( D ) Increased appearance of GFP - LC3 positive vesicles in PI4K2A K / O cells and reduced acidification of the LC3 - positive vesicles using the GFP - mCherry - LC3 reporter . Cells were transfected with the indicated constructs for one day and observed by confocal microscopy . Scale bars : 20 µm . ( E ) Quantification of the LC3 positive vesicles . ( n = 67 , 34 , 26 , 13 , 30 , 27 for the various groups , respectively from left to right ) . * * * designates P < 0 . 0001 using unpaired t - test . The same data set was also analyzed with the non - parametric Mann - Whitney test showing a P = 0 . 0004 and 0 . 0012 for # 20 and # 26 clones relative to control , respectively . ( F ) Co - localization analysis using the Pearson coefficient ( n = 31 , 21 and 14 cells analyzed for the different groups from left to right ) . * * * designates P < 0 . 0001 using unpaired t - test . The same data set was also analyzed with the non - parametric Mann - Whitney test showing a P value < 0 . 0001 for both groups .

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(D) Increased appearance of GFP-LC3 positive vesicles in PI4K2A K/O cells and reduced acidification of the LC3-positive vesicles using the GFP-mCherry-LC3 reporter. Cells were transfected with the indicated constructs for one day and observed by confocal microscopy. Scale bars: 20 µm. (E) Quantification of the LC3 positive vesicles. (n = 67, 34, 26, 13, 30, 27 for the various groups, respectively from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P = 0.0004 and 0.0012 for #20 and #26 clones relative to control, respectively. (F) Co-localization analysis using the Pearson coefficient (n = 31, 21 and 14 cells analyzed for the different groups from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P value < 0.0001 for both groups.


( A , B ) Distribution of mCherry - GFP - LC3 in HEK293 - AT1 cells ( without starvation ) after treatment with control siRNA ( A ) or siRNA for PIP5Kγ ( PIP5K1C ) . Scale bars : 20 µm . Note the massive tubulation of the LC3 compartment in some of the knocked - down cells ( B , lower images , scale bar : 10 µm ) . ( C ) Comparison of Pearson coefficients from cells treated with control or PIP5Kγ RNAi . For statistical analysis the unpaired t - test was used ( n = 50 and 45 cells for control a PIP5Kγ knock down groups , respectively ; * * * P < 0 . 0001 ) . ( D ) Representative confocal images of live HEK293 - AT1 cells expressing GFP - Rab7 treated with control or PIP5Kγ RNAi . Note the tubulation in the PIP5Kγ RNAi - treated cells . Scale bars : 10 µm .

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(A, B) Distribution of mCherry-GFP-LC3 in HEK293-AT1 cells (without starvation) after treatment with control siRNA (A) or siRNA for PIP5Kγ (PIP5K1C). Scale bars: 20 µm. Note the massive tubulation of the LC3 compartment in some of the knocked-down cells (B, lower images, scale bar: 10 µm). (C) Comparison of Pearson coefficients from cells treated with control or PIP5Kγ RNAi. For statistical analysis the unpaired t-test was used (n = 50 and 45 cells for control a PIP5Kγ knock down groups, respectively; *** P< 0.0001). (D) Representative confocal images of live HEK293-AT1 cells expressing GFP-Rab7 treated with control or PIP5Kγ RNAi. Note the tubulation in the PIP5Kγ RNAi-treated cells. Scale bars: 10 µm.


( A ) HEK293 - AT1 cells were transfected with PLEKHM1 - GFP - Rab7 along with CFP - FKBP - PIP5Kγ and iRFP - FRB - Rab7 constructs for one day and examined live by confocal microscopy . Representative cells show reduced PLEKHM1 localization when PIP5Kγ was recruited to the Rab7 endosomes ( lower row images ) . ( B ) This response is reduced in PI4K2A K / O cells . ( Data information : Scale bars : 20 µm .

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(A) HEK293-AT1 cells were transfected with PLEKHM1-GFP-Rab7 along with CFP-FKBP-PIP5Kγ and iRFP-FRB-Rab7 constructs for one day and examined live by confocal microscopy. Representative cells show reduced PLEKHM1 localization when PIP5Kγ was recruited to the Rab7 endosomes (lower row images). (B) This response is reduced in PI4K2A K/O cells. ( Data information: Scale bars: 20 µm.


( C ) Quantification of these changes by BRET analysis where the full - length PLEKHM1 was fused to Sluc and Venus was fused to Rab7 in the BRET construct . For PIP5Kγ recruitment a mutant dark CFP ( W66A ) - FKBP - PIP5Kγ was used with an iRFP - FRB - Rab7 . BRET values were normalized to those of DMSO treated cells . Comparisons were made for parental ( blue ) and PI4K2A K / O ( green and orange ) cells . ( D ) Similar BRET analysis where the PLEKHM1 was replaced by either RILP or Vps35 in the BRET construct . D Data information Means ± S . E . M . are shown from three experiments performed in triplicates .

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(C) Quantification of these changes by BRET analysis where the full-length PLEKHM1 was fused to Sluc and Venus was fused to Rab7 in the BRET construct. For PIP5Kγ recruitment a mutant dark CFP(W66A)-FKBP-PIP5Kγ was used with an iRFP-FRB-Rab7. BRET values were normalized to those of DMSO treated cells. Comparisons were made for parental (blue) and PI4K2A K/O (green and orange) cells. (D) Similar BRET analysis where the PLEKHM1 was replaced by either RILP or Vps35 in the BRET construct. D Data information Means ± S.E.M. are shown from three experiments performed in triplicates.


( A ) Scheme of individual actin filaments polymerizing on functionalized glass surfaces from profilin - actin in solution . ( B ) Time - lapse TIRF microscopy of single filament growth from Alexa488phalloidin - stabilized seeds ( green ) in the presence of 5μM profilin - actin .

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(A) Scheme of individual actin filaments polymerizing on functionalized glass surfaces from profilin-actin in solution. (B) Time-lapse TIRF microscopy of single filament growth from Alexa488phalloidin-stabilized seeds (green) in the presence of 5μM profilin-actin.


( C ) Scheme of branched actin filaments polymerizing within dendritic actin networks assembled from NPF - and lipid - coated microspheres with profilin - actin , CP and Arp2 / 3 in solution . ( D ) Widefield epifluorescence images of indicated network components of bead - attached dendritic networks after kinetic arrest at indicated times .

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(C) Scheme of branched actin filaments polymerizing within dendritic actin networks assembled from NPF- and lipid-coated microspheres with profilin-actin, CP and Arp2/3 in solution. (D) Widefield epifluorescence images of indicated network components of bead-attached dendritic networks after kinetic arrest at indicated times.


Biochemical conditions are matching ( B ) with the exception of the additional presence of 200nM CP and 50nM Arp2 / 3 ( E ) Scheme of dendritic network growth from square WAVE1 - micropatterns on a 2 - dimensional coverslip with profilin - actin , CP and Arp2 / 3 in solution . ( F ) Reconstructed axial view for indicated network components from confocal imaging of dendritic network growth . Conditions are as in ( D )

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Biochemical conditions are matching (B) with the exception of the additional presence of 200nM CP and 50nM Arp2/3 (E) Scheme of dendritic network growth from square WAVE1-micropatterns on a 2-dimensional coverslip with profilin-actin, CP and Arp2/3 in solution. (F) Reconstructed axial view for indicated network components from confocal imaging of dendritic network growth. Conditions are as in (D)


( B ) TIRF microscopy of actin monomers ( blue , 1uM total , 30 % Cy5 labeled ) in the presence of equimolar profilin recruited to a WAVE1 pattern ( magenta ) .

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(B) TIRF microscopy of actin monomers (blue, 1uM total, 30% Cy5 labeled) in the presence of equimolar profilin recruited to a WAVE1 pattern (magenta).


( C ) TIRF microscopy of actin polymerization in - ( blue arrows ) and outside ( red arrows ) of WAVE1 - micropatterns ( magenta ) in the presence of 1uM profilin - actin visualized by a filament - binding probe ( 5nM Alexa488 - UTRNN ) .

(C) TIRF microscopy of actin polymerization in- (blue arrows) and outside (red arrows) of WAVE1-micropatterns (magenta) in the presence of 1uM profilin-actin visualized by a filament-binding probe (5nM Alexa488-UTRNN).


( D ) Kymograph of a growing filament barbed end entering a WAVE1 micropattern . Conditions as in ( C )

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(D) Kymograph of a growing filament barbed end entering a WAVE1 micropattern. Conditions as in (C)


( G ) Surface mCherry - NPF fluorescence and corresponding kymographs of barbed end growth for indicated WAVE1 , N - WASP or dark mCherry control constructs in 1uM profilin - actin .

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(G) Surface mCherry-NPF fluorescence and corresponding kymographs of barbed end growth for indicated WAVE1, N-WASP or dark mCherry control constructs in 1uM profilin-actin.


( B ) Scheme of domain architecture of surface - immobilized WAVE1 variants ( top panel ) , time averages ( middle panels , 7 min total ) and representative kymographs ( lower panel ) from multi - color TIRF imaging of actin polymerization on WAVE1 - coated coverslips . Conditions are 1uM actin , 5nM Alexa488 - UTRNN .

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(B) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of actin polymerization on WAVE1-coated coverslips. Conditions are 1uM actin, 5nM Alexa488-UTRNN.


( E ) TIRF microscopy of actin polymerization from 2uM actin in the absence ( upper panel ) or presence of near - saturating amounts of WAVE1 WCA .

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(E) TIRF microscopy of actin polymerization from 2uM actin in the absence (upper panel) or presence of near-saturating amounts of WAVE1 WCA.


( F ) Barbed end polymerization rates as a function of actin and WAVE1 - actin ratios . The red line is a fit to a minimal kinetic model assuming two polymerization competent actin species that associate to filament ends with different on - rates as indicated ( see Methods ) .

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(F) Barbed end polymerization rates as a function of actin and WAVE1-actin ratios. The red line is a fit to a minimal kinetic model assuming two polymerization competent actin species that associate to filament ends with different on-rates as indicated (see Methods).


( B ) Widefield epifluorescence images ( Alexa488 - actin ) of bead - attached dendritic networks following kinetic arrest after 10 min of network growth for spike - in experiments ( i . e . 60 % his10 - darkCherry - PWCA , 40 % his10 - Cherry - W ; molar percentage ) using indicated WAVE1 variants . Conditions are 5μM profilin - actin , 200nM CP and 50nM Arp2 / 3 . For all conditions , actin network assembly was quenched by adding 5M excess Latrunculin B and phalloidin 10 minutes after initiating the reaction .

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(B) Widefield epifluorescence images (Alexa488-actin) of bead-attached dendritic networks following kinetic arrest after 10 min of network growth for spike-in experiments (i.e. 60% his10-darkCherry-PWCA, 40% his10-Cherry-W; molar percentage) using indicated WAVE1 variants. Conditions are 5μM profilin-actin, 200nM CP and 50nM Arp2/3. For all conditions, actin network assembly was quenched by adding 5M excess Latrunculin B and phalloidin 10 minutes after initiating the reaction.


( C ) Average network growth rates for indicated WAVE1 variants or mCherry as a negative control ( N = 57 - 73 beads per condition ) . Errors bars represent SD .

(C) Average network growth rates for indicated WAVE1 variants or mCherry as a negative control (N= 57-73 beads per condition). Errors bars represent SD.


( D ) Scheme of domain architecture of surface - immobilized WAVE1 variants ( top panel ) , time averages ( middle panels , 7 min total ) and representative kymographs ( lower panel ) from multi - color TIRF imaging of polymerization from profilin - actin on WAVE1 - coated coverslips . Conditions are 2µM actin , 3µM profilin , 5nM Alexa488 - UTRNN .

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(D) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of polymerization from profilin-actin on WAVE1-coated coverslips. Conditions are 2µM actin, 3µM profilin, 5nM Alexa488-UTRNN.


( E ) Average barbed end polymerization rates for indicated WAVE1 variants . Errors bars represent SD .

(E) Average barbed end polymerization rates for indicated WAVE1 variants. Errors bars represent SD.


( A ) The weighted mass average of WAVE1 PRD ( 15 μM ) determined by sedimentation equilibrium ultracentrifugation with LatB - stabilized monomers ( 15 μM ) and various profilin concentrations as indicated . The lower dashed line indicates the expected mass of WAVE1 PRD ; the 5 - 7 kDa upshift is due to non - specific actin binding ( WAVE1 PRD [ null ] , black symbols ) . The upper dashed line mark the equilibrium mass observed for 15 μM WAVE1 PRD [ B ] in the presence of 90 μM profilin alone .

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(A) The weighted mass average of WAVE1 PRD (15 μM) determined by sedimentation equilibrium ultracentrifugation with LatB-stabilized monomers (15 μM) and various profilin concentrations as indicated. The lower dashed line indicates the expected mass of WAVE1 PRD; the 5-7 kDa upshift is due to non-specific actin binding (WAVE1 PRD[null], black symbols). The upper dashed line mark the equilibrium mass observed for 15 μM WAVE1 PRD[B] in the presence of 90 μM profilin alone.


( C ) FRET efficiencies for donor - ( Alexa488 - ) labeled WAVE1 variants ( c = 7 . 5nM , PWCA = blue , WCA = red , P _ CA = grey ) as a function of LatB - stabilized , quencher - ( Atto540Q - ) labeled actin monomers concentration . Lines are fits to a single site binding curve .

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(C) FRET efficiencies for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P_CA=grey) as a function of LatB-stabilized, quencher- (Atto540Q-) labeled actin monomers concentration. Lines are fits to a single site binding curve.


( D ) WH2 occupancies ( left axis , Methods ) and FRET efficiencies ( right axis ) for donor - ( Alexa488 - ) labeled WAVE1 variants ( c = 7 . 5nM , PWCA = blue , WCA = red , P * WCA ( null ) = cyan , P _ CA = grey ) as a function of profilin concentration . Concentration of LatB - stabilized , Atto540Q - actin monomers was constant ( 175nM ) at all conditions . Lines are fits to a competitive inhibition model ( see Methods ) .

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(D) WH2 occupancies (left axis, Methods) and FRET efficiencies (right axis) for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P*WCA(null)=cyan, P_CA=grey) as a function of profilin concentration. Concentration of LatB-stabilized, Atto540Q-actin monomers was constant (175nM) at all conditions. Lines are fits to a competitive inhibition model (see Methods).


, Scaled reduced cost analysis for uptake rates of amino acids at different growth rates . Colour represents the scaled reduced cost value . A higher value indicates a greater impact of a given increase in the amino acid uptake on growth rate .

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, Scaled reduced cost analysis for uptake rates of amino acids at different growth rates. Colour represents the scaled reduced cost value. A higher value indicates a greater impact of a given increase in the amino acid uptake on growth rate.


, Protein fold changes of the CcpA mutant over wild type . Log2 fold changes were calculated from LFQ values p values were calculated based on 2 - sided two sample t - tests with a FDR threshold of 0 . 05 and S 0 equal to 1 ( Tusher et al , 2001 ) . Proteins outside the significance lines ( FDR = 0 . 05 , S 0 = 1 ) are significantly changed in expression . Example sets of proteins are highlighted and are labeled with their gene names .

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, Protein fold changes of the CcpA mutant over wild type. Log2 fold changes were calculated from LFQ values p values were calculated based on 2-sided two sample t-tests with a FDR threshold of 0.05 and S0 equal to 1 (Tusher et al, 2001). Proteins outside the significance lines (FDR = 0.05, S0 = 1) are significantly changed in expression. Example sets of proteins are highlighted and are labeled with their gene names.


The differentially expressed RBPs during embryoid body ( EB ) differentiation , neural differentiation and cardiac differentiation , which are classified as different clusters . The right line plots show the dynamic expression pattern of each cluster , gray lines represent the expression of all genes in individual cluster , while the red line represents the average expression level . The number on the graph indicates the number of RBPs in the cluster . The expression value was firstly z - score scaled before plotting . EBD4 : embryoid body at day 4 ; EBD8 : embryoid body at day8 ; NSC : Neural Stem Cells ; NPC : Neural Progenitor Cell ; MES : mesodermal cells ; CP : Cardiac Precursors ; CM : Cardiomyocytes .

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The differentially expressed RBPs during embryoid body (EB) differentiation, neural differentiation and cardiac differentiation, which are classified as different clusters. The right line plots show the dynamic expression pattern of each cluster, gray lines represent the expression of all genes in individual cluster, while the red line represents the average expression level. The number on the graph indicates the number of RBPs in the cluster. The expression value was firstly z-score scaled before plotting. EBD4: embryoid body at day 4; EBD8: embryoid body at day8; NSC: Neural Stem Cells; NPC: Neural Progenitor Cell; MES: mesodermal cells; CP: Cardiac Precursors; CM: Cardiomyocytes.


Heatmap showing the expression pattern of the 29 RBPs during EB differentiation .

Heatmap showing the expression pattern of the 29 RBPs during EB differentiation.


AP staining of 11 RBP - KO ES cell lines maintained under ES cells culture conditions . Scale bar , 200 μm .

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AP staining of 11 RBP-KO ES cell lines maintained under ES cells culture conditions. Scale bar, 200 μm.


EB formation from WT and RBP - KO ES cell lines . WT ES cell - derived EBs formed cystic structures by day 8 of differentiation ; most of RBP - KO ES cell - derived EBs were more compact . Scale bar , 600 μm .

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EB formation from WT and RBP-KO ES cell lines. WT ES cell-derived EBs formed cystic structures by day 8 of differentiation; most of RBP-KO ES cell-derived EBs were more compact. Scale bar, 600 μm.


Time - course analysis of Oct4 expression and three lineage differentiation markers ( Hand1 , Brachyury and FGF5 ) during EB formation . It was quantified by qPCR , the relative expression of genes was calculated by the double delta Ct ( ΔΔCt ) method , normalized to the expression value at day 0 . The relative expression data was represented as the heatmap .

Time-course analysis of Oct4 expression and three lineage differentiation markers (Hand1, Brachyury and FGF5) during EB formation. It was quantified by qPCR, the relative expression of genes was calculated by the double delta Ct (ΔΔCt) method, normalized to the expression value at day 0. The relative expression data was represented as the heatmap.


AP staining of WT and 11 RBP - KO ES cell lines after LIF withdrawal . Scale bar , 200μm .

AP staining of WT and 11 RBP-KO ES cell lines after LIF withdrawal. Scale bar, 200μm.


RBP ranking based on ES cell abilities to self - renewal ( determined by colony formation ( ± LIF ) and Oct4 expression ) and exit - from - pluripotency ( determined by AP staining , EB formation and marker genes expression ) in the context of RBP KO . The expression value of Oct4 , FGF5 , Hand1 and Brachyury was represented as the relative level of these genes compared to their expressions in WT ES cells . The quantitation of other phenotypes , such as colony formation , AP staining and EB formation was manually defined .

RBP ranking based on ES cell abilities to self-renewal (determined by colony formation (±LIF) and Oct4 expression) and exit-from-pluripotency (determined by AP staining, EB formation and marker genes expression) in the context of RBP KO. The expression value of Oct4, FGF5, Hand1 and Brachyury was represented as the relative level of these genes compared to their expressions in WT ES cells. The quantitation of other phenotypes, such as colony formation, AP staining and EB formation was manually defined.


Immunofluorescence staining of Oct4 and hnRNPLL in WT and hnRNPLL - KO ES cells from two clones ( H8 and G6 ) . hnRNPLL was not expressed in either of the hnRNPLL - KO clones . DAPI stained the nuclei . Scale bar , 100 μm .

Immunofluorescence staining of Oct4 and hnRNPLL in WT and hnRNPLL-KO ES cells from two clones (H8 and G6). hnRNPLL was not expressed in either of the hnRNPLL-KO clones. DAPI stained the nuclei. Scale bar, 100 μm.


EB formation from WT and the two hnRNPLL - KO ES cell clones . WT ES cell - derived EBs formed cystic structures by day 8 of differentiation ; While both hnRNPLL - KO ES cell clones - derived EBs were more compact . Scale bar , 200 μm .

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EB formation from WT and the two hnRNPLL-KO ES cell clones. WT ES cell-derived EBs formed cystic structures by day 8 of differentiation; While both hnRNPLL-KO ES cell clones-derived EBs were more compact. Scale bar, 200 μm.


AP staining of WT and both hnRNPLL - KO ES cell clones after LIF withdrawal . Scale bar , 200 μm .

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AP staining of WT and both hnRNPLL-KO ES cell clones after LIF withdrawal. Scale bar, 200 μm.


Cumulative distribution showing the relative expression of WT - differentially expressed genes in hnRNPLL - KO ES cells during EB differentiation . Red lines and green lines represent the up - and down - regulated genes during EB differentiation of WT ES cells , while the black line stands for all expressed genes in WT and hnRNPLL - KO ES cells . Kolmogorov - Smirnov test was used to determine the significance . The relative expression ratio was log2 - transformed before plotting .

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Cumulative distribution showing the relative expression of WT-differentially expressed genes in hnRNPLL-KO ES cells during EB differentiation. Red lines and green lines represent the up- and down-regulated genes during EB differentiation of WT ES cells, while the black line stands for all expressed genes in WT and hnRNPLL-KO ES cells. Kolmogorov-Smirnov test was used to determine the significance. The relative expression ratio was log2-transformed before plotting.


Heatmap of key pluripotency and differentiation associated gene expression during EB formation from WT and hnRNPLL - KO clones ( H8 and G6 ) .

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Heatmap of key pluripotency and differentiation associated gene expression during EB formation from WT and hnRNPLL-KO clones (H8 and G6).


Hematoxylin and eosin labeling and Oct4 immunohistochemical staining of teratomas derived from WT and hnRNPLL - KO ES cells ( left ) . Representative Oct4 - positive regions are shown . The percentage of Oct4 positive region ( % ) is plotted ( right ) . The data represent the means ± SD , ( n = 3 , from biological repeats ) . * P ＜ 0 . 05 ; * * * P ＜ 0 . 001 , T - test was used to determine the significance . Scale bar , 100 μm .

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Hematoxylin and eosin labeling and Oct4 immunohistochemical staining of teratomas derived from WT and hnRNPLL-KO ES cells (left). Representative Oct4-positive regions are shown. The percentage of Oct4 positive region (%) is plotted (right). The data represent the means ± SD, (n=3, from biological repeats). *P＜0.05; ***P＜0.001, T-test was used to determine the significance. Scale bar, 100 μm.


AP staining of WT and hnRNPLL - overexpression ES cell lines maintained under ES cells culture conditions and colony formation assay . Scale bar , 200 μm . The data represent the means ± SD ( n = 3 biological repeats ) .

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AP staining of WT and hnRNPLL-overexpression ES cell lines maintained under ES cells culture conditions and colony formation assay. Scale bar, 200 μm. The data represent the means ± SD (n=3 biological repeats).


Relative expression of Oct4 and Sox2 after hnRNPLL overexpression . The data represent the means ± SD ( n = 3 , from biological repeats ) . * * * P ＜ 0 . 001 . T - test was used to determine the significance .

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Relative expression of Oct4 and Sox2 after hnRNPLL overexpression. The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. T-test was used to determine the significance.


Birth rate of the different hnRNPLL genotypes , the birth rate of hnRNPLL - / - mice was significantly lower than that of WT hnRNPLL + / + mice . The data represent the means ± SD . T - test was used to determine the significance .

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Birth rate of the different hnRNPLL genotypes, the birth rate of hnRNPLL-/- mice was significantly lower than that of WT hnRNPLL+/+ mice. The data represent the means ± SD. T-test was used to determine the significance.


Representative images of hnRNPLL + / + and hnRNPLL - / - littermates at 3 weeks .

Representative images of hnRNPLL+/+ and hnRNPLL-/- littermates at 3 weeks.


Body weight growth curve of mice at different postnatal days . The data represent the means ± SD ( n = 5 - 10 ) . * * * P ＜ 0 . 001 . T - test was used to determine the significance .

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Body weight growth curve of mice at different postnatal days. The data represent the means ± SD (n=5-10). ***P＜0.001. T-test was used to determine the significance.


Survival curve analysis for hnRNPLL + / + , hnRNPLL + / - and hnRNPLL - / - mice ( ten mice in hnRNPLL + / + / + / - group and seven mice in hnRNPLL - / - group ) .

Survival curve analysis for hnRNPLL+/+, hnRNPLL+/-and hnRNPLL-/-mice (ten mice in hnRNPLL+/+/+/-group and seven mice in hnRNPLL-/-group).


Genotypes and representative images of hnRNPLL + / + embryo and retarded hnRNPLL - / - embryos at E11 . 5 which generated from hnRNPLL + / - intercrosses . Scale bar , 1mm .

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Genotypes and representative images of hnRNPLL+/+ embryo and retarded hnRNPLL-/- embryos at E11.5 which generated from hnRNPLL+/- intercrosses. Scale bar, 1mm.


Silver nitrate - stained NuPAGE gel of hnRNPLL interacting proteins . Red star indicate hnRNPLL specific interacting proteins compared to IgG .

Silver nitrate-stained NuPAGE gel of hnRNPLL interacting proteins. Red star indicate hnRNPLL specific interacting proteins compared to IgG.


Mass spectrum identification of the hnRNPLL interactome in ES cells . RNA splicing associated proteins are well enriched by hnRNPLL IP . The enrichment score was log2 - transformed before plotting .

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Mass spectrum identification of the hnRNPLL interactome in ES cells. RNA splicing associated proteins are well enriched by hnRNPLL IP. The enrichment score was log2-transformed before plotting.


Western blot validation the interactions between hnRNPLL and the indicated splicing associated proteins .

Western blot validation the interactions between hnRNPLL and the indicated splicing associated proteins.


Cumulative distribution of PSI with exon skipping events between WT and hnRNPLL - KO ES cells . Kolmogorov - Smirnov test was used to determine the significance .

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Cumulative distribution of PSI with exon skipping events between WT and hnRNPLL-KO ES cells. Kolmogorov-Smirnov test was used to determine the significance.


Volcano plot of differential exon skipping events between WT and hnRNPLL - KO ES cells . Indicated AS events were labeled in purple ( up - regulated ) and green ( down - regulated ) with gene names .

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Volcano plot of differential exon skipping events between WT and hnRNPLL-KO ES cells. Indicated AS events were labeled in purple (up-regulated) and green (down-regulated) with gene names.


Western blot analysis of hnRNPLL protein during EB formation of WT ES cells at indicated days .

Western blot analysis of hnRNPLL protein during EB formation of WT ES cells at indicated days.


Heatmap of the 38 and 25 divergent exon skipping events in Figure 5I . * indicates the different exon skipping events of the same gene .

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Heatmap of the 38 and 25 divergent exon skipping events in Figure 5I. * indicates the different exon skipping events of the same gene.


RT - PCR validation of partial divergent exon skipping events shown in Figure 5J . Red boxes represent ESC preferred exons , blue boxes indicate EB preferred exons .

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RT-PCR validation of partial divergent exon skipping events shown in Figure 5J. Red boxes represent ESC preferred exons, blue boxes indicate EB preferred exons.


RIP - PCR validation of the transcripts that interact with hnRNPLL . The left panel shows the results of western blots verification of hnRNPLL Immunoprecipitation efficiency . The data represent the means ± SD ( n = 3 , from biological repeats ) . * * * P ＜ 0 . 001 . T - test was used to determine the significance .

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RIP-PCR validation of the transcripts that interact with hnRNPLL. The left panel shows the results of western blots verification of hnRNPLL Immunoprecipitation efficiency. The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. T-test was used to determine the significance.


Purification of hnRNPLL - RNA complex and detection of the RNA targets in the complex by PCR . The left panel shows the membrane position labeled as the yellow square which was cut to extract the hnRNPLL - RNA complex . The bands around 70KD indicate hnRNPLL proteins , the band around 50KD indicate the heavy chain of antibodies used in Immunoprecipitation . The right plot indicates the enrichment of these RNA targets in the hnRNPLL - RNA complex . The data represent the means ± SD ( n = 3 , from biological repeats ) . * P ＜ 0 . 05 ; * * * P ＜ 0 . 001 . T - test was used to determine the significance .

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Purification of hnRNPLL-RNA complex and detection of the RNA targets in the complex by PCR. The left panel shows the membrane position labeled as the yellow square which was cut to extract the hnRNPLL-RNA complex. The bands around 70KD indicate hnRNPLL proteins, the band around 50KD indicate the heavy chain of antibodies used in Immunoprecipitation. The right plot indicates the enrichment of these RNA targets in the hnRNPLL-RNA complex. The data represent the means ± SD (n=3, from biological repeats). *P＜0.05; ***P＜0.001. T-test was used to determine the significance.


Detection of Bptf and Tbx3 exon skipping events during EB formation in WT and hnRNPLL - KO ES cells ( left ) and in hnRNPLL - overexpression ES cells under ES cells culture conditions ( right ) by RT - PCR . OE , hnRNPLL overexpression .

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Detection of Bptf and Tbx3 exon skipping events during EB formation in WT and hnRNPLL-KO ES cells (left) and in hnRNPLL-overexpression ES cells under ES cells culture conditions (right) by RT-PCR.OE, hnRNPLL overexpression.


RT - PCR validation of Bptf exon24a and Tbx3 exon2a deletion in WT ES cells .

RT-PCR validation of Bptf exon24a and Tbx3 exon2a deletion in WT ES cells.


AP staining of Bptf E24a KO or Tbx3 E2a KO ES cells maintained under normal ES cells culture conditions ( upper ) . Scale bar , 200 μm . Colony formation assay of Bptf E24a KO or Tbx3 E2a KO ES cells under ES cells culture condition ( lower left ) . The data represent the means ± SD ( n = 3 , from biological repeats ) . * * * P ＜ 0 . 001 . Colony diameter of Tbx3 E2a KO ES cells ( lower right ) . The data represent the means ± SD ( n ＞ 80 ) . * * * P ＜ 0 . 001 . T - test was used to determine the significance .

AP staining of Bptf E24a KO or Tbx3 E2a KO ES cells maintained under normal ES cells culture conditions (upper). Scale bar, 200 μm. Colony formation assay of Bptf E24a KO or Tbx3 E2a KO ES cells under ES cells culture condition (lower left). The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. Colony diameter of Tbx3 E2a KO ES cells (lower right). The data represent the means ± SD (n＞80). ***P＜0.001. T-test was used to determine the significance.


Relative expression of Oct4 and Rex1 , determined by qRT - PCR . The data represent the means ± SD ( n = 3 , from biological repeats ) . * * * P ＜ 0 . 001 . T - test was used to determine the significance .

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Relative expression of Oct4 and Rex1, determined by qRT-PCR. The data represent the means ± SD (n=3, from biological repeats). ***P＜0.001. T-test was used to determine the significance.


RT - PCR validation of Bptf and Tbx3 alternative exon deletion , separately in hnRNPLL - KO H8 cells ( upper ) and simultaneously in hnRNPLL - KO H8 cells ( lower ) .

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RT-PCR validation of Bptf and Tbx3 alternative exon deletion, separately in hnRNPLL-KO H8 cells (upper) and simultaneously in hnRNPLL-KO H8 cells (lower).


AP staining of H8 - Bptf or H8 - Tbx3 exon - KO ES cells after LIF withdrawal . hnRNPLL - H8 , hnRNPLL - KO ES cells ; H8 - Bptf KO , alternative Bptf exon was deleted in hnRNPLL - KO H8 cells ; H8 - Tbx3 KO , alternative Tbx3 exon was deleted in hnRNPLL - KO H8 cells ; H8 - Bptf / Tbx3 KO , alternative Bptf or Tbx3 exons were simultaneously deleted in hnRNPLL - KO H8 cells . Scale bar , 200 μm .

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AP staining of H8-Bptf or H8-Tbx3 exon-KO ES cells after LIF withdrawal. hnRNPLL-H8, hnRNPLL-KO ES cells; H8-Bptf KO, alternative Bptf exon was deleted in hnRNPLL-KO H8 cells; H8-Tbx3 KO, alternative Tbx3 exon was deleted in hnRNPLL-KO H8 cells; H8-Bptf/Tbx3 KO, alternative Bptf or Tbx3 exons were simultaneously deleted in hnRNPLL-KO H8 cells. Scale bar, 200 μm.


Colony formation assay of H8 - Bptf or H8 - Tbx3 exon - KO ES cells after LIF withdrawal . The data represent the means ± SD ( n = 3 , from biological repeats ) .

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Colony formation assay of H8-Bptf or H8-Tbx3 exon-KO ES cells after LIF withdrawal. The data represent the means± SD (n=3, from biological repeats).


Hematoxylin and eosin labeling of teratomas derived from WT and H8 - Bptf or H8 - Tbx3 exon - KO ES cells . The blue triangle represent ectoderm , the dark one represent mesoderm and the green one represent endoderm . Scale bar , 100 μm .

Hematoxylin and eosin labeling of teratomas derived from WT and H8-Bptf or H8-Tbx3 exon-KO ES cells. The blue triangle represent ectoderm, the dark one represent mesoderm and the green one represent endoderm. Scale bar, 100 μm.


Oct4 immunohistochemical staining of teratomas derived from WT and H8 - Bptf or H8 - Tbx3 exon - KO ES cells . Representative Oct4 - positive regions are shown ( left ) . Scale bar , 100 µm . The percentage Oct4 - positive region ( % ) is shown ( right ) . The data represent the means ± SD ( n = 3 biological repeats ) . * * P ＜ 0 . 01 ; ns , not significant . T - test was used to determine the significance .

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Oct4 immunohistochemical staining of teratomas derived from WT and H8-Bptf or H8-Tbx3 exon-KO ES cells. Representative Oct4-positive regions are shown (left). Scale bar, 100 µm. The percentage Oct4-positive region (%) is shown (right). The data represent the means± SD (n=3 biological repeats). **P＜0.01; ns, not significant. T-test was used to determine the significance.


HCT - 116 ( A ) and HT - 29 ( B ) cells were treated with the indicated concentrations of sodium butyrate ( NaB ) for 24 ( grey bars ) , 48 ( pink bars ) , or 72 ( yellow bars ) hours and cell proliferation was assessed . HCT -

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HCT-116 (A) and HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 (grey bars), 48 (pink bars), or 72 (yellow bars) hours and cell proliferation was assessed. HCT-116


( C , E ) and HT - 29 ( D , F ) cells were treated with the indicated concentrations of NaB for 24 h . C . D . The percentage of apoptotic cells in HCT - 116 ( C ) and HT - 29 ( D ) cells was quantified in three independent experiments using an annexinV / PI assay ( annexinV + / PI - ) by flow cytometry and expressed as mean ± SD . One - way ANOVA was used to compare between the control cells and NaB treatments . * p < 0 . 05 , * * p < 0 . 01 compared to control . E . F . Representative Western blots of full - length and cleaved PARP products in HCT - 116 ( E ) and HT - 29 ( F ) cells .

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(C, E) and HT-29 (D, F) cells were treated with the indicated concentrations of NaB for 24 h. C.D. The percentage of apoptotic cells in HCT-116 (C) and HT-29 (D) cells was quantified in three independent experiments using an annexinV/PI assay (annexinV+/PI-) by flow cytometry and expressed as mean ± SD. One-way ANOVA was used to compare between the control cells and NaB treatments. *p<0.05, ** p<0.01 compared to control. E.F. Representative Western blots of full-length and cleaved PARP products in HCT-116 (E) and HT-29 (F) cells.


HCT - 116 ( A , C ) and HT - 29 ( B , D ) cells were treated with 2mM sodium butyrate ( NaB ) for 24 h and then stained with acridine orange ( AO ; A - B ) or monodansylcadaverine ( MDC ; C - D ) .

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HCT-116 (A, C) and HT-29 (B, D) cells were treated with 2mM sodium butyrate (NaB) for 24 h and then stained with acridine orange (AO; A-B) or monodansylcadaverine (MDC; C-D).


Flow cytometry measured the percentage of AO + cells and the results of three independent AO fluorescence experiments for HTC - 116 ( E ) and HT - 29 ( F ) cells were expressed as mean ± SD . One - way ANOVA was used to compare control cells and NaB treatments . * p < 0 . 05 , * * p < 0 . 01 when compared to control .

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Flow cytometry measured the percentage of AO+ cells and the results of three independent AO fluorescence experiments for HTC-116 (E) and HT-29 (F) cells were expressed as mean ± SD. One-way ANOVA was used to compare control cells and NaB treatments. *p<0.05, ** p<0.01 when compared to control.


HCT - 116 ( A ) or HT - 29 ( B ) cells were treated with the indicated concentrations of sodium butyrate ( NaB ) for 24 h . Representative blots showed expression of Beclin1 , ATG3 , and LC3 as well as GAPDH , the loading control .

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HCT-116 (A) or HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 h. Representative blots showed expression of Beclin1, ATG3, and LC3 as well as GAPDH, the loading control.


C . Electron microscopy images of control and NaB ( 2mM ) treated HCT - 116 cells . ER , endoplasmic reticulum ; Mt , mitochondrion ; AL .

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C. Electron microscopy images of control and NaB (2mM) treated HCT-116 cells. ER, endoplasmic reticulum; Mt, mitochondrion; AL.


HCT - 116 or HT - 29 cells were treated with 5mM 3 - MA or 5μM chloroquine ( CQ ) for 30 min , and then with 2mM sodium butyrate ( NaB ) for 24 h . Representative Western blots of LC3 - I and LC3 - II expression were quantified by densitometry and normalized to GAPDH ( ratio of LC3 : GAPDH ) . The fold change from control is indicated as the mean ± SD of three independent experiments . One - way ANOVA was used for statistical analysis . * * p < 0 . 01 , compared to the respective control group . ( A ) HCT - 116 cells treated with 3 - MA and NaB ;

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HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (A) HCT-116 cells treated with 3-MA and NaB;


HCT - 116 or HT - 29 cells were treated with 5mM 3 - MA or 5μM chloroquine ( CQ ) for 30 min , and then with 2mM sodium butyrate ( NaB ) for 24 h . Representative Western blots of LC3 - I and LC3 - II expression were quantified by densitometry and normalized to GAPDH ( ratio of LC3 : GAPDH ) . The fold change from control is indicated as the mean ± SD of three independent experiments . One - way ANOVA was used for statistical analysis . * * p < 0 . 01 , compared to the respective control group . ( B ) HT - 29 cells treated with 3 - MA and

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HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (B) HT-29 cells treated with 3-MA and NaB


HCT - 116 or HT - 29 cells were treated with 5mM 3 - MA or 5μM chloroquine ( CQ ) for 30 min , and then with 2mM sodium butyrate ( NaB ) for 24 h . Representative Western blots of null and LC3 - II expression were quantified by densitometry and normalized to GAPDH ( ratio of LC3 : GAPDH ) . The fold change from control is indicated as the mean ± SD of three independent experiments . One - way ANOVA was used for statistical analysis . * * p < 0 . 01 , compared to the respective control group . ( C ) HCT - 116 cells treated with CQ and NaB ;

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HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of null and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (C) HCT-116 cells treated with CQ and NaB;


HCT - 116 or HT - 29 cells were treated with 5mM 3 - MA or 5μM chloroquine ( CQ ) for 30 min , and then with 2mM sodium butyrate ( NaB ) for 24 h . Representative Western blots of LC3 - I and LC3 - II expression were quantified by densitometry and normalized to GAPDH ( ratio of LC3 : GAPDH ) . The fold change from control is indicated as the mean ± SD of three independent experiments . One - way ANOVA was used for statistical analysis . * * p < 0 . 01 , compared to the respective control group . ( D ) HT - 20 cells treated with CQ and NaB .

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HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group.(D) HT-20 cells treated with CQ and NaB.


HCT - 116 ( A ) and HT - 29 ( B ) cells were treated with the indicated concentrations of sodium butyrate ( NaB ) for 24 h . Representative Western blots show the expression of IRE - 1a , BIP , PDI , and CHOP . GAPDH was used as the loading control .

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HCT-116 (A) and HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 h. Representative Western blots show the expression of IRE-1a, BIP, PDI, and CHOP. GAPDH was used as the loading control.


HCT - 116 or HT - 29 cells were treated with 10 μg / mL cycloheximide for 30 min followed by sodium butyrate ( NaB ) for 24 h . Representative Western blots showed the expression of IRE - 1a , BIP , PDI , and GADPH ( loading control ) in HCT - 116 ( A ) or HT - 29 ( C ) cells . Protein expression was quantified by densitometry and normalized to GAPDH ( ratio of protein : GAPDH ) . The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT - 116 ( B ) and HT - 29 ( D ) cells . are shown . One - way ANOVA was used for statistical analysis . * P < 0 . 05 , * * p < 0 . 01 , compared to the respective control group .

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HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide for 30 min followed by sodium butyrate (NaB) for 24 h. Representative Western blots showed the expression of IRE-1a, BIP, PDI, and GADPH (loading control) in HCT-116 (A) or HT-29 (C) cells. Protein expression was quantified by densitometry and normalized to GAPDH (ratio of protein:GAPDH). The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT-116 (B) and HT-29 (D) cells. are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.


HCT - 116 ( A , B ) or HT - 29 ( C , D ) cells were treated with 0 . 1μM mithramycin for 30 min followed by sodium butyrate ( NaB ) for 24 h . Representative Western blots showing the expression of IRE - 1a , BIP , and PDI in HCT - 116 ( A ) or HT - 29 ( C ) cells are shown . GAPDH was used as loading control . Protein expression ( IRE - 1a , BIP , and PDI ) was quantified by densitometry and normalized to GAPDH ( ratio of protein : GAPDH ) . The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT - 116 ( B ) and HT - 29 ( D ) cells . One - way ANOVA was used for statistical analysis . * P < 0 . 05 , * * p < 0 . 01 , compared to the respective control group .

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

HCT-116 (A, B) or HT-29 (C,D) cells were treated with 0.1μM mithramycin for 30 min followed by sodium butyrate (NaB) for 24 h. Representative Western blots showing the expression of IRE-1a, BIP, and PDI in HCT-116 (A) or HT-29 (C) cells are shown. GAPDH was used as loading control. Protein expression (IRE-1a, BIP, and PDI) was quantified by densitometry and normalized to GAPDH (ratio of protein:GAPDH). The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT-116 (B) and HT-29 (D) cells. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.


HCT - 116 or HT - 29 cells were treated with 10 μg / mL cycloheximide or 0 . 1μM mithramycin for 30 min and then with 2mM sodium butyrate ( NaB ) for 24 h . Representative Western blots of the expression of LC3 - II are shown . The level of LC3 - II expression was quantified by densitometry and normalized to GAPDH ( ratio of LC3 - II : GAPDH ) . The fold change from control cells is shown . Means and standard deviation ( SD ) of three independent experiments are shown . One - way ANOVA was used for statistical analysis . * P < 0 . 05 , * * p < 0 . 01 , compared to the control group . ( A - B ) HCT - 116 cells treated with cyclohexamide ; ( E - F ) HT - 29 cells treated with cyclohexamide ;

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

HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide or 0.1μM mithramycin for 30 min and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of the expression of LC3-II are shown. The level of LC3-II expression was quantified by densitometry and normalized to GAPDH (ratio of LC3-II:GAPDH). The fold change from control cells is shown. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the control group. (A-B) HCT-116 cells treated with cyclohexamide; (E-F) HT-29 cells treated with cyclohexamide;


HCT - 116 or HT - 29 cells were treated with 10 μg / mL cycloheximide or 0 . 1μM mithramycin for 30 min and then with 2mM sodium butyrate ( NaB ) for 24 h . Representative Western blots of the expression of LC3 - II are shown . The level of LC3 - II expression was quantified by densitometry and normalized to GAPDH ( ratio of LC3 - II : GAPDH ) . The fold change from control cells is shown . Means and standard deviation ( SD ) of three independent experiments are shown . One - way ANOVA was used for statistical analysis . * P < 0 . 05 , * * p < 0 . 01 , compared to the control group . ( C - D ) HCT - 116 cells treated with mithramycin ; ( G - H ) HT - 29 cells treated with mithramycin . cancel

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-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR O O

HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide or 0.1μM mithramycin for 30 min and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of the expression of LC3-II are shown. The level of LC3-II expression was quantified by densitometry and normalized to GAPDH (ratio of LC3-II:GAPDH). The fold change from control cells is shown. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the control group. (C-D) HCT-116 cells treated with mithramycin;  (G-H) HT-29 cells treated with mithramycin. cancel


HCT - 116 cells were transfected with BIP or CHOP specific siRNAs for 48 h and then treated with or without 2mM sodium butyrate ( NaB ) for 24 h . Negative control ( NC ) scramble siRNA was used the negative control for the transfection . Representative Western blots are shown for BIP siRNA ( A ) and CHOP siRNA ( C ) . The expression level of each protein was determined by densitometry and normalized to GAPDH ( ratio of protein : GAPDH ) . ( B ) Normalized expression levels of BIP and LC3 - II in HCT - 116 cells treated with BIP specific siRNA . ( C ) Normalized expression levels of CHOP and LC3 - II in HCT - 116 cells treated with CHOP specific siRNA . Means and standard deviation ( SD ) of three independent experiments are shown . One - way ANOVA was used for statistical analysis . * P < 0 . 05 , * * p < 0 . 01 , compared to the respective control group .

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

HCT-116 cells were transfected with BIP or CHOP specific siRNAs for 48 h and then treated with or without 2mM sodium butyrate (NaB) for 24 h. Negative control (NC) scramble siRNA was used the negative control for the transfection. Representative Western blots are shown for BIP siRNA (A) and CHOP siRNA (C). The expression level of each protein was determined by densitometry and normalized to GAPDH (ratio of protein:GAPDH). (B) Normalized expression levels of BIP and LC3-II in HCT-116 cells treated with BIP specific siRNA. (C) Normalized expression levels of CHOP and LC3-II in HCT-116 cells treated with CHOP specific siRNA. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.


HCT - 116 ( A ) and HT - 29 ( B ) cells were exposed to 5mM 3 - MA or 5μM chloroquine ( CQ ) for 30 min and then treated with 2mM sodium butyrate ( NaB ) for 24 h . The percentage of apoptotic cells ( annexinV + / PI - ) was quantified by flow cytometry using an annexinV / PI assay and expressed as mean ± SD of three independent experiments . One - way ANOVA was used for statistical analysis to compare control cells and NaB treatments . * p < 0 . 05 , * * p < 0 . 01 compared to control . ( C . D . ) Cells were treated as described above and the proteins were harvested for Western blot analysis . Expression of full - length and cleaved PARP in HCT - 116 ( C ) or HT - 29 ( D ) cells was compared to GAPDH , the loading control . The ratio of cleaved PARP to total PARP was calculated and the fold change in normalized PARP expression relative to cells treated with 2mM NaB is shown as the mean ± SD of three independent experiments . One - way ANOVA was used for statistical analysis . * P < 0 . 05 , * * p < 0 . 01 , compared to the 2mM NaB group .

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

HCT-116 (A) and HT-29 (B) cells were exposed to 5mM 3-MA or 5μM chloroquine (CQ) for 30 min and then treated with 2mM sodium butyrate (NaB) for 24 h. The percentage of apoptotic cells (annexinV+/PI-) was quantified by flow cytometry using an annexinV/PI assay and expressed as mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis to compare control cells and NaB treatments. *p<0.05, ** p<0.01 compared to control. (C.D.) Cells were treated as described above and the proteins were harvested for Western blot analysis. Expression of full-length and cleaved PARP in HCT-116 (C) or HT-29 (D) cells was compared to GAPDH, the loading control. The ratio of cleaved PARP to total PARP was calculated and the fold change in normalized PARP expression relative to cells treated with 2mM NaB is shown as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. *P<0.05, ** p<0.01, compared to the 2mM NaB group.


( A ) Western blot analysis of supernatants containing long and short mouse and human DNGR - 1 ECD proteins under reducing and non - reducing conditions .

O O O O 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) Western blot analysis of supernatants containing long and short mouse and human DNGR-1 ECD proteins under reducing and non-reducing conditions.


( B ) Western blot analysis of supernatants after protein production of WT and C94S mutant long mouse DNGR - 1 ECD proteins under reducing and non - reducing conditions . C94S mutant - containing supernatant was 200x concentrated before analysis .

O O O O O O O O O O O O O O O O O O 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) Western blot analysis of supernatants after protein production of WT and C94S mutant long mouse DNGR-1 ECD proteins under reducing and non-reducing conditions. C94S mutant-containing supernatant was 200x concentrated before analysis.


( C ) Western blot analysis of supernatants containing the indicated DNGR - 1 ECD proteins after treatment with PNGase F overnight . HRP - conjugated anti - FLAG antibody was used for detection of all proteins . Numbers on the side of blots indicate positions of molecular weight markers .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of supernatants containing the indicated DNGR-1 ECD proteins after treatment with PNGase F overnight. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers.


( A ) 0 . 25 μg of purified long mouse DNGR - 1 ECD was diluted into 10 mM Tris buffer of the indicated pH ( left panel ) or into 10 mM Tris pH 7 . 4 buffer supplemented with increasing amounts of NaCl ( 1 - 250 mM ; right panel ) , and reduction sensitivity of DNGR - 1 was assessed by reducing SDS - PAGE and Western blot . ( B ) 0 . 25 μg of purified DNGR - 1 long mouse ECD was diluted into buffers of different pH and ionic strength , and its reduction sensitivity was assessed by reducing SDS - PAGE and Western blot . The intensity of bands corresponding to dimer and monomer was determined densitometrically , and the ratio was plotted as a function of buffer ionic strength and pH .

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

(A) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into 10 mM Tris buffer of the indicated pH (left panel) or into 10 mM Tris pH 7.4 buffer supplemented with increasing amounts of NaCl (1 - 250 mM; right panel), and reduction sensitivity of DNGR-1 was assessed by reducing SDS-PAGE and Western blot. (B) 0.25 μg of purified DNGR-1 long mouse ECD was diluted into buffers of different pH and ionic strength, and its reduction sensitivity was assessed by reducing SDS-PAGE and Western blot. The intensity of bands corresponding to dimer and monomer was determined densitometrically, and the ratio was plotted as a function of buffer ionic strength and pH.


( C ) 0 . 25 μg of purified long mouse DNGR - 1 ECD was diluted into PBS or 10 mM MES pH 6 . 1 buffer , and its reduction sensitivity was tested under mildly ( Laemmli buffer ) or strongly denaturing ( 8 M urea ) conditions by reducing SDS - PAGE and Western blot .

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

(C) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffer, and its reduction sensitivity was tested under mildly (Laemmli buffer) or strongly denaturing (8 M urea) conditions by reducing SDS-PAGE and Western blot.


( D ) 0 . 25 μg of purified long mouse DNGR - 1 ECD was diluted into PBS or 10 mM MES pH 6 . 1 buffers , and its reduction sensitivity after different lengths of heat - denaturation in Laemmli buffer was tested by reducing SDS - PAGE and Western blot . HRP - conjugated anti - FLAG antibody was used for detection of all proteins . Numbers on the side of blots indicate positions of molecular weight markers . Reduction - resistant dimers are indicated with an * and reduction - sensitive protein with a + .

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 O

(D) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffers, and its reduction sensitivity after different lengths of heat-denaturation in Laemmli buffer was tested by reducing SDS-PAGE and Western blot. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( A ) 1 μg of purified long mouse DNGR - 1 ECD was transferred into 10mM MES pH 6 . 1 and 0 . 5 μg into PBS . Half of the MES sample and the whole PBS sample were dialyzed against 2 l of PBS overnight at 4 ° C . After dialysis , all samples were prepared for reducing SDS - PAGE and Western blot .

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 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

(A) 1 μg of purified long mouse DNGR-1 ECD was transferred into 10mM MES pH 6.1 and 0.5 μg into PBS. Half of the MES sample and the whole PBS sample were dialyzed against 2 l of PBS overnight at 4°C. After dialysis, all samples were prepared for reducing SDS-PAGE and Western blot.


( B ) Supernatants after production of indicated proteins in 293F cells were harvested and diluted into indicated buffers at a 2 : 3 ratio and analyzed using reducing SDS - PAGE and Western blot . HRP - conjugated anti - FLAG antibody was used for detection of all proteins . Numbers on the side of blots indicate positions of molecular weight markers . Reduction - resistant dimers are indicated with an * and reduction - sensitive protein with a + .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Supernatants after production of indicated proteins in 293F cells were harvested and diluted into indicated buffers at a 2:3 ratio and analyzed using reducing SDS-PAGE and Western blot. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( B ) Supernatants containing ECD of long mouse DNGR - 1 , Dectin - 1 and the chimeric receptor were prepared for SDS - PAGE and Western blot under non - reducing conditions .

O O O O O O O O O O O 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) Supernatants containing ECD of long mouse DNGR-1, Dectin-1 and the chimeric receptor were prepared for SDS-PAGE and Western blot under non-reducing conditions.


( C ) Supernatants containing indicated proteins were diluted into PBS or pH 6 . 1 MES buffer and analyzed using SDS - PAGE and Western blot under reducing conditions . HRP - conjugated anti - FLAG antibody was used for detection of all proteins . Numbers on the side of blots indicate positions of molecular weight markers . Reduction - resistant dimers are indicated with an * and reduction sensitive protein with a + .

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) Supernatants containing indicated proteins were diluted into PBS or pH 6.1 MES buffer and analyzed using SDS-PAGE and Western blot under reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction sensitive protein with a +.


( A ) Long mouse and human DNGR - 1 ECD proteins were diluted in PBS or 10 mM MES pH 6 . 1 buffer and 20 independent far - UV spectra were acquired for each condition ( red and black lines depict the composite curve for each condition )

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

(A) Long mouse and human DNGR-1 ECD proteins were diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent far-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition)


( B ) Long mouse DNGR - 1 ECD was diluted in PBS or 10 mM MES pH 6 . 1 buffer and 20 independent near - UV spectra were acquired for each condition ( red and black lines depict the composite curve for each condition and the blue line shows the difference between the two ) .

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

(B) Long mouse DNGR-1 ECD was diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent near-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition and the blue line shows the difference between the two).


( B ) and ( C ) Supernatants containing the indicated ECD proteins ( mouse , long isoform ) were diluted 1 : 2 into the indicated buffers and analyzed by SDS - PAGE and Western blot under reducing or non reducing conditions . HRP - conjugated anti - FLAG antibody was used for detection of all proteins . Numbers on the side of blots indicate positions of molecular weight markers . Reduction - resistant dimers are indicated with an * and reduction - sensitive protein with a + .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) and (C) Supernatants containing the indicated ECD proteins (mouse, long isoform) were diluted 1:2 into the indicated buffers and analyzed by SDS-PAGE and Western blot under reducing or non reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( D ) Phoenix cells expressing the indicated proteins were lysed in 1 % SDS in PBS or 10mM MES pH 6 . 1 buffers and the lysates were analyzed by SDS - PAGE and Western blot under reducing conditions . Anti - DNGR - 1 antibody ( clone 397 ) followed by HRP - conjugated secondary anti - rat antibody was used for detection . Numbers on the side of blots indicate positions of molecular weight markers . Reduction - resistant dimers are indicated with an * and reduction - sensitive protein with a + .

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 O

(D) Phoenix cells expressing the indicated proteins were lysed in 1% SDS in PBS or 10mM MES pH 6.1 buffers and the lysates were analyzed by SDS-PAGE and Western blot under reducing conditions. Anti-DNGR-1 antibody (clone 397) followed by HRP-conjugated secondary anti-rat antibody was used for detection. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( A ) Decreasing amounts of F - actin were spotted onto a nitrocellulose membrane and binding of indicated DNGR - 1 ECD ( mouse , long isoform ) proteins was tested in PBS or MES pH 6 . 1 buffers . HRP - conjugated anti - FLAG antibody was used for detection . The signal was quantified by densitometry and quantitation of four independent experiments is 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 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

(A) Decreasing amounts of F-actin were spotted onto a nitrocellulose membrane and binding of indicated DNGR-1 ECD (mouse, long isoform) proteins was tested in PBS or MES pH 6.1 buffers. HRP-conjugated anti-FLAG antibody was used for detection. The signal was quantified by densitometry and quantitation of four independent experiments is shown.


( B and C ) B3Z - Syk reporter cells expressing indicated mutant or WT DNGR - 1 receptors ( mouse , long isoform ) were incubated with UV - irradiated 293T cells ( ratio dead : reporter cells is indicated ) , or with plate - bound anti - DNGR - 1 antibody or medium alone at 37 ° C overnight . Activation of the NFAT reporter was measured at the end of the incubation period . Data are plotted as mean ± SD of experimental duplicates . One representative of three experiments is 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C) B3Z-Syk reporter cells expressing indicated mutant or WT DNGR-1 receptors (mouse, long isoform) were incubated with UV-irradiated 293T cells (ratio dead:reporter cells is indicated), or with plate-bound anti-DNGR-1 antibody or medium alone at 37°C overnight. Activation of the NFAT reporter was measured at the end of the incubation period. Data are plotted as mean ± SD of experimental duplicates. One representative of three experiments is shown.


( D ) Phoenix cells expressing indicated mutant or WT DNGR - 1 proteins ( mouse , long isoform ) were treated with F - actin , F - actin buffer , anti - DNGR - 1 antibody or isotype - matched antibody of irrelevant specificity for 45 minutes , fixed in PFA , surface - stained for DNGR - 1 and analyzed by flow cytometry . One representative of three experiments is 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 O O O O O O O O O O O O O O O O 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) Phoenix cells expressing indicated mutant or WT DNGR-1 proteins (mouse, long isoform) were treated with F-actin, F-actin buffer, anti-DNGR-1 antibody or isotype-matched antibody of irrelevant specificity for 45 minutes, fixed in PFA, surface-stained for DNGR-1 and analyzed by flow cytometry. One representative of three experiments is shown.


( A ) DNGR - 1 KO MuTu DCs were transduced to express indicated DNGR - 1 proteins , FACS - sorted and expanded . Surface ( left panel ) and total ( right panel ) DNGR - 1 expression was determined by flow cytometry .

O O O O O O O O O O O O O O O O O O O O O O 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) DNGR-1 KO MuTu DCs were transduced to express indicated DNGR-1 proteins, FACS-sorted and expanded. Surface (left panel) and total (right panel) DNGR-1 expression was determined by flow cytometry.


( B and C ) Ovalbumin - expressing mouse embryonic fibroblasts ( bm1 T OVA MEFs ) were UV - irradiated , left to undergo secondary necrosis and incubated overnight with MuTu cells transduced with indicated WT or mutant DNGR - 1 proteins or an empty - vector control ( ratio dead cells : MuTu cells is indicated ) and OVA - specific pre - activated OT - I CD8 + T - lymphocytes . The amount of IFNγ accumulating in the culture medium was assessed using ELISA . One representative of two ( B ) or four ( C ) experiments is 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C) Ovalbumin-expressing mouse embryonic fibroblasts (bm1 T OVA MEFs) were UV-irradiated, left to undergo secondary necrosis and incubated overnight with MuTu cells transduced with indicated WT or mutant DNGR-1 proteins or an empty-vector control (ratio dead cells:MuTu cells is indicated) and OVA-specific pre-activated OT-I CD8+ T-lymphocytes. The amount of IFNγ accumulating in the culture medium was assessed using ELISA. One representative of two (B) or four (C) experiments is shown.


C ) ykt6ts pho8 ∆ 60 cells containing an empty plasmid , 3HA - Ykt6 or 3HA - tagged Ykt6 mutant variants as indicated were analyzed as in A ) .

O O O O O O O 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) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6 or 3HA-tagged Ykt6 mutant variants as indicated were analyzed as in A).


D ) ykt6ts pho8 ∆ 60 cells containing an empty plasmid , 3HA - Ykt6 , or ADH1 promoter driven 3HA - Ykt6 wild type or 3HA - tagged Ykt6 mutant variants as indicated were grown and Pho8 ∆ 60 activity was monitored as described in A ) .

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D) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6, or ADH1 promoter driven 3HA-Ykt6 wild type or 3HA-tagged Ykt6 mutant variants as indicated were grown and Pho8∆60 activity was monitored as described in A).


E ) Ykt6 levels were analyzed from the strains shown in D ) and corresponding cell extracts were analyzed by anti - HA and anti - Pgk1 Western blotting .

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E) Ykt6 levels were analyzed from the strains shown in D) and corresponding cell extracts were analyzed by anti-HA and anti-Pgk1 Western blotting.


A ) ykt6ts cells containing centromeric plasmids as indicated were grown at permissive temperature ( 23 ºC ) , shifted to the restrictive temperature at 37 ºC for 1 hour before starvation for 1 hour at 37 ºC . Cells were lysed and the total extract ( T ) separated into a cytoplasmic ( S ) and a 100 ' 000x g membrane pellet ( P ) fraction . Fractions were analyzed by anti - HA , anti - Tom70 and anti - Pgk1 Western blotting . One representative experiment out of three biological replicates is shown .

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A) ykt6ts cells containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 1 hour before starvation for 1 hour at 37 ºC. Cells were lysed and the total extract (T) separated into a cytoplasmic (S) and a 100'000x g membrane pellet (P) fraction. Fractions were analyzed by anti-HA, anti-Tom70 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.


B ) vam3 ∆ or ykt6ts vam3 ∆ cells containing the indicated centromeric plasmids were grown at permissive temperature ( 23 ºC ) , shifted to the restrictive temperature at 37 ° C for 1 hour followed by 1 hour starvation in SD - N at 37 ºC . After cell lysis , samples were subjected to proteinase K ( ProtK ) and Triton X - 100 ( TX100 ) treatment as indicated and analyzed by anti - Ape1 Western blotting . Ape1 * : proteinase K - resistant fragment of Ape1 . One representative experiment out of three biological replicates is shown .

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B) vam3∆ or ykt6ts vam3∆ cells containing the indicated centromeric plasmids were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by 1 hour starvation in SD-N at 37 ºC. After cell lysis, samples were subjected to proteinase K (ProtK) and Triton X-100 (TX100) treatment as indicated and analyzed by anti-Ape1 Western blotting. Ape1*: proteinase K-resistant fragment of Ape1. One representative experiment out of three biological replicates is shown.


C ) Trichloroacetic acid extracts were prepared from ykt6ts cells containing an empty plasmid , 3HA - Ykt6 wild type , or 3HA - tagged Ykt6 mutant variants . Cells were grown at the permissive temperature ( 23 ºC ) to late exponential phase in selective medium , shifted to the restrictive temperature ( 37 ° C ) for 1 h preincubation and continued to grow at the restrictive temperature for another 4 hours before harvesting . Samples were analyzed by anti - CPY and anti - Pgk1 Western blotting . One representative experiment out of three biological replicates is shown .

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C) Trichloroacetic acid extracts were prepared from ykt6ts cells containing an empty plasmid, 3HA-Ykt6 wild type, or 3HA-tagged Ykt6 mutant variants. Cells were grown at the permissive temperature (23 ºC) to late exponential phase in selective medium, shifted to the restrictive temperature (37 °C) for 1 h preincubation and continued to grow at the restrictive temperature for another 4 hours before harvesting. Samples were analyzed by anti-CPY and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.


D ) Exponentially growing ykt6ts cells at permissive temperature ( 23 ºC ) containing centromeric plasmids as indicated were shifted to the restrictive temperature ( 37 ºC ) for 1 hour before starvation for 4 hours in SD - N . Trichloroacetic acid extracts were prepared . Lipidation of Atg8 was analyzed on a 15 % SDS - PAGE containing 6 M urea by anti - Atg8 and anti - Pgk1 Western blotting . One representative experiment out of three biological replicates is shown .

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D) Exponentially growing ykt6ts cells at permissive temperature (23 ºC) containing centromeric plasmids as indicated were shifted to the restrictive temperature (37 ºC) for 1 hour before starvation for 4 hours in SD-N. Trichloroacetic acid extracts were prepared. Lipidation of Atg8 was analyzed on a 15 % SDS-PAGE containing 6 M urea by anti-Atg8 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.


A and B ) GFP - Atg8 atg1 ∆ , GFP - Atg8 atg1 ∆ vam3 ∆ , GFP - Atg8 wild type , GFP - Atg8 vam3 ∆ , GFP - Atg8 ykt6ts or GFP - Atg8 ykt6ts vam3 ∆ cell containing centromeric plasmids as indicated were grown at permissive temperature ( 23 ºC ) , shifted to the restrictive temperature at 37 ºC for 2 hours before starvation for 1 hour at 37 ºC in SD - N . Representative microscopy images are shown in A ) . GFP - Atg8 puncta per cell were quantified from 3 biological replicates ( n ≥ 100 cells ) and the mean was plotted in B ) . Error bars are SD . Scale bar : 5 µm .

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A and B) GFP-Atg8 atg1∆, GFP-Atg8 atg1∆ vam3∆, GFP-Atg8 wild type, GFP-Atg8 vam3∆, GFP-Atg8 ykt6ts or GFP-Atg8 ykt6ts vam3∆ cell containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 2 hours before starvation for 1 hour at 37 ºC in SD-N. Representative microscopy images are shown in A). GFP-Atg8 puncta per cell were quantified from 3 biological replicates (n≥100 cells) and the mean was plotted in B). Error bars are SD. Scale bar: 5 µm.


Autophagosomes were prepared from GFP - Atg8 vam3 ∆ pep4 ∆ or GFP - Atg8 ykt6ts vam3 ∆ pep4 ∆ cells containing centromeric plasmids as indicated . Cells were starved for 16 hours at the permissive temperature ( 23 ° C ) to allow formation of mature , closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting . Vacuoles were isolated from Vph1 - 4xmCherry atg15 ∆ pep4 ∆ cells grown under rich conditions at 30 ° C . Fusion reactions were incubated at the restrictive temperature ( 30 ° C ) for 2 hours . Representative images are shown in C ) .

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Autophagosomes were prepared from GFP-Atg8 vam3∆ pep4∆ or GFP-Atg8 ykt6ts vam3∆ pep4∆ cells containing centromeric plasmids as indicated. Cells were starved for 16 hours at the permissive temperature (23 °C) to allow formation of mature, closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting. Vacuoles were isolated from Vph1-4xmCherry atg15∆ pep4∆ cells grown under rich conditions at 30 °C. Fusion reactions were incubated at the restrictive temperature (30 °C) for 2 hours. Representative images are shown in C).


Autophagosomes were prepared from GFP - Atg8 vam3 ∆ pep4 ∆ or GFP - Atg8 ykt6ts vam3 ∆ pep4 ∆ cells containing centromeric plasmids as indicated . Cells were starved for 16 hours at the permissive temperature ( 23 ° C ) to allow formation of mature , closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting . Vacuoles were isolated from Vph1 - 4xmCherry atg15 ∆ pep4 ∆ cells grown under rich conditions at 30 ° C . Fusion reactions were incubated at the restrictive temperature ( 30 ° C ) for 2 hours . The graph in D ) shows the mean from at least three independent biological experiments . Error bars represent the standard deviation .

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Autophagosomes were prepared from GFP-Atg8 vam3∆ pep4∆ or GFP-Atg8 ykt6ts vam3∆ pep4∆ cells containing centromeric plasmids as indicated. Cells were starved for 16 hours at the permissive temperature (23 °C) to allow formation of mature, closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting. Vacuoles were isolated from Vph1-4xmCherry atg15∆ pep4∆ cells grown under rich conditions at 30 °C. Fusion reactions were incubated at the restrictive temperature (30 °C) for 2 hours. The graph in D) shows the mean from at least three independent biological experiments. Error bars represent the standard deviation.


The indicated strains were grown to late exponential phase at permissive temperature ( 23 ºC ) , shifted to the restrictive temperature at 37 ° C for 1 hour followed by starvation in SD - N for 1 hour at 37 ºC . 3HA - Ykt6 and its mutant variants expressed from a centromeric plasmid were immunoprecipitated ( IP ) and analyzed by anti - HA Western blotting . Co - precipitating proteins were analyzed by anti - Vam3 and anti - Vti1 Western blotting . One representative experiment out of three biological replicates is shown .

The indicated strains were grown to late exponential phase at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by starvation in SD-N for 1 hour at 37 ºC. 3HA-Ykt6 and its mutant variants expressed from a centromeric plasmid were immunoprecipitated (IP) and analyzed by anti-HA Western blotting. Co-precipitating proteins were analyzed by anti-Vam3 and anti-Vti1 Western blotting. One representative experiment out of three biological replicates is shown.


A . 3D image of an EGFP - CaaX - expressing Ramos B cell embedded in Matrigel ( left ) and segmentation of the inner dome ( center ) using our own script based on Watershed segmentation and segmentation of the cell surface ( right ) using an nnU - Net model that we trained based on our own - generated ground truth .

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A. 3D image of an EGFP-CaaX-expressing Ramos B cell embedded in Matrigel (left) and segmentation of the inner dome (center) using our own script based on Watershed segmentation and segmentation of the cell surface (right) using an nnU-Net model that we trained based on our own-generated ground truth.


C . Cluster density at surface features . IgM - BCR cluster density within different topographical features quantified as cluster count per surface feature volume .

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C. Cluster density at surface features. IgM-BCR cluster density within different topographical features quantified as cluster count per surface feature volume.


F , G , H . Continuous analysis of IgM - BCR cluster density using geodesic distance maps initiated from ( F ) microvilli tips towards microvilli roots , ( G ) centerline of ridges towards cell body surface , and ( H ) nodes towards cell body surface .

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F, G, H. Continuous analysis of IgM-BCR cluster density using geodesic distance maps initiated from (F) microvilli tips towards microvilli roots, (G) centerline of ridges towards cell body surface, and (H) nodes towards cell body surface.


A , B . Sum projections of the skeletonized ridge network and microvilli ( A ) and segmented IgM - BCR clusters ( B ) in an untreated and 100 µM CK - 666 - treated cell for 15 consecutive time frames amounting to 37 . 5 s .

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A, B. Sum projections of the skeletonized ridge network and microvilli (A) and segmented IgM-BCR clusters (B) in an untreated and 100 µM CK-666-treated cell for 15 consecutive time frames amounting to 37.5 s.


G . Continuous analysis of IgM - BCR cluster density in 15 - 30 min 100 µM CK - 666 - treated cells using geodesic distance maps initiating from distinct surface features ( centerline of ridges , nodes and microvilli tips ) ( to compare to untreated in Fig 3F ) . Data points and the dotted lines represent mean ± SD . H . Time - course images of EGFP - CaaX and IgM - BCR intensity in an elongating microvillus ( purple , MV mask ) .

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G. Continuous analysis of IgM-BCR cluster density in 15-30 min 100 µM CK-666-treated cells using geodesic distance maps initiating from distinct surface features (centerline of ridges, nodes and microvilli tips) (to compare to untreated in Fig 3F). Data points and the dotted lines represent mean ± SD. H. Time-course images of EGFP-CaaX and IgM-BCR intensity in an elongating microvillus (purple, MV mask).


A . IgM - BCR cluster distribution upon Ag stimulation . Ramos B cells in the Matrigel were stimulated with 50 ng / ml NIP - 15 - BSA Ag for at least 15 minutes . Ag - MC on the B cell surface were segmented and overlaid with the ridge network ( including microvilli ) with a 1 . 25x and 3 . 0x local intensity cut - off for cluster detection .

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A. IgM-BCR cluster distribution upon Ag stimulation. Ramos B cells in the Matrigel were stimulated with 50 ng/ml NIP-15-BSA Ag for at least 15 minutes. Ag-MC on the B cell surface were segmented and overlaid with the ridge network (including microvilli) with a 1.25x and 3.0x local intensity cut-off for cluster detection.


G , H . Staticity scores for different surface features ( R + N , MV ) ( G ) and for segmented IgM - BCR clusters assigned to different surface features ( H ) in unstimulated and Ag - stimulated cells . Violin plot solid lines represent the median and dotted lines represent the quartiles .

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G, H. Staticity scores for different surface features (R+N, MV) (G) and for segmented IgM-BCR clusters assigned to different surface features (H) in unstimulated and Ag-stimulated cells. Violin plot solid lines represent the median and dotted lines represent the quartiles.


A . Phalloidin - Alexa555 staining of fixed and permeabilized Ramos B cells expressing EGFP - CaaX and IgM - BCR with or without 30 min 2 µM LatA treatment .

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A. Phalloidin-Alexa555 staining of fixed and permeabilized Ramos B cells expressing EGFP-CaaX and IgM-BCR with or without 30 min 2 µM LatA treatment.


D . Surface - masked EGFP - CaaX and IgM - BCR signal and overlay of segmented elevations ( yellow ) with the surface - masked IgM - BCR signal in cells treated with LatA and LatA + Ag ( 50 ng / ml NIP - 15 - BSA ) . E . Time frames from a LatA + Ag cell showing directed movement of Ag - MC towards the elevations ( yellow ) . Each Ag - MC shown with a different colored arrow .

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D. Surface-masked EGFP-CaaX and IgM-BCR signal and overlay of segmented elevations (yellow) with the surface-masked IgM-BCR signal in cells treated with LatA and LatA+Ag (50 ng/ml NIP-15-BSA). E. Time frames from a LatA+Ag cell showing directed movement of Ag-MC towards the elevations (yellow). Each Ag-MC shown with a different colored arrow.


F . IgM - BCR intensity enrichment within all the segmented clusters in LatA and Lat + Ag cells . G . Cluster density at surface features . Quantification of the IgM - BCR cluster density in terms of cluster count per surface feature volume for all segmented IgM - BCR clusters ( left panel , local contrast cutoff = 1x ) and for Ag - MC ( right panel , local contrast cutoff = 3x ) .

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F. IgM-BCR intensity enrichment within all the segmented clusters in LatA and Lat+Ag cells. G. Cluster density at surface features. Quantification of the IgM-BCR cluster density in terms of cluster count per surface feature volume for all segmented IgM-BCR clusters (left panel, local contrast cutoff = 1x) and for Ag-MC (right panel, local contrast cutoff = 3x). H


I . Surface - masked EGFP - CaaX and IgM - BCR signal for a LatA and LatA + Ag cell and the sum projections of their segmented IgM - BCR clusters ( duration : 37 . 5 s ) . IgM - BCR clusters are color - coded ( green and yellow ) according to their assigned surface region .

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I. Surface-masked EGFP-CaaX and IgM-BCR signal for a LatA and LatA+Ag cell and the sum projections of their segmented IgM-BCR clusters (duration: 37.5 s). IgM-BCR clusters are color-coded (green and yellow) according to their assigned surface region.


A . Immunoblot analysis of ACE2 expression in the lysate of human lung A549 cells overexpressing ACE2 compared to non - transduced ( - ) A549 cells . Calnexin served as loading control .

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A. Immunoblot analysis of ACE2 expression in the lysate of human lung A549 cells overexpressing ACE2 compared to non-transduced (-) A549 cells. Calnexin served as loading control.