was measured by real - time quantitative PCR . Data are presented as mean + standard error of the mean of three biological replicates .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, 4T1 cells were pretreated with AZD6738 at indicated concentrations for 2 hours, and then irradiated with 10 Gy. 4 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.


B , Ifnb1 mRNA levels were measured by real - time quantitative PCR in WT , Sting KO , Mavs KO , and Irf3 KO 4T1 cells . Data are presented as mean + standard error of the mean of three biological replicates .

B, Ifnb1 mRNA levels were measured by real-time quantitative PCR in WT, Sting KO, Mavs KO, and Irf3 KO 4T1 cells. Data are presented as mean + standard error of the mean of three biological replicates.


C , MC38 cells were pretreated with AZD6738 at indicated concentrations for 2 hours , and then irradiated with 20 Gy . 3 days later , Ifnb1 mRNA level was measured by real - time quantitative PCR . Data are presented as mean + standard error of the mean of three biological replicates .

C, MC38 cells were pretreated with AZD6738 at indicated concentrations for 2 hours, and then irradiated with 20 Gy. 3 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.


D , Ifnb1 mRNA levels were measured by real - time quantitative PCR in WT , Sting KO , Mavs KO , and Irf3 KO MC38 cells . Data are presented as mean + standard error of the mean of three biological replicates .

D, Ifnb1 mRNA levels were measured by real-time quantitative PCR in WT, Sting KO, Mavs KO, and Irf3 KO MC38 cells. Data are presented as mean + standard error of the mean of three biological replicates.


A , MCF10A WT , cGAS KO , STING KO , and MAVS KO cells were treated with poly ( dA - dT ) / LyoVec at indicated concentrations for 24 hours . Immunoblotting was performed with indicated antibodies .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

A, MCF10A WT, cGAS KO, STING KO, and MAVS KO cells were treated with poly(dA-dT)/LyoVec at indicated concentrations for 24 hours. Immunoblotting was performed with indicated antibodies.


B , 4T1 WT , Sting KO , Mavs KO , and Irf3 KO cells were treated with poly ( dA - dT ) / LyoVec at indicated concentrations for 24 hours . Immunoblotting was performed with indicated antibodies .

B, 4T1 WT, Sting KO, Mavs KO, and Irf3 KO cells were treated with poly(dA-dT)/LyoVec at indicated concentrations for 24 hours. Immunoblotting was performed with indicated antibodies.


C , END - seq was used to map double - strand breaks ( DSBs ) associated with TA dinucleotide repeats in MCF10A cells with the indicated treatments . ATR inhibitor ( ATRi ) AZD6738 ( 250nM ) was used with ionizing radiation ( IR , 10 Gy ) for 6 hours . NT , not treated .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

C, END-seq was used to map double-strand breaks (DSBs) associated with TA dinucleotide repeats in MCF10A cells with the indicated treatments. ATR inhibitor (ATRi) AZD6738 ( 250nM) was used with ionizing radiation (IR, 10 Gy) for 6 hours. NT, not treated.


D , MCF10A WT , MDA5 KO , RIG - I KO , and MAVS KO cells were transfected with AT - rich oligos ( 2 μg / ml ) with LyoVec for 24 hours . Immunoblotting was performed with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

D, MCF10A WT, MDA5 KO, RIG-I KO, and MAVS KO cells were transfected with AT-rich oligos (2 μg/ml) with LyoVec for 24 hours. Immunoblotting was performed with indicated antibodies.


E , MCF10A WT and STING KO cells were transfected with AT - rich and non - AT - rich oligos ( 2 μg / ml ) with lipofectamine 3000 for 4 hours . Immunoblotting was performed with indicated antibodies .

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

E, MCF10A WT and STING KO cells were transfected with AT-rich and non-AT-rich oligos (2 μg/ml) with lipofectamine 3000 for 4 hours. Immunoblotting was performed with indicated antibodies.


F , 4T1 WT , Sting KO , and Irf3 KO cells were transfected with AT - rich and non - AT - rich oligos ( 2 μg / ml ) with LyoVec for 24 hours . Immunoblotting was performed with indicated antibodies .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

F, 4T1 WT, Sting KO, and Irf3 KO cells were transfected with AT-rich and non-AT-rich oligos (2 μg/ml) with LyoVec for 24 hours. Immunoblotting was performed with indicated antibodies.


A cdc14 ‐ 3 temperature ‐ sensitive cells reconstituted with CDC14 or CDC14 ‐ NLS were released from a G1 arrest at the restrictive temperature and stained for Cdc14 and the spindle ( Tub1 ) once cells had reached late anaphase .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A cdc14‐3 temperature‐sensitive cells reconstituted with CDC14 or CDC14‐NLS were released from a G1 arrest at the restrictive temperature and stained for Cdc14 and the spindle (Tub1) once cells had reached late anaphase.


B Wild ‐ type , cdc14 ‐ 3 , + CDC14 and + CDC14 ‐ NLS cells were released from an α ‐ factor ‐ induced G1 arrest and re ‐ arrested in the next G1 . Samples were harvested at the indicated time points and stained with DAPI and for the nucleolar protein Nop1 ( n > 100 ) . Photographs of examples of cells without ( top ) and with ( bottom ) rDNA segregation defects at 80 min after release are shown together with quantification over time .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Wild‐type, cdc14‐3, +CDC14 and +CDC14‐NLS cells were released from an α‐factor‐induced G1 arrest and re‐arrested in the next G1. Samples were harvested at the indicated time points and stained with DAPI and for the nucleolar protein Nop1 (n > 100). Photographs of examples of cells without (top) and with (bottom) rDNA segregation defects at 80 min after release are shown together with quantification over time.


C Cells from the experiment in ( B ) were stained for the spindle ( n > 100 ) . Cells without ( top ) and with ( bottom ) anaphase spindles at 100 min after release are shown together with their quantification over time .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Cells from the experiment in (B) were stained for the spindle (n > 100). Cells without (top) and with (bottom) anaphase spindles at 100 min after release are shown together with their quantification over time.


D FACS analysis of DNA content and Western blot analysis of cell cycle markers of the samples from ( B ) . Tub1 served as a loading control .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

D FACS analysis of DNA content and Western blot analysis of cell cycle markers of the samples from (B). Tub1 served as a loading control.


A + CDC14 and + CDC14 ‐ NLS cells were grown at 35 . 5 ° C for 5 h and sonicated , and differential interference contrast ( DIC ) images were acquired .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A +CDC14 and +CDC14‐NLS cells were grown at 35.5°C for 5 h and sonicated, and differential interference contrast (DIC) images were acquired.


B Cells from a synchronous culture ( as in Fig B ) were treated with zymolyase to remove the cell wall . The fraction of remaining budded cells is shown over time ( n = 100 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Cells from a synchronous culture (as in Fig B) were treated with zymolyase to remove the cell wall. The fraction of remaining budded cells is shown over time (n = 100).


C Cells expressing the plasma membrane marker GFP ‐ Spo2051 - 91 , exemplifying the indicated bud neck phenotypes and stages of cytokinesis . D Bud neck phenotypes of synchronized GFP ‐ Spo2051 - 91 ‐ expressing + CDC14 or + CDC14 ‐ NLS cells during the progression of a cell cycle ( n > 75 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Cells expressing the plasma membrane marker GFP‐Spo2051-91, exemplifying the indicated bud neck phenotypes and stages of cytokinesis.D Bud neck phenotypes of synchronized GFP‐Spo2051-91‐expressing +CDC14 or +CDC14‐NLS cells during the progression of a cell cycle (n > 75).


E + CDC14 or + CDC14 ‐ NLS cells expressing Spc42 ‐ YFP and GFP fusions with the indicated cytokinetic protein were synchronized in G1 using α ‐ factor . Upon release at 35 . 5 ° C , cells were imaged every 2 min and the time from anaphase onset to the indicated events was recorded . Error bars represent SD ; Student ' s t ‐ test was applied to analyze differences between the indicated conditions ; * * P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 . For representative image sequences , see Supplementary Fig S2C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E +CDC14 or +CDC14‐NLS cells expressing Spc42‐YFP and GFP fusions with the indicated cytokinetic protein were synchronized in G1 using α‐factor. Upon release at 35.5°C, cells were imaged every 2 min and the time from anaphase onset to the indicated events was recorded. Error bars represent SD; Student's t‐test was applied to analyze differences between the indicated conditions; **P ≤ 0.01; ****P ≤ 0.0001. For representative image sequences, see Supplementary Fig S2C.


A MET3pr ‐ CDC20cdc15 ‐ as1 cells were arrested in metaphase by Cdc20 depletion and subsequently treated with 1NM ‐ PP1 to inhibit Cdc15 ‐ as1 . Cells were released from the metaphase arrest by Cdc20 re ‐ induction ( left graph ) , or Cdc14 and / or Sic1 expression were induced from the GAL1 promoter while cells remained arrested ( right graph ) . The fraction of large ‐ budded cells after spheroplastation ( n = 100 ) was quantified . Representative photographs 120 min after galactose addition are shown . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

A MET3pr‐CDC20cdc15‐as1 cells were arrested in metaphase by Cdc20 depletion and subsequently treated with 1NM‐PP1 to inhibit Cdc15‐as1. Cells were released from the metaphase arrest by Cdc20 re‐induction (left graph), or Cdc14 and/or Sic1 expression were induced from the GAL1 promoter while cells remained arrested (right graph). The fraction of large‐budded cells after spheroplastation (n = 100) was quantified. Representative photographs 120 min after galactose addition are shown. Scale bar, 10 μm.


B As ( A ) , the plasma membrane marker GFP ‐ Spo2051 - 91 was visualized following Cdc14 induction ( n = 100 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O

B As (A), the plasma membrane marker GFP‐Spo2051-91 was visualized following Cdc14 induction (n = 100).


C Samples from the experiment in ( B ) were processed for Western blot analysis of the indicated cell cycle proteins . Arrowheads indicate endogenous and exogenous forms of Cdc14 . Tub1 served as a loading control . A schematic of downstream processing for the phosphoproteome analysis is shown on the right .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Samples from the experiment in (B) were processed for Western blot analysis of the indicated cell cycle proteins. Arrowheads indicate endogenous and exogenous forms of Cdc14. Tub1 served as a loading control. A schematic of downstream processing for the phosphoproteome analysis is shown on the right.


A Phospho ‐ peptide abundance during Cdc14 ‐ induced mitotic exit . Examples of phospho ‐ peptides that disappear with early ( red ) , intermediate ( orange ) and late ( green ) timing or that remain stable ( black ) are shown . This color coding is used throughout all panels of this figure . B Numbers of unique peptides and corresponding proteins that do or do not contain a minimal Cdk consensus motif are shown , categorized according to timing of disappearance as described in ( A ) . C Protein stability data for proteins corresponding to disappearing phospho ‐ peptides ( blue ) and for all other proteins ( black ) . D The fractions of peptides that contain a minimal Cdk consensus motif ( [ S / T ] ‐ P ) within the timing categories shown in ( B ) were calculated . The ratio and P ‐ value of enrichment of each category relative to the combined stable / rest category ( gray ) are shown . E Similar to ( D ) , the enrichment for reported physical Cdc14 interactors is assessed within [ S / T ] ‐ P ‐ containing phospho ‐ peptides . F Peptides that contain the indicated Cdk consensus sequence motifs and that have disappeared over time are represented as a fraction of all phospho ‐ peptides containing that motif .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Phospho‐peptide abundance during Cdc14‐induced mitotic exit. Examples of phospho‐peptides that disappear with early (red), intermediate (orange) and late (green) timing or that remain stable (black) are shown. This color coding is used throughout all panels of this figure.B Numbers of unique peptides and corresponding proteins that do or do not contain a minimal Cdk consensus motif are shown, categorized according to timing of disappearance as described in (A).C Protein stability data for proteins corresponding to disappearing phospho‐peptides (blue) and for all other proteins (black).D The fractions of peptides that contain a minimal Cdk consensus motif ([S/T]‐P) within the timing categories shown in (B) were calculated. The ratio and P‐value of enrichment of each category relative to the combined stable/rest category (gray) are shown.E Similar to (D), the enrichment for reported physical Cdc14 interactors is assessed within [S/T]‐P‐containing phospho‐peptides.F Peptides that contain the indicated Cdk consensus sequence motifs and that have disappeared over time are represented as a fraction of all phospho‐peptides containing that motif.


G Unique phospho ‐ peptides of Spa2 , as an example , are categorized based on their dephosphorylation timing ( graph ) . Enrichment of clustered timing for all proteins covered by phospho ‐ peptides was compared to random for Cdk or non ‐ Cdk consensus site ‐ containing peptides ( table ) . Fisher ' s exact tests were used to determine significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Unique phospho‐peptides of Spa2, as an example, are categorized based on their dephosphorylation timing (graph). Enrichment of clustered timing for all proteins covered by phospho‐peptides was compared to random for Cdk or non‐Cdk consensus site‐containing peptides (table). Fisher's exact tests were used to determine significance.


B Western blot analysis of cells expressing conditional Clb2m ( + ) or Clb2mΔCdk ( Δ ) fusions to the indicated proteins , before ( − ) and 5 h after ( + ) β ‐ estradiol treatment . Tub1 served as a loading control . c , control cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

B Western blot analysis of cells expressing conditional Clb2m (+) or Clb2mΔCdk (Δ) fusions to the indicated proteins, before (−) and 5 h after (+) β‐estradiol treatment. Tub1 served as a loading control. c, control cells.


C FACS analysis of DNA content after conditional Clb2m or Clb2mΔCdk fusion to the indicated proteins . Quantification of the fraction of cells with > 2C DNA content is shown in the bar graph for two independent biological replicates , with red dots representing individual and bars representing average values . Bnr1 fusions were included as controls for cytokinetic defects . Student ' s t ‐ test was applied to compare matched Clb2m versus Clb2mΔCdk fusion strains . ‐ P > 0 . 05 ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C FACS analysis of DNA content after conditional Clb2m or Clb2mΔCdk fusion to the indicated proteins. Quantification of the fraction of cells with > 2C DNA content is shown in the bar graph for two independent biological replicates, with red dots representing individual and bars representing average values. Bnr1 fusions were included as controls for cytokinetic defects. Student's t‐test was applied to compare matched Clb2m versus Clb2mΔCdk fusion strains. ‐P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.


D Serial dilutions of cells from ( C ) were plated 6 h following β ‐ estradiol treatment and grown for 1 day at 36 ° C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Serial dilutions of cells from (C) were plated 6 h following β‐estradiol treatment and grown for 1 day at 36°C.


E The length of cell chains containing a common cytoplasm was scored 6 h after β ‐ estradiol treatment using the GFP ‐ Spo2051 - 91 plasma membrane marker ( n > 150 ) . c , control cells ; scale bars , 10 μm .

E The length of cell chains containing a common cytoplasm was scored 6 h after β‐estradiol treatment using the GFP‐Spo2051-91 plasma membrane marker (n > 150). c, control cells; scale bars, 10 μm.


A Genes corresponding to the indicated proteins were replaced by wild ‐ type ( wt ) or Cdk phospho ‐ site mutant ( xA ) sequences and then conditionally fused to Clb2m or Clb2mΔCdk . Cytokinetic defects were scored by FACS analysis of DNA content as in Fig C . The individual values from two independent clones ( red dots ) as well as their average ( bars ) are shown . Student ' s t ‐ test was applied to compare matched wt and xA strains . c , control cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Genes corresponding to the indicated proteins were replaced by wild‐type (wt) or Cdk phospho‐site mutant (xA) sequences and then conditionally fused to Clb2m or Clb2mΔCdk. Cytokinetic defects were scored by FACS analysis of DNA content as in Fig C. The individual values from two independent clones (red dots) as well as their average (bars) are shown. Student's t‐test was applied to compare matched wt and xA strains. c, control cells.


B Cell chain formation in the samples from ( A ) . The fraction of cells with > 2 cell bodies for each clone are represented with red dots and their averages by the black bar ( n > 150 ) . Significance is determined by Student ' s t ‐ tests for the indicated conditions . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Cell chain formation in the samples from (A). The fraction of cells with > 2 cell bodies for each clone are represented with red dots and their averages by the black bar (n > 150). Significance is determined by Student's t‐tests for the indicated conditions. Scale bars, 5 μm.


C The indicated strains were spotted in serial dilutions onto auxin ‐ containing plates and grown for 2 days .

C The indicated strains were spotted in serial dilutions onto auxin‐containing plates and grown for 2 days.


D Auxin was added to cultures of the indicated strains , and after 6 h , cells were sonicated and the number of attached cell bodies was counted ( n > 200 ) . Significance was calculated using a Wilcoxon rank ‐ sum test .

O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Auxin was added to cultures of the indicated strains, and after 6 h, cells were sonicated and the number of attached cell bodies was counted (n > 200). Significance was calculated using a Wilcoxon rank‐sum test.


E The fraction of cells in asynchronously proliferating cultures of the indicated strains with a DNA content of > 2C was determined by FACS analysis ( n = 3 ) . Error bars represent SD .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

E The fraction of cells in asynchronously proliferating cultures of the indicated strains with a DNA content of > 2C was determined by FACS analysis (n = 3). Error bars represent SD.


A Cyk3 ‐ GFP signal intensity at the GFP relative to the onset of actomyosin ring contraction ( t = 0 ) . The mean and SEM from at least 20 cells are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Cyk3‐GFP signal intensity at the GFP relative to the onset of actomyosin ring contraction (t = 0). The mean and SEM from at least 20 cells are shown.


B Inn1 ‐ Clb2m , Inn1 ‐ Clb2mΔCdk and control cells before Clb2m fusion were arrested in G1 using α ‐ factor , released and harvested 90 - 100 min later at the time of cytokinesis . Indirect immunofluorescence microscopy was used to determine Inn1bud neck staining and the spindle status . Representative images of full spindles and partially and fully disassembled spindles are shown . nr , non ‐ recombined cells . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Inn1‐Clb2m, Inn1‐Clb2mΔCdk and control cells before Clb2m fusion were arrested in G1 using α‐factor, released and harvested 90-100 min later at the time of cytokinesis. Indirect immunofluorescence microscopy was used to determine Inn1bud neck staining and the spindle status. Representative images of full spindles and partially and fully disassembled spindles are shown. nr, non‐recombined cells. Scale bars, 5 μm.


C The time from anaphase onset until the GFP appearance of Inn1 was determined by filming synchronized Inn1 ‐ Clb2m ‐ GFP and Inn1 ‐ Clb2mΔCdk ‐ GFP cells containing Spc42 ‐ YFP ‐ labeled spindle pole bodies . Error bars represent SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C The time from anaphase onset until the GFP appearance of Inn1 was determined by filming synchronized Inn1‐Clb2m‐GFP and Inn1‐Clb2mΔCdk‐GFP cells containing Spc42‐YFP‐labeled spindle pole bodies. Error bars represent SD.


D As in ( C ) , now the persistence of a full actomyosin ring was measured in cells expressing Inn1 ‐ Clb2m , Inn1 ‐ Clb2mΔCdk or their respective non ‐ phosphorylatable mutants together with Myo1 ‐ GFP and Spc42 ‐ YFP . Error bars represent SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D As in (C), now the persistence of a full actomyosin ring was measured in cells expressing Inn1‐Clb2m, Inn1‐Clb2mΔCdk or their respective non‐phosphorylatable mutants together with Myo1‐GFP and Spc42‐YFP. Error bars represent SD.


E Montage of medial view projections of actomyosin rings in Inn1 ‐ Clb2m and Inn1 ‐ Clb2mΔCdk cells . Frames are shown in 2 ‐ min intervals . F Actomyosin ring contraction phenotypes from at least 21 cells filmed as in ( E ) were blindly scored as having a normal ( " constriction " ) or aberrant ( " disintegration " ) cytokinetic phenotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Montage of medial view projections of actomyosin rings in Inn1‐Clb2m and Inn1‐Clb2mΔCdk cells. Frames are shown in 2‐min intervals.F Actomyosin ring contraction phenotypes from at least 21 cells filmed as in (E) were blindly scored as having a normal ("constriction") or aberrant ("disintegration") cytokinetic phenotype.


G Cytokinetic defect and cell viability of Inn1 ‐ Clb2m or Inn1 ‐ Clb2mΔCdk cells in the absence or presence of Chs2V377I . In the bar graph , red dots represent values of duplicate biological replicates with bars representing the average . Significance is calculated using Student ' s t ‐ test . 10 ‐ fold spot dilution assays are represented , together with the Western blotting analysis for Inn1 before and after its fusion to Clb2m . Tub1 served as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Cytokinetic defect and cell viability of Inn1‐Clb2m or Inn1‐Clb2mΔCdk cells in the absence or presence of Chs2V377I. In the bar graph, red dots represent values of duplicate biological replicates with bars representing the average. Significance is calculated using Student's t‐test. 10‐fold spot dilution assays are represented, together with the Western blotting analysis for Inn1 before and after its fusion to Clb2m. Tub1 served as a loading control.


( A ) Overlay of 15N - 1H HSQC spectra of 15N - labeled Ca2 + / CaM by itself ( red peaks ) and after addition of saturating , unlabeled PSD - 95 ( 1 - 21 ) ( blue peaks ) or saturating , unlabeled PSD - 95 _ pT19 ( green peaks ) . All NMR experiments were recorded at 600 MHz 1H frequency .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Overlay of 15N-1H HSQC spectra of 15N-labeled Ca2+/CaM by itself (red peaks) and after addition of saturating, unlabeled PSD-95 (1-21) (blue peaks) or saturating, unlabeled PSD-95_pT19 (green peaks). All NMR experiments were recorded at 600 MHz 1H frequency.


( B ) Chemical shift perturbation of CaM amide resonances in the absence vs . presence of PSD - 95 _ pT19 . Chemical shift difference ( CSD ) plotted on the vertical axis was defined as CSD = { ( HNA - HNB ) 2 + ( 15NA - 15NB ) 2 } 1 / 2 , where " A " and " B " designate free and bound states of CaM . HN and 15N represent amide 1H and 15N chemical shifts , respectively . Perturbations greater than the average were used as active ambiguous restraints in the Haddock docking calculation ( see Methods ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Chemical shift perturbation of CaM amide resonances in the absence vs. presence of PSD-95_pT19. Chemical shift difference (CSD) plotted on the vertical axis was defined as CSD = {(HNA - HNB)2 + (15NA - 15NB)2}1/2 , where "A" and "B" designate free and bound states of CaM. HN and 15N represent amide 1H and 15N chemical shifts, respectively. Perturbations greater than the average were used as active ambiguous restraints in the Haddock docking calculation (see Methods).


( C ) Representative data from IPAP [ 15N - 1H ] - HSQC spectra of 15N - labeled Ca2 + / CaM bound to PSD - 95 _ pT19 in the absence ( blue ) and presence of 17 mg / ml filamentous bacteriophage Pf1 ( red ) . Residual dipolar couplings ( RDCs ) for backbone HN resonances are calculated ( in Hertz ) as the difference in splitting for the 15N - { 1H } doublet components ( red peaks ) relative to the isotropic 1JNH coupling ( blue peaks ) . RDC values are calculated to be + 40 , + 30 , + 20 , + 20 , and - 20 Hz for I100 , I27 , N137 , D64 , and I130 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative data from IPAP [15N-1H]-HSQC spectra of 15N-labeled Ca2+/CaM bound to PSD-95_pT19 in the absence (blue) and presence of 17 mg/ml filamentous bacteriophage Pf1 (red). Residual dipolar couplings (RDCs) for backbone HN resonances are calculated (in Hertz) as the difference in splitting for the 15N-{1H} doublet components (red peaks) relative to the isotropic 1JNH coupling (blue peaks). RDC values are calculated to be +40, +30, +20, +20, and -20 Hz for I100, I27, N137, D64, and I130, respectively.


( D ) Correlation between observed RDCs ( panel C ) versus RDCs calculated from the final NMR derived structure of Ca2 + / CaM bound to PSD - 95 _ pT19 . The correlation coefficient ( R ) equals 0 . 99 and Q is 0 . 11 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Correlation between observed RDCs (panel C) versus RDCs calculated from the final NMR derived structure of Ca2+/CaM bound to PSD-95_pT19. The correlation coefficient (R) equals 0.99 and Q is 0.11.


( A ) NMR structure of Ca2 + / CaM ( grey ) bound to unphosphorylated PSD - 95 . PSD - 95 residues D2 ( depicted as starting point ) to R13 are resolved and form an - helix ( cyan ) ( Zhang et al . , 2014 ) . Residues 14 - 19 of unphosphorylated PSD - 95 are unstructured and not shown . Ca2 + ions bound to CaM are depicted by orange spheres .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

(A) NMR structure of Ca2+/CaM (grey) bound to unphosphorylated PSD-95. PSD-95 residues D2 (depicted as starting point) to R13 are resolved and form an -helix (cyan) (Zhang et al., 2014). Residues 14-19 of unphosphorylated PSD-95 are unstructured and not shown. Ca2+ ions bound to CaM are depicted by orange spheres.


( B ) NMR structure of Ca2 + / CaM bound to PSD - 95 phosphorylated at T19 ( PDB accession no . 5J7J ) . Residues 14 - 19 of phosphorylated PSD - 95 adopt a hook - like structure ( highlighted in red ) not seen in unphosphorylated PSD - 95 . This hook - like structure is stabilized by PSD - 95 residues E17 and pT19 forming salt bridges with R126 and K115 , respectively . Side - chain atoms are depicted by a stick representation for PSD - 95 residues Q15 , E17 and T19 ( red ) and CaM residues K115 , T117 , and R126 ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) NMR structure of Ca2+/CaM bound to PSD-95 phosphorylated at T19 (PDB accession no. 5J7J). Residues 14-19 of phosphorylated PSD-95 adopt a hook-like structure (highlighted in red) not seen in unphosphorylated PSD-95. This hook-like structure is stabilized by PSD-95 residues E17 and pT19 forming salt bridges with R126 and K115, respectively. Side-chain atoms are depicted by a stick representation for PSD-95 residues Q15, E17 and T19 (red) and CaM residues K115, T117, and R126 (blue).


( A ) FP titrations showing the binding of wild - type ( WT ) CaM to PSD - 95 ( 1 - 21 ) peptides , either WT ( blue ) , Q15A ( salmon ) , E17R ( black ) , T19E ( green ) , or T19K ( yellow ) . The solid lines represent optimal theoretical curves calculated using a one - site binding model described in Methods . The calculated dissociation constants in each case are given in Table 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) FP titrations showing the binding of wild-type (WT) CaM to PSD-95 (1-21) peptides, either WT (blue), Q15A (salmon), E17R (black), T19E (green), or T19K (yellow). The solid lines represent optimal theoretical curves calculated using a one-site binding model described in Methods. The calculated dissociation constants in each case are given in Table 1.


( B ) FP titrations showing the binding of R126E CaM to PSD - 95 peptides , either WT ( blue ) or E17R ( black ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) FP titrations showing the binding of R126E CaM to PSD-95 peptides, either WT (blue) or E17R (black).


( C ) FP titrations showing the binding of K115E CaM to PSD - 95 peptides T19K ( yellow ) or T19E ( green ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) FP titrations showing the binding of K115E CaM to PSD-95 peptides T19K (yellow) or T19E (green).


Cultured cortical neurons were treated with BIC ( 50 μM ) at 15 DIV for 48h or at 17 DIV for 2 h or left untreated before harvesting , extraction , and analysis of palmitoylation by the biotin switch method and pull down with NeutrAvidin - agarose beads . ( A ) Representative immunoblots of pull down samples ( top two panels ) and total lysate ( bottom two panels ) for PSD - 95 and , as control , the trimeric G protein subunit G - i3 . Omission of NH2OH before biotinylation resulted in no NeutrAvidin pull down as negative control for non - specific pull down .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

Cultured cortical neurons were treated with BIC (50 μM) at 15 DIV for 48h or at 17 DIV for 2 h or left untreated before harvesting, extraction, and analysis of palmitoylation by the biotin switch method and pull down with NeutrAvidin-agarose beads. (A) Representative immunoblots of pull down samples (top two panels) and total lysate (bottom two panels) for PSD-95 and, as control, the trimeric G protein subunit G-i3. Omission of NH2OH before biotinylation resulted in no NeutrAvidin pull down as negative control for non-specific pull down.


( B ) Quantification of PSD - 95 palmitoylation . Seven samples from three independent sets of cultures were analyzed per condition ( * * p < 0 . 01 ; t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Quantification of PSD-95 palmitoylation. Seven samples from three independent sets of cultures were analyzed per condition (**p<0.01; t-test).


Cultured hippocampal neurons were infected at 14 DIV with lentivirus for expression of both shRNA against PSD - 95 ( sh95 ) and sh - resistant PSD - 95 - EGFP . Thus endogenous PSD - 95 was replaced with either wild - type ( WT ) , E17R , or T19K PSD - 95 . Cultures were treated with or without BIC ( 50 μM ) at 17 DIV for 24 - 48 h or left untreated before fixation and staining for surface GluA1 . ( A ) Representative confocal microscopic images of PSD - 95 - EGFP fluorescence ( green ) and surface GluA1 immunostaining ( red ) at low ( top panels ; scale bar : 20 µm ) and high enlargement ( bottom panels ; scale bar : 5 µm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cultured hippocampal neurons were infected at 14 DIV with lentivirus for expression of both shRNA against PSD-95 (sh95) and sh-resistant PSD-95-EGFP. Thus endogenous PSD-95 was replaced with either wild-type (WT), E17R, or T19K PSD-95. Cultures were treated with or without BIC (50 μM) at 17 DIV for 24-48 h or left untreated before fixation and staining for surface GluA1. (A) Representative confocal microscopic images of PSD-95-EGFP fluorescence (green) and surface GluA1 immunostaining (red) at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm).


( B ) Quantification of surface GluA1 intensity from ( A ) showed reduction in AMPAR surface levels during scaling down in neurons expressing WT PSD - 95 but not in those expressing either E17R or T19K mutant . Three dendritic segments from each of 31 - 42 neurons from three independent sets of cultures were analyzed per condition ( * * p < 0 . 01 ; n . s . , not significant ; one - way ANOVA followed by Bonferroni ' s post hoc test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Quantification of surface GluA1 intensity from (A) showed reduction in AMPAR surface levels during scaling down in neurons expressing WT PSD-95 but not in those expressing either E17R or T19K mutant. Three dendritic segments from each of 31-42 neurons from three independent sets of cultures were analyzed per condition (**p<0.01; n.s., not significant; one-way ANOVA followed by Bonferroni's post hoc test).


Cultured hippocampal neurons were co - transfected with mCherry and EGFP - tagged PSD - 95 wild - type ( WT ) , E17R , or T19K at 10 - 11 DIV and treated with BIC ( 50 μM ) or left untreated at 17 DIV for 24 - 48 h before fixation . ( A ) Representative confocal microscopic images of PSD - 95 - EGFP ( green ) and mCherry ( red ) used as " fill " to visualize the dendrite with spine heads ( scale bar : 2 µm ) after control ( top ) and BIC treatment ( bottom ) . Individual channels are shown in grayscale .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

Cultured hippocampal neurons were co-transfected with mCherry and EGFP-tagged PSD-95 wild-type (WT), E17R, or T19K at 10-11 DIV and treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation. (A) Representative confocal microscopic images of PSD-95-EGFP (green) and mCherry (red) used as "fill" to visualize the dendrite with spine heads (scale bar: 2 µm) after control (top) and BIC treatment (bottom). Individual channels are shown in grayscale.


( B ) BIC treatment reduced spine enrichment of WT but not E17R or T19K PSD - 95 as quantified as the ratio of EGFP intensity in spine heads relative to that in the adjacent dendritic shaft . Values were normalized to control treatments , which were set to equal 100 % for each PSD - 95 construct .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) BIC treatment reduced spine enrichment of WT but not E17R or T19K PSD-95 as quantified as the ratio of EGFP intensity in spine heads relative to that in the adjacent dendritic shaft. Values were normalized to control treatments, which were set to equal 100% for each PSD-95 construct.


( C ) mCherry showed no enrichment in spines and BIC had no effect on its localization .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

(C) mCherry showed no enrichment in spines and BIC had no effect on its localization.


( D - F ) Spine enrichment and puncta size and intensity of PSD - 95 were comparable under basal conditions for WT , E17R , and T19K PSD - 95 ( A . U . , arbitrary unit ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-F) Spine enrichment and puncta size and intensity of PSD-95 were comparable under basal conditions for WT, E17R, and T19K PSD-95 (A.U., arbitrary unit).


( G - I ) Spine density , head diameter , and length were comparable under basal conditions for neurons expressing WT , E17R , and T19K PSD - 95 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-I) Spine density, head diameter, and length were comparable under basal conditions for neurons expressing WT, E17R, and T19K PSD-95.


Cultured hippocampal neurons were transfected at 10 - 11 DIV with the indicated CaM - IRES - ECFP plasmids and infected at 14 DIV with lentivirus expressing shRNA against PSD - 95 ( sh95 ) and sh - resistant wild - type ( WT ) or E17R PSD - 95 - EGFP . Cultures were treated with BIC ( 50 μM ) or left untreated at 17 DIV for 24 - 48 h before fixation . ( A ) Representative confocal microscopic images of intrinsic ECFP and EGFP fluorescence , and surface GluA1 immunostaining at low ( top panels ; scale bar : 20 µm ) and high enlargement ( bottom panels ; scale bar : 5 µm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cultured hippocampal neurons were transfected at 10-11 DIV with the indicated CaM-IRES-ECFP plasmids and infected at 14 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. Cultures were treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation. (A) Representative confocal microscopic images of intrinsic ECFP and EGFP fluorescence, and surface GluA1 immunostaining at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm).


( B ) Quantification of surface GluA1 intensity from ( A ) showed impaired AMPAR downscaling in neurons expressing PSD - 95 - E17R in presence of wild - type CaM ( CaM - WT ) that was rescued by coexpression of CaM - R126E . Three dendritic segments from each of 18 - 26 neurons from three independent sets of cultures were analyzed per condition ( * * p < 0 . 01 , * p < 0 . 05 vs . untreated basal levels ; one - way ANOVA followed by Bonferroni ' s post hoc test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Quantification of surface GluA1 intensity from (A) showed impaired AMPAR downscaling in neurons expressing PSD-95-E17R in presence of wild-type CaM (CaM-WT) that was rescued by coexpression of CaM-R126E. Three dendritic segments from each of 18-26 neurons from three independent sets of cultures were analyzed per condition (**p<0.01,*p<0.05 vs. untreated basal levels; one-way ANOVA followed by Bonferroni's post hoc test).


Cultured hippocampal neurons were co - transfected at 10 - 11 DIV with RFP and the indicated CaM - IRES - ECFP plasmids and infected at 12 - 13 DIV with lentivirus expressing shRNA against PSD - 95 ( sh95 ) and sh - resistant wild - type ( WT ) or E17R PSD - 95 - EGFP . mEPSC recordings were performed at DIV 17 - 19 , following 48 h of control or BIC ( 50 μM ) treatment . ( A ) Representative traces of AMPAR mEPSC recordings ( left ) and averages of all mEPSC events from single representative neurons ( right ) drawn to scale ( top ) and normalized to peak ( bottom ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cultured hippocampal neurons were co-transfected at 10-11 DIV with RFP and the indicated CaM-IRES-ECFP plasmids and infected at 12-13 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. mEPSC recordings were performed at DIV 17-19, following 48 h of control or BIC (50 μM) treatment. (A) Representative traces of AMPAR mEPSC recordings (left) and averages of all mEPSC events from single representative neurons (right) drawn to scale (top) and normalized to peak (bottom).


( B ) Quantification of mEPSC amplitude revealed downscaling by BIC in neurons expressing PSD - 95WT / CaMWT or PSD - 95E17R / CaMR126E but not PSD - 95WT / CaMR126E ( left ) over the full range of amplitude distributions ( right ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Quantification of mEPSC amplitude revealed downscaling by BIC in neurons expressing PSD-95WT / CaMWT or PSD-95E17R / CaMR126E but not PSD-95WT / CaMR126E (left) over the full range of amplitude distributions (right).


( C , D ) Mini - EPSC frequency ( C ) and decay time ( D ) were comparable among all conditions ( n = 11 - 25 neurons per condition from 8 - 14 independent sets of cultures prepared from the embryos of 8 - 14 dams ; * p < 0 . 05 ; two - way ANOVA followed by Bonferroni ' s test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C,D) Mini-EPSC frequency (C) and decay time (D) were comparable among all conditions (n=11-25 neurons per condition from 8-14 independent sets of cultures prepared from the embryos of 8-14 dams;* p<0.05; two-way ANOVA followed by Bonferroni's test).


A . Expression levels of various GPI - APs in different clones of PIGG - KO HEK293 cells . Fluorescence intensities of NT5E in PIGG - KO clones # 4 , # 10 , # 17 , # 19 and wild type cells are 575 , 910 , 696 , 530 and 1548 while those of EphrinA5 in clones # 4 , # 17 , # 19 and wild type cells are 1431 , 2080 , 1868 and 3336 , respectively . Data information : Each FACS analysis was performed at least three times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Expression levels of various GPI-APs in different clones of PIGG-KO HEK293 cells. Fluorescence intensities of NT5E in PIGG-KO clones #4, #10, #17, #19 and wild type cells are 575, 910, 696, 530 and 1548 while those of EphrinA5 in clones #4, #17, #19 and wild type cells are 1431, 2080, 1868 and 3336, respectively. Data information: Each FACS analysis was performed at least three times.


B . Expression levels of GPI - APs in PIGG - KO clone # 19 cells stably transfected with PIGG cDNA compared to levels in empty vector transfected cells . Fluorescence intensities of NT5E in PIGG cDNA and in vector transfected cells are 1332 and 886 , respectively . Data information : Each FACS analysis was performed at least three times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Expression levels of GPI-APs in PIGG-KO clone #19 cells stably transfected with PIGG cDNA compared to levels in empty vector transfected cells. Fluorescence intensities of NT5E in PIGG cDNA and in vector transfected cells are 1332 and 886, respectively. Data information: Each FACS analysis was performed at least three times.


A . Expression levels of different GPI - APs in PIGG - KO HEK293 cells transiently co - transfected with PIGG cDNA or empty vector . Vertical bars represent % of levels of GPI - APs expression in non - rescued PIGG - KO cells relative to those in PIGG cDNA rescued PIGG - KO cells . Analysis was repeated 3 to 6 times and SD bars were shown .

A. Expression levels of different GPI-APs in PIGG-KO HEK293 cells transiently co-transfected with PIGG cDNA or empty vector. Vertical bars represent % of levels of GPI-APs expression in non-rescued PIGG-KO cells relative to those in PIGG cDNA rescued PIGG-KO cells. Analysis was repeated 3 to 6 times and SD bars were shown.


B . HA tagged NTNG2 and FLAG tagged DAF were transiently co - transfected together with PIGG cDNA or empty vector to PIGG - KO cells and were analyzed by FACS . Fluorescence intensity levels of NTNG2 in PIGG - KO cells and in PIGG rescued cells are 639 and 3426 , while those of DAF in PIGG - KO cells and in PIGG rescued cells are 562 and 599 , respectively . Each FACS analysis was performed at least three times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. HA tagged NTNG2 and FLAG tagged DAF were transiently co-transfected together with PIGG cDNA or empty vector to PIGG-KO cells and were analyzed by FACS. Fluorescence intensity levels of NTNG2 in PIGG-KO cells and in PIGG rescued cells are 639 and 3426, while those of DAF in PIGG-KO cells and in PIGG rescued cells are 562 and 599, respectively. Each FACS analysis was performed at least three times.


( C ) Exemplary images of COS - 7 cells expressing PEX - mRFP - FKBP before ( upper panels ) and 30 minutes after ( middle panels ) rapalog addition in the presence of a translocating motor - KIF1C - MDC - FRB and a non - translocating motor - KIF3B - MDC - FRB . Yellow lines indicate COS7 cell outline . Lower panel represents the overlay of sequential binarized images color coded by time ( see also Appendix Figure S1 ) . Scale bar = 20

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Exemplary images of COS-7 cells expressing PEX-mRFP-FKBP before (upper panels) and 30 minutes after (middle panels) rapalog addition in the presence of a translocating motor - KIF1C-MDC-FRB and a non-translocating motor - KIF3B-MDC-FRB. Yellow lines indicate COS7 cell outline. Lower panel represents the overlay of sequential binarized images color coded by time (see also Appendix Figure S1). Scale bar = 20 µm


( D ) Plots representing intensity time traces of R90 % for COS - 7 cells transfected with PEX - mRFP - FKBP and KIF1C - MDC - FRB ( n = 13 ) or KIF3B - MDC - FRB ( n = 10 ) . R90 % is the radius for each time point that includes 90 % of the total fluorescent intensity . Error bars indicate SEM .

(D) Plots representing intensity time traces of R90% for COS-7 cells transfected with PEX-mRFP-FKBP and KIF1C-MDC-FRB (n =13) or KIF3B-MDC-FRB (n =10). R90% is the radius for each time point that includes 90% of the total fluorescent intensity. Error bars indicate SEM.


E ) Quantitative representation of average peroxisome displacement 30 min after addition of rapalog for COS - 7 cells transfected with kinesin constructs summarized in ( A ) . The number of analyzed cells is summarized in Appendix Table S1 . Kinesins were considered translocators , if they displaced peroxisomes ≥ 5 µm ( marked with a blue dashed line ) in 30 minutes after rapalog addition . Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Quantitative representation of average peroxisome displacement 30 min after addition of rapalog for COS-7 cells transfected with kinesin constructs summarized in (A). The number of analyzed cells is summarized in Appendix Table S1. Kinesins were considered translocators, if they displaced peroxisomes ≥ 5 µm (marked with a blue dashed line) in 30 minutes after rapalog addition. Error bars indicate SEM.


( F ) Quantitative representation of average dispersion speed of peroxisomes recruited to translocating kinesins as calculated from time traces of R90 % ( summarized in Appendix figure S2 , Appendix Table S1 ) . Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Quantitative representation of average dispersion speed of peroxisomes recruited to translocating kinesins as calculated from time traces of R90% (summarized in Appendix figure S2, Appendix Table S1). Error bars indicate SEM.


( A ) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV14 hippocampal neurons expressing PEX - mRFP - FKBP , KIF1C - MDC - FRB , KIF21A - MDC - FRB or KIF5B - MDC - FRB . The morphology of transfected cell is visualized using a BFP fill . Axons are marked with a blue line . Arrows mark peroxisome targeting to dendrites ( gray ) and axon ( blue ) 30 min after rapalog addition . Scale bar = 20 µm .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(A) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV14 hippocampal neurons expressing PEX-mRFP-FKBP, KIF1C-MDC-FRB, KIF21A-MDC-FRB or KIF5B-MDC-FRB. The morphology of transfected cell is visualized using a BFP fill. Axons are marked with a blue line. Arrows mark peroxisome targeting to dendrites (gray) and axon (blue) 30 min after rapalog addition. Scale bar = 20 µm.


( B ) Plots representing intensity time traces of dendrites and axons before and after rapalog addition for KIF1C - MDC - FRB ( n = 6 neurons ) , KIF21A - MDC - FRB ( n = 4 ) and KIF5B - MDC - FRB ( n = 8 ) . Traces were normalized to the average intensity before rapalog addition , and to the background .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

(B) Plots representing intensity time traces of dendrites and axons before and after rapalog addition for KIF1C-MDC-FRB (n = 6 neurons), KIF21A-MDC-FRB (n = 4) and KIF5B-MDC-FRB (n = 8). Traces were normalized to the average intensity before rapalog addition, and to the background.


C ) Quantitative representation of percentage of DIV14 neurons transfected as in ( A ) with constructs showed in Figure 1A , in which peroxisomes after addition of rapalog redistributed to either axons ( " a " , blue bars ) or to axons and at least 2 dendrites ( " a + d " , red bars ) . Kinesins were considered " dendrite targeting " if dendrites were targeted in ≥ 50 % of cells and are highlighted in red . Kinesin constructs are transfected as indicated ; the number of analyzed cells is summarized in Appendix Table S1 . Error bars indicate

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Quantitative representation of percentage of DIV14 neurons transfected as in (A) with constructs showed in Figure 1A, in which peroxisomes after addition of rapalog redistributed to either axons ("a", blue bars) or to axons and at least 2 dendrites ("a+d", red bars). Kinesins were considered "dendrite targeting" if dendrites were targeted in ≥ 50% of cells and are highlighted in red. Kinesin constructs are transfected as indicated; the number of analyzed cells is summarized in Appendix Table S1. Error bars indicate SEM


( D ) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV2 hippocampal neurons transfected as in ( A ) . Scale bars : 20

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(D) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV2 hippocampal neurons transfected as in (A). Scale bars: 20 µm


( E ) Percentage of DIV2 neurons transfected with KIF1C - MDC - FRB ( n = 9 neurons ) , KIF21A - MDC - FRB ( n = 14 ) and KIF5B - MDC - FRB ( n = 14 ) in which peroxisomes redistributed into at least 2 neurites after addition of rapalog and ( F ) Percentage of neurites of DIV2 neurons transfected as in ( E ) targeted with peroxisomes after addition of rapalog . Error bars indicate SEM ; Scale bars : 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(E) Percentage of DIV2 neurons transfected with KIF1C-MDC-FRB (n=9 neurons), KIF21A-MDC-FRB (n=14) and KIF5B-MDC-FRB (n=14) in which peroxisomes redistributed into at least 2 neurites after addition of rapalog and (F) Percentage of neurites of DIV2 neurons transfected as in (E) targeted with peroxisomes after addition of rapalog. Error bars indicate SEM; Scale bars: 20 µm.


( A ) Representative confocal images of a hippocampal neuron ( DIV14 ) immunostained for MAP2 , β - III - tubulin and DCLK1 . Scale bar = 20

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative confocal images of a hippocampal neuron (DIV14) immunostained for MAP2, β-III-tubulin and DCLK1. Scale bar = 20 µm


( B ) Quantification of the average ratio of DCLK1 , MAP1A , MAP1B and MAP2 to total tubulin immunofluorescence intensities in dendrites compared to axons . Per condition 6 - 9 cells were analyzed . Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Quantification of the average ratio of DCLK1, MAP1A, MAP1B and MAP2 to total tubulin immunofluorescence intensities in dendrites compared to axons. Per condition 6-9 cells were analyzed. Error bars indicate SEM.


( C ) Representative images of DIV10 hippocampal neurons transfected for 4 days with plasmids encoding β - gal for visualizing the transfected cells and three different DCLK1 shRNAs or DCLK2 shRNA and immunostained for β - gal ( red ) and DCLK1 ( green ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative images of DIV10 hippocampal neurons transfected for 4 days with plasmids encoding β-gal for visualizing the transfected cells and three different DCLK1 shRNAs or DCLK2 shRNA and immunostained for β-gal (red) and DCLK1 (green).


( D ) Quantitative analysis of normalized average intensity of DCLK1 in regions of interest of DIV10 neurons transfected with DCLK1 sh # 1 ( n = 16 neurons ) , DCLK1 sh # 2 ( n = 16 ) and DCLK1 sh # 3 ( n = 18 ) . Non - transfected cells were taken as a control ( n = 48 ) . N = 2 . Error bars indicate SEM ; * * * - p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison Test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantitative analysis of normalized average intensity of DCLK1 in regions of interest of DIV10 neurons transfected with DCLK1 sh#1 (n = 16 neurons), DCLK1 sh#2 (n=16) and DCLK1 sh#3 (n=18). Non-transfected cells were taken as a control (n = 48). N=2. Error bars indicate SEM; *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test).


( E ) Example of Western blot analysis of protein extracts of DIV4 cortical neurons electroporated on DIV0 with pSUPER ( control ) or pSUPER encoding one of three DCLK1 shRNAs . Levels of actin were used as an equal loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Example of Western blot analysis of protein extracts of DIV4 cortical neurons electroporated on DIV0 with pSUPER (control) or pSUPER encoding one of three DCLK1 shRNAs. Levels of actin were used as an equal loading control.


( F ) Quantification of protein levels of DCLK1 of samples represented in ( E ) . N = 3 , error bars indicate SEM , * * * - p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison Test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Quantification of protein levels of DCLK1 of samples represented in (E). N = 3, error bars indicate SEM, *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test).


( G ) Representative maximum projections of peroxisome distribution in DIV14 neurons transfected with PEX - mRFP - FKBP , KIF1C ( MDC ) - FRB and the indicated shRNA before and 30 minutes after rapalog addition . BFP fill was co - transected to visualize the morphology of transfected neuron . Axons are marked with a blue line . Arrows mark dendrite ( gray ) and axon ( blue ) targeting of peroxisomes after rapalog addition . Scale bar = 20

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

(G) Representative maximum projections of peroxisome distribution in DIV14 neurons transfected with PEX-mRFP-FKBP, KIF1C (MDC)-FRB and the indicated shRNA before and 30 minutes after rapalog addition. BFP fill was co-transected to visualize the morphology of transfected neuron. Axons are marked with a blue line. Arrows mark dendrite (gray) and axon (blue) targeting of peroxisomes after rapalog addition. Scale bar = 20 µm


( H ) Quantitative representation of the percentage of DIV14 neurons transfected with PEX - RFP together with either KIF1C , KIF21A or KIF5B and an indicated shRNA . as in ( A ) in which after addition of rapalog peroxisomes redistributed to axons ( " a " , blue bars ) , to both axons and dendrites ( " a + d " , red bars ) or did not redistribute into any neuronal compartment ( " nt " - no targeting , gray bars ) . Per condition 19 - 44 neurons were analyzed ; N = 2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Quantitative representation of the percentage of DIV14 neurons transfected with PEX-RFP together with either KIF1C, KIF21A or KIF5B and an indicated shRNA. as in (A) in which after addition of rapalog peroxisomes redistributed to axons ("a", blue bars), to both axons and dendrites ("a+d", red bars) or did not redistribute into any neuronal compartment ("nt"-no targeting, gray bars). Per condition 19-44 neurons were analyzed; N = 2.


( A - G ) Representative frame ( A , D ) and a kymograph ( B , F ) of a simultaneous two - color movie of a hippocampal neuron expressing NPY - mCherry and either KIF1A - GFP ( A , B ) , BDNF - GFP ( D ) or TfR - GFP ( F ) . Scale bar = 20 µm ( A , D ) , 5 µm ( B , F ) . ( C ) Quantification of vesicle colocalization in cells described in ( A , B ) The quantifications indicate the % of KIF1Avesicles that are NPY positive - left bar quantified from images of fixed cells presented in ( A ) and right bar quantified from kymographs as presented in ( B ) ( only moving fraction ) . ( E ) Quantification of vesicle colocalization in cells described in ( D ) - left bar indicates the % of NPYvesicles that are BDNF positive , the right bar indicates the % of BDNFvesicles that are NPY positive . ( G ) Quantification of vesicle colocalization in cells described in ( F ) . The left bar indicates the % of NPYvesicles that are TfR positive , the right bar indicates the % of TfR that are NPY positive .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-G) Representative frame (A, D) and a kymograph (B, F) of a simultaneous two-color movie of a hippocampal neuron expressing NPY-mCherry and either KIF1A-GFP (A, B), BDNF-GFP (D) or TfR-GFP (F). Scale bar = 20 µm (A, D), 5 µm (B, F). (C) Quantification of vesicle colocalization in cells described in (A, B) The quantifications indicate the % of KIF1Avesicles that are NPY positive - left bar quantified from images of fixed cells presented in (A) and right bar quantified from kymographs as presented in (B) (only moving fraction). (E) Quantification of vesicle colocalization in cells described in (D) - left bar indicates the % of NPYvesicles that are BDNF positive, the right bar indicates the % of BDNFvesicles that are NPY positive. (G) Quantification of vesicle colocalization in cells described in (F). The left bar indicates the % of NPYvesicles that are TfR positive, the right bar indicates the % of TfR that are NPY positive.


( H , I ) Quantification of run lengths ( H ) and velocities ( I ) of vesicles in dendrites of hippocampal neurons transfected at DIV10 - 14 for 4 days with either NPY - GFP ( n = 924 movements ) or TfR - GFP ( n = 286 ) or KIF1A - GFP ( n = 174 ) together with a morphology marker BFP . N = 2 . Error bars indicate SEM ; * * * p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison Test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H, I) Quantification of run lengths (H) and velocities (I) of vesicles in dendrites of hippocampal neurons transfected at DIV10-14 for 4 days with either NPY-GFP (n = 924 movements) or TfR-GFP (n = 286) or KIF1A-GFP (n = 174) together with a morphology marker BFP. N = 2. Error bars indicate SEM; *** p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test ).


( K ) Representative kymographs of NPY - GFP - labeled dense core vesicle motility in primary dendrites of transfected neurons . DIV10 - 14 hippocampal neurons were cotransfected , for 4 days , with plasmids encoding NPY - GFP , BFP fill and a KIF21 shRNA mix ( n = 30 ) , KIF1shRNA mix ( n = 31 ) , KIF1A shRNA ( n = 19 ) , KIF1B shRNA ( n = 19 ) or KIF1C shRNA ( n = 11 ) . pSUPER was used as a control ( n = 15 ) . Scale bar = 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Representative kymographs of NPY-GFP-labeled dense core vesicle motility in primary dendrites of transfected neurons. DIV10-14 hippocampal neurons were cotransfected, for 4 days, with plasmids encoding NPY-GFP, BFP fill and a KIF21 shRNA mix (n = 30), KIF1shRNA mix (n = 31), KIF1A shRNA (n = 19), KIF1B shRNA (n = 19) or KIF1C shRNA (n = 11). pSUPER was used as a control (n = 15). Scale bar = 5 µm.


( L ) Quantification of the average number of dense core vesicle entries into the primary dendrite in 100 s after photobleaching . N = 2 - 3 . Error bars indicate SEM ; * * * - p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison Test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) Quantification of the average number of dense core vesicle entries into the primary dendrite in 100 s after photobleaching. N = 2-3. Error bars indicate SEM; *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test).


( M ) Representative kymographs of TfR - GFP - labeled recycling endosome motility in primary dendrites . DIV10 - 14 Hippocampal neurons were cotransfected with TfR - GFP together with a BFP fill and a KIF1shRNA mix ( n = 11 ) . pSUPER was used as a control ( n = 20 ) . Scale bar = 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Representative kymographs of TfR-GFP-labeled recycling endosome motility in primary dendrites. DIV10-14 Hippocampal neurons were cotransfected with TfR-GFP together with a BFP fill and a KIF1shRNA mix (n = 11). pSUPER was used as a control (n = 20). Scale bar = 5 µm.


( N ) Quantification of the average number of recycling endosome entries into the primary dendrite in 50 s . N = 2 . Error bars indicate SEM . ns - not significant ( Mann - Whitney test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(N) Quantification of the average number of recycling endosome entries into the primary dendrite in 50 s. N = 2. Error bars indicate SEM. ns - not significant (Mann-Whitney test).


( O ) Representative images of hippocampal neurons ( DIV4 ) cotransfected with an empty pSUPER ( control ) or pSUPER encoding an indicated shRNA together with a GFP fill fixed at DIV8 and immunostained for MAP2 and sodium channels ( AIS ) . Scale bar = 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(O) Representative images of hippocampal neurons (DIV4) cotransfected with an empty pSUPER (control) or pSUPER encoding an indicated shRNA together with a GFP fill fixed at DIV8 and immunostained for MAP2 and sodium channels (AIS). Scale bar = 50 µm.


( P ) Sholl analysis of cells described in ( O ) Error bars indicate SEM ; * - p < 0 . 05 , * * - p < 0 . 01 , and * * * - p < 0 . 001 ( two - way ANOVA test followed by Bonferroni ' s post hoc test ) . ( R ) Analysis of the total dendritic length of cells described in ( O ) Error bars indicate SEM ; * - p < 0 . 05 , * * - p < 0 . 01 and * * * - p < 0 . 001 ( 1 - way ANOVA followed by a Tukey ' s Multiple Comparison Test ) . Per condition 21 - 24 neurons ( n ) were analyzed ; N = 2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(P) Sholl analysis of cells described in (O) Error bars indicate SEM; * - p < 0.05, ** - p < 0.01, and *** - p < 0.001 (two-way ANOVA test followed by Bonferroni's post hoc test). (R) Analysis of the total dendritic length of cells described in (O) Error bars indicate SEM; * - p < 0.05, ** - p < 0.01 and *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test). Per condition 21-24 neurons (n) were analyzed; N = 2.


( B ) Representative kymographs of dense - core vesicle motility in proximal dendrites of transfected neurons . DIV 10 - 14 hippocampal neurons were co - transfected for 4 days with NPY - GFP and pSUPER ( control ) or pSUPER encoding DCLK1 shRNA in the absence or presence of DCLK1 truncation constructs and dense core vesicle motility in dendrites was recorded during live - cell imaging . Scale bar = 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Representative kymographs of dense-core vesicle motility in proximal dendrites of transfected neurons. DIV 10-14 hippocampal neurons were co-transfected for 4 days with NPY-GFP and pSUPER (control) or pSUPER encoding DCLK1 shRNA in the absence or presence of DCLK1 truncation constructs and dense core vesicle motility in dendrites was recorded during live-cell imaging. Scale bar = 5 µm.


( C ) Quantification of the average number of dense - core vesicle entries into dendrites of neurons transfected as in ( B ) during 100 s of the live recording after photobleaching . Per condition 10 - 32 cells were analyzed . Error bars indicate SEM ; * * * - p < 0 . 001 ( Kruskal - Wallis test followed by post hoc Dunn ' s test ) . N = 2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of the average number of dense-core vesicle entries into dendrites of neurons transfected as in (B) during 100 s of the live recording after photobleaching. Per condition 10-32 cells were analyzed. Error bars indicate SEM; *** - p < 0.001 (Kruskal-Wallis test followed by post hoc Dunn's test). N = 2.


( D , E ) DIV10 - 14 hippocampal neurons were co - transfected for 4 days with either pSUPER ( control , n = 20 neurons ) or DCLK1 shRNA together with TfR - GFP ( n = 16 ) . ( D ) Representative kymographs of recycling endosome motility in dendrites . Scale bar = 5 µm . ( E ) Quantification of the average number of recycling endosome entries into dendrites during 50 s of the live recording after photobleaching . Error bars indicate SEM ; ns - not significant ( Mann - Whitney test ) . N = 2 .

(D, E) DIV10-14 hippocampal neurons were co-transfected for 4 days with either pSUPER (control, n = 20 neurons) or DCLK1 shRNA together with TfR-GFP (n = 16).(D) Representative kymographs of recycling endosome motility in dendrites. Scale bar = 5 µm. (E) Quantification of the average number of recycling endosome entries into dendrites during 50 s of the live recording after photobleaching. Error bars indicate SEM; ns - not significant (Mann-Whitney test). N = 2.


( A ) Example of Western blot analysis of DCLK1 and DCX levels in protein extracts obtained from DIV1 , DIV7 , DIV14 and DIV21 hippocampal neurons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Example of Western blot analysis of DCLK1 and DCX levels in protein extracts obtained from DIV1, DIV7, DIV14 and DIV21 hippocampal neurons.


( B ) Representative image of a hippocampal neuron ( DIV14 ) immunostained for DCLK1 and β - III - tubulin . Scale bar = 20 µm .

(B) Representative image of a hippocampal neuron (DIV14) immunostained for DCLK1 and β-III-tubulin. Scale bar = 20 µm.


( C , D ) Representative image of a COS - 7 cell transfected with DCLK1 - GFP ( C ) or DCLK1 ( ΔKD ) - GFP ( D ) and stained for detyrosinated , tyrosinated or α - tubulin . ( E ) Representative image of a COS - 7 cell transfected with DCLK1 - GFP and TagRFP - EB3 and recorded during live - cell imaging . Representative GFP was drawn ( over a region marked with white boxes ) showing that DCLK1 - GFP colocalizes with EB3 - TagRFP - T . Scale bars = 20 um .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Representative image of a COS-7 cell transfected with DCLK1-GFP (C) or DCLK1(ΔKD)-GFP (D) and stained for detyrosinated, tyrosinated or α-tubulin.(E) Representative image of a COS-7 cell transfected with DCLK1-GFP and TagRFP-EB3 and recorded during live-cell imaging. Representative GFP was drawn (over a region marked with white boxes) showing that DCLK1-GFP colocalizes with EB3-TagRFP-T. Scale bars = 20 um.


( A ) Extracts of DIV 7 hippocampal neurons electroporated with pSuper control or with three different DCLK1 shRNAs at DIV0 . Samples were analyzed by western blot with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Extracts of DIV 7 hippocampal neurons electroporated with pSuper control or with three different DCLK1 shRNAs at DIV0. Samples were analyzed by western blot with indicated antibodies.


( B , E ) Representative images of a hippocampal cell transfected at DIV10 with RFP ( to visualize transfected cells ) , pSUPER ( control ) or DCLK1 shRNA , fixed at DIV14 and stained for EB3 . Black lines indicate the neuronal soma outline . Scale bars = 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, E) Representative images of a hippocampal cell transfected at DIV10 with RFP (to visualize transfected cells), pSUPER (control) or DCLK1 shRNA, fixed at DIV14 and stained for EB3. Black lines indicate the neuronal soma outline. Scale bars = 10 µm.


( C , D ) Quantification of the number of EB3 comets in the dendrite ( C ) and in the soma ( D ) . 25 - 26 neurons were analyzed per condition , N = 2 . Error bars indicate SEM , * * * - p < 0 . 001 , * * - p < 0 , 01 ( unpaired t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Quantification of the number of EB3 comets in the dendrite (C) and in the soma (D). 25-26 neurons were analyzed per condition, N=2. Error bars indicate SEM, *** - p < 0.001, ** - p<0,01 (unpaired t-test).


( F - I ) Hippocampal neurons were co - transfected with MACF18 - GFP and pSUPER ( control ) or DCLK1 shRNA and the dynamics of microtubules in dendrites was traced using life - cell microscopy . ( F ) Representative kymographs of dynamic microtubules ends growth in dendrites with and without photoablation . Scale bar = 10 µm ( C ) . ( G , I ) Quantification of the fraction of MACF18 events moving retrogradely and anterogradely in a dendrite before ( G ) and after ( I ) photoablation . Eighteen neurons were analyzed per condition ; N = 2 . ( H ) Scheme of live - cell imaging after photoablation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F-I) Hippocampal neurons were co-transfected with MACF18-GFP and pSUPER (control) or DCLK1 shRNA and the dynamics of microtubules in dendrites was traced using life-cell microscopy. (F) Representative kymographs of dynamic microtubules ends growth in dendrites with and without photoablation. Scale bar = 10 µm (C). (G, I) Quantification of the fraction of MACF18 events moving retrogradely and anterogradely in a dendrite before (G) and after (I) photoablation. Eighteen neurons were analyzed per condition; N=2. (H) Scheme of live-cell imaging after photoablation.


( J , K ) Biotin pull - downs ( PD ) from extracts of HEK293 transfected with biotin - tagged DCLK1 and ( J ) KIF1A - MDC - HA - FRB , KIF1C - MDC - HA - FRB , KIF5B - MDC - HA - FRB constructs and probed for HA / DCLK1 , ( K ) different GFP - tagged KIF1A truncation constructs and probed for GFP / DCLK1 . The ratio input / pellet is 2 % for all pull - down experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J, K) Biotin pull-downs (PD) from extracts of HEK293 transfected with biotin-tagged DCLK1 and (J) KIF1A-MDC-HA-FRB, KIF1C-MDC-HA-FRB, KIF5B-MDC-HA-FRB constructs and probed for HA/DCLK1, (K) different GFP-tagged KIF1A truncation constructs and probed for GFP/DCLK1. The ratio input/pellet is 2% for all pull-down experiments.


( L ) GFP pull - down from extracts of HEK293 transfected with DCLK1 fragments and KIF1A - MDC - HA - FRB , KIF1C - MDC - HA - FRB and KIF1C - MD - HA and probed for HA / GFP . LacZ - HA was used as a negative control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) GFP pull-down from extracts of HEK293 transfected with DCLK1 fragments and KIF1A-MDC-HA-FRB, KIF1C-MDC-HA-FRB and KIF1C-MD-HA and probed for HA/GFP. LacZ-HA was used as a negative control.


B . IHLs were isolated from the liver of Tie2 - GFP mice intrasplenically injected with NaCl ( n = 3 ) or with 5x103 CT26 at the specified time points ( day 25 post - injection n = 6 , day 35 post - injection n = 5 ) . The number of GFP + TEMs was estimated by flow cytometry as 7AAD - / CD45 + / CD11b + / CD11c - / GFP + cells per liver ; data pooled from three independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. IHLs were isolated from the liver of Tie2-GFP mice intrasplenically injected with NaCl (n=3) or with 5x103 CT26 at the specified time points (day 25 post-injection n=6, day 35 post-injection n=5). The number of GFP+ TEMs was estimated by flow cytometry as 7AAD-/CD45+/CD11b+/CD11c-/GFP+ cells per liver; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.