( E ) MEFs that had been transfected with the control siRNA or Rab12 siRNA were cultured as in ( C , D ) , and their lysates were analysed by immunoblotting with the antibodies indicated .

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(E) MEFs that had been transfected with the control siRNA or Rab12 siRNA were cultured as in (C,D), and their lysates were analysed by immunoblotting with the antibodies indicated.


( G ) Control and Rab12 ‐ knockdown MEFs were cultured under N or S conditions in the absence or presence of 100 μM bafilomycin A1 for 1 h . Cell lysates were analysed by immunoblotting with the antibodies indicated , and the intensity of the LC3 ‐ II band was quantified . The normalized amount ( arbitrary units ) of LC3 ‐ II in lanes 1 , 2 , 3 , 4 , 5 and 6 is 1 . 0 , 2 . 3 , 4 . 3 , 0 . 9 , 1 . 6 and 3 . 8 , respectively .

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(G) Control and Rab12‐knockdown MEFs were cultured under N or S conditions in the absence or presence of 100 μM bafilomycin A1 for 1 h. Cell lysates were analysed by immunoblotting with the antibodies indicated, and the intensity of the LC3‐II band was quantified. The normalized amount (arbitrary units) of LC3‐II in lanes 1, 2, 3, 4, 5 and 6 is 1.0, 2.3, 4.3, 0.9, 1.6 and 3.8, respectively.


( H ) MEFs transiently expressing mStr ‐ Rab12 were cultured under starved conditions and immunostained with the antibodies indicated . Scale bar , 20 μm . MEFs , mouse embryonic fibroblasts ; N , nutrient ‐ rich ; S , starved ; siRNA , short interfering RNA . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 .

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(H) MEFs transiently expressing mStr‐Rab12 were cultured under starved conditions and immunostained with the antibodies indicated. Scale bar, 20 μm. MEFs, mouse embryonic fibroblasts; N, nutrient‐rich; S, starved; siRNA, short interfering RNA. *P0.05; **P0.01; ***P0.005.


( A ) Control and Rab12 ‐ knockdown MEFs were cultured under nutrient ‐ rich or starved conditions , fixed and then immunostained with the antibodies indicated . Scale bar , 20 μm .

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(A) Control and Rab12‐knockdown MEFs were cultured under nutrient‐rich or starved conditions, fixed and then immunostained with the antibodies indicated. Scale bar, 20 μm.


( B ) Control and Rab12 ‐ knockdown MEFs were cultured as in ( A ) , and their lysates were analysed by immunoblotting with the antibodies indicated .

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(B) Control and Rab12‐knockdown MEFs were cultured as in (A), and their lysates were analysed by immunoblotting with the antibodies indicated.


( C ) Lysates of MEFs that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated .

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(C) Lysates of MEFs that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated.


( D , E ) Quantification of the phospho ‐ S6K levels and phospho ‐ Akt levels shown in ( C ) . MEFs , mouse embryonic fibroblasts ; N , nutrient ‐ rich ; S , starved ; siRNA , short interfering RNA . * P0 . 05 ; * * P0 . 01 .

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(D,E) Quantification of the phospho‐S6K levels and phospho‐Akt levels shown in (C). MEFs, mouse embryonic fibroblasts; N, nutrient‐rich; S, starved; siRNA, short interfering RNA. *P0.05; **P0.01.


( A ) MEFs transiently co ‐ expressing Myc ‐ PAT4 and mStr ‐ Rab12 were immunostained with anti ‐ Myc antibody .

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(A) MEFs transiently co‐expressing Myc‐PAT4 and mStr‐Rab12 were immunostained with anti‐Myc antibody.


( B ) MEFs transiently expressing Myc ‐ PAT4 were immunostained with the antibodies indicated . Scale bars , 20 μm .

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(B) MEFs transiently expressing Myc‐PAT4 were immunostained with the antibodies indicated. Scale bars, 20 μm.


( C ) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA ‐ PAT4 were analysed by immunoblotting with the antibodies indicated .

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(C) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA‐PAT4 were analysed by immunoblotting with the antibodies indicated.


( D ) Lysates of MEFs stably expressing HA ‐ PAT4 that had been transfected with control or Rab12 siRNAs were analysed by immunoblotting with the antibodies indicated .

(D) Lysates of MEFs stably expressing HA‐PAT4 that had been transfected with control or Rab12 siRNAs were analysed by immunoblotting with the antibodies indicated.


( E ) Quantification of ( D ) . * * * P0 . 005 .

(E) Quantification of (D). ***P0.005.


( F ) PAT4 mRNA levels from MEFs transfected with control or Rab12 siRNA as revealed by reverse ‐ transcription PCR analyses . Gapdh was used as an internal control .

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(F) PAT4 mRNA levels from MEFs transfected with control or Rab12 siRNA as revealed by reverse‐transcription PCR analyses. Gapdh was used as an internal control.


( G ) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA ‐ PAT4 that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated . Note that the amounts of plasma ‐ membrane ‐ localized PAT4 protein and TfR protein were higher in Rab12 ‐ knockdown MEFs , whereas the total amounts of EGFR protein and plasma membrane ‐ localized EGFR protein in these cells were slightly lower , but the mechanisms responsible for these changes are unknown . GAPDH , glyceraldehyde ‐ 3 ‐ phosphate dehydrogenase ; MEFs , mouse embryonic fibroblasts ; PAT4 , proton ‐ coupled amino ‐ acid transporter 4 ; siRNA , short interfering RNA .

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(G) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA‐PAT4 that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated. Note that the amounts of plasma‐membrane‐localized PAT4 protein and TfR protein were higher in Rab12‐knockdown MEFs, whereas the total amounts of EGFR protein and plasma membrane‐localized EGFR protein in these cells were slightly lower, but the mechanisms responsible for these changes are unknown. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; MEFs, mouse embryonic fibroblasts; PAT4, proton‐coupled amino‐acid transporter 4; siRNA, short interfering RNA.


( A ) Lysates of MEFs transfected with the siRNAs indicated were analysed by immunoblotting with the antibodies indicated .

(A) Lysates of MEFs transfected with the siRNAs indicated were analysed by immunoblotting with the antibodies indicated.


( B ) Quantification of the phospho ‐ S6K levels shown in ( A ) .

(B) Quantification of the phospho‐S6K levels shown in (A).


( C ) MEFs transfected with the siRNAs indicated were cultured under nutrient ‐ rich ( N ) or starved ( S ) conditions and were immunostained with anti ‐ LC3 antibody . The mean numbers of LC3 ‐ positive dots per cell are shown .

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(C) MEFs transfected with the siRNAs indicated were cultured under nutrient‐rich (N) or starved (S) conditions and were immunostained with anti‐LC3 antibody. The mean numbers of LC3‐positive dots per cell are shown.


( A - C ) 10X phase contrast images of vero monolayers at 3 days post - inoculation . Panels shown are ( A ) mock , ( B ) NP , and ( C ) OP .

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(A-C) 10X phase contrast images of vero monolayers at 3 days post-inoculation. Panels shown are (A) mock, (B) NP, and (C) OP.


( D ) Electron microscopic image of the viral isolate showing extracellular spherical particles with cross sections through the nucleocapsids , seen as black dots .

(D) Electron microscopic image of the viral isolate showing extracellular spherical particles with cross sections through the nucleocapsids, seen as black dots.


A ) Two SARS - CoV - 2 passage 4 stocks ( black and gray ) were quantified utilizing plaque assay at day two ( closed circles ) and day three ( open circles ) post infection of Vero E6 and Vero CCL81 .

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A) Two SARS-CoV-2 passage 4 stocks (black and gray) were quantified utilizing plaque assay at day two (closed circles) and day three (open circles) post infection of Vero E6 and Vero CCL81.


B ) Plaque morphology on Vero E6 and Vero CCL81 at day 2 and day 3 post inoculation .

B) Plaque morphology on Vero E6 and Vero CCL81 at day 2 and day 3 post inoculation.


C ) Cell monolayers two days post infection in Vero E6 ( top ) and Vero CCL81 ( bottom row ) at multiple dilutions .

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C) Cell monolayers two days post infection in Vero E6 (top) and Vero CCL81 (bottom row) at multiple dilutions.


Viral titer for SARS - CoV - 2 was quantitated by plaque assay on Vero E6 cells two days post inoculation .

Viral titer for SARS-CoV-2 was quantitated by plaque assay on Vero E6 cells two days post inoculation.


Protein lysates were probed by western blot with B ) anti - SARS nucleocapsid ( N ) antibody or C ) anti - SARS - CoV spike ( S ) antibody .

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Protein lysates were probed by western blot with B) anti-SARS nucleocapsid (N) antibody or C) anti- SARS-CoV spike (S) antibody.


The VPR and DPR ( right ) were dissected from E10 . 5 Pdx1 - GFP embryos ( left ) . Scale bar : 500 µm . DPR , dorsal Pdx1 - expressing region ; VPR , ventral Pdx1 - expressing region ; Duo , duodenum bud .

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The VPR and DPR (right) were dissected from E10.5 Pdx1-GFP embryos (left). Scale bar: 500 µm. DPR, dorsal Pdx1-expressing region; VPR, ventral Pdx1-expressing region; Duo, duodenum bud.


FACS gating for sorting GFPlow and GFPhigh cells from the VPR and DPR .

FACS gating for sorting GFPlow and GFPhigh cells from the VPR and DPR.


The ratios of GFPhigh cells to GFP + cells in the VPR and DPR . The data show the mean ± SEM ; n = number of independent biological replicates , unpaired t - test . * * * p - value < 0 . 001 .

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The ratios of GFPhigh cells to GFP+ cells in the VPR and DPR. The data show the mean ± SEM; n = number of independent biological replicates, unpaired t-test. ***p-value < 0.001.


Hierarchical clustering of 815 genes correlating with the first two PCs identified three groups of cell types and four major distinct clusters ( a - d ) of genes ( p < 1 × 10 - 9 , cell cycle - related genes are excluded ) . Each column is a single - cell , and each row represents one gene . Cluster - specific TFs are listed on the right . The genes in bold are known to have roles in pancreatic development . L , GFPlow ; H , GFPhigh .

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Hierarchical clustering of 815 genes correlating with the first two PCs identified three groups of cell types and four major distinct clusters (a-d) of genes (p < 1 ×10-9, cell cycle-related genes are excluded). Each column is a single-cell, and each row represents one gene. Cluster-specific TFs are listed on the right. The genes in bold are known to have roles in pancreatic development. L, GFPlow; H, GFPhigh.


Expression levels of group - specific genes in individual cells . The gene expression values were projected onto the PCA ( left ) and box ( right ) plots . Each point represents a cell . Points were colored based on the hierarchical clustering in Fig 1E . The y - axes of the box plots represent the expression levels ( RPKM ) . Central line in the box represents median ; box limits represent the first and third quartile ; whiskers represent 1 . 5 × interquartile range . Unpaired Wilcoxon rank - sum test . * p - value < 1 x 10 - 3 ; * * p - value < 1 x 10 - 6 ; * * * p - value < 1 x 10 - 9 .

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Expression levels of group-specific genes in individual cells. The gene expression values were projected onto the PCA (left) and box (right) plots. Each point represents a cell. Points were colored based on the hierarchical clustering in Fig 1E. The y-axes of the box plots represent the expression levels (RPKM). Central line in the box represents median; box limits represent the first and third quartile; whiskers represent 1.5 × interquartile range. Unpaired Wilcoxon rank-sum test. *p-value < 1 x 10-3; **p-value <1 x 10-6; ***p-value <1 x 10-9.


Flow cytometric analysis of BrdU incorporation rates in GFPlow and GFPhigh cells from the DPR and VPR . Statistical analysis of BrdU + in different cell populations . The Data show the mean ± SEM ; n = number of independent biological replicates , unpaired t - test . * p - value < 0 . 05 ; * * * p - value < 0 . 001 .

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Flow cytometric analysis of BrdU incorporation rates in GFPlow and GFPhigh cells from the DPR and VPR. Statistical analysis of BrdU+ in different cell populations. The Data show the mean ± SEM; n = number of independent biological replicates, unpaired t-test. *p-value < 0.05; ***p-value < 0.001.


Flow cytometric analysis of endocrine progenitors and β - cells in the ventral pancreases ( V . P . ) and dorsal pancreases ( D . P . ) . ( A , D and G ) Dissected ventral and dorsal pancreatic tissues . Scale bar : 500 µm . ( B , E and H ) FACS gating for sorting Ngn3 - GFP + or Ins1 - RFP + cells from the V . P . and D . P . ( C and F ) statistical analysis of percentages of Ngn3 - GFPhigh cells among Ngn3 - GFP + cells in the V . P . and D . P . ( I ) Statistical analysis of percentages of Ins1 - RFP + cells among total pancreatic cells in the V . P . and D . P .

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Flow cytometric analysis of endocrine progenitors and β-cells in the ventral pancreases (V. P.) and dorsal pancreases (D. P.). (A, D and G) Dissected ventral and dorsal pancreatic tissues. Scale bar: 500 µm. (B, E and H) FACS gating for sorting Ngn3-GFP+ or Ins1-RFP+ cells from the V. P. and D. P. (C and F) statistical analysis of percentages of Ngn3-GFPhigh cells among Ngn3-GFP+ cells in the V. P. and D. P. (I) Statistical analysis of percentages of Ins1-RFP+ cells among total pancreatic cells in the V. P. and D. P.


Differentially expressed genes among cells from VPR , DPR , the V . P . and D . P . at various developmental stages . The numbers indicate significantly highly expressed genes in the ventral ( blue ) or dorsal ( orange ) regions . The dotted lines represent the two - fold change threshold .

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Differentially expressed genes among cells from VPR, DPR, the V. P. and D. P. at various developmental stages. The numbers indicate significantly highly expressed genes in the ventral (blue) or dorsal (orange) regions. The dotted lines represent the two-fold change threshold.


Flow cytometric analyses of Liv2 + cells in the VPR , DPR and the liver buds ( LB ) . Liv2 + cells from the VPR - L ( brown arrow ) , Liv2 - cells from the VPR - L ( yellow arrow ) and Liv2 + cells from the LB ( black arrow ) were sorted for single - cell RNA - seq .

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Flow cytometric analyses of Liv2+ cells in the VPR, DPR and the liver buds (LB). Liv2+ cells from the VPR-L (brown arrow), Liv2- cells from the VPR-L (yellow arrow) and Liv2+ cells from the LB (black arrow) were sorted for single-cell RNA-seq.


Immunofluorescence of PDX1 and Liv2 on sagittal section of E10 . 5 mouse embryo . The white and yellow arrows denote PDX1low / Liv2 + cells in in the liver and EHBD regions , respectively . Scale bar : 50 µm .

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Immunofluorescence of PDX1 and Liv2 on sagittal section of E10.5 mouse embryo. The white and yellow arrows denote PDX1low/Liv2+ cells in in the liver and EHBD regions, respectively. Scale bar: 50 µm.


Hierarchical clustering of 694 genes correlated with the first two PCs identified three major distinct cell lineages ( p < 1 × 10 - 6 ) . Each column represents a single - cell and each row represents one gene . Cells were re - ordered according to the value of PC1 or PC2 . Genes in clusters ' a , b ' and ' c , d ' were re - ordered by correlations with PC2 and PC1 , respectively . The color of the cell type and cell source are indicated in ( C ) . Lineage - specific TFs are listed on the right .

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Hierarchical clustering of 694 genes correlated with the first two PCs identified three major distinct cell lineages (p < 1 × 10-6). Each column represents a single-cell and each row represents one gene. Cells were re-ordered according to the value of PC1 or PC2. Genes in clusters 'a, b' and 'c, d' were re-ordered by correlations with PC2 and PC1, respectively. The color of the cell type and cell source are indicated in (C). Lineage-specific TFs are listed on the right.


The gene expression values of lineage marker genes were projected onto the PCA ( E ) and box ( F ) plots . Each point represents a cell . The y - axes of the box plots represent the expression levels ( RPKM ) . Central line in the box represents median ; box limits represent the first and third quartile ; whiskers represent 1 . 5 × interquartile range . Unpaired Wilcoxon rank - sum test . * * p - value < 1 x 10 - 5 ; * * * p - value < 1 x 10 - 10 .

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The gene expression values of lineage marker genes were projected onto the PCA (E) and box (F) plots. Each point represents a cell. The y-axes of the box plots represent the expression levels (RPKM). Central line in the box represents median; box limits represent the first and third quartile; whiskers represent 1.5 × interquartile range. Unpaired Wilcoxon rank-sum test. **p-value <1 x 10-5; ***p-value <1 x 10-10.


Pdx1 and Sox17 expression levels in E9 . 5 VPR and DPR cells .

Pdx1 and Sox17 expression levels in E9.5 VPR and DPR cells.


Sagittal section of E12 . 5 Pdx1 - Cre ; Rosa26 - LacZ mouse embryo counterstained for ALB ( brown ) . X - gal staining ( blue ) showing cells derived from Pdx1 - expressing cells . The arrowheads indicate cells co - stained with X - gal and ALB ( See Fig EV5D for more images of different sections ) . Scale bar : 50 µm ( left ) or 20 µm ( right ) .

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Sagittal section of E12.5 Pdx1-Cre; Rosa26-LacZ mouse embryo counterstained for ALB (brown). X-gal staining (blue) showing cells derived from Pdx1-expressing cells. The arrowheads indicate cells co-stained with X-gal and ALB (See Fig EV5D for more images of different sections). Scale bar: 50 µm (left) or 20 µm (right).


F . BAZ2A read coverage quantification at ± 1Kb from peak summit of BAZ2A - bound regions identified in PC3 cells and the corresponding reads for BAZ2A - BRD mutant ( Y1775F ) in PC3 cells . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * * * < 0 . 001 ) . Data information : Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1 . 5 times the interquartile range .

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F. BAZ2A read coverage quantification at ±1Kb from peak summit of BAZ2A-bound regions identified in PC3 cells and the corresponding reads for BAZ2A-BRD mutant (Y1775F) in PC3 cells. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001). Data information: Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.


G . Regions bound by BAZ2A in PC3 cells are enriched in H3K14ac . Read coverage quantification for H3K14ac levels at regions bound by BAZ2A in PC3 cells ( BAZ2Awt and BAZ2A - BRD mutant ( Y1775F ) ) at ± 1Kb from BAZ2A peak summits . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * * * < 0 . 001 ) . Data information : Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1 . 5 times the interquartile range .

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G. Regions bound by BAZ2A in PC3 cells are enriched in H3K14ac. Read coverage quantification for H3K14ac levels at regions bound by BAZ2A in PC3 cells (BAZ2Awt and BAZ2A-BRD mutant (Y1775F)) at ±1Kb from BAZ2A peak summits. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001). Data information: Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.


B . EP300 acetylates H3K14 in PC3 cells . Western blot showing H3K14ac levels in PC3 cells treated for three days without and with the selective catalytic EP300 inhibitor A - 485 ( 10 μM ) . Histone H3 serves as protein loading control .

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B. EP300 acetylates H3K14 in PC3 cells. Western blot showing H3K14ac levels in PC3 cells treated for three days without and with the selective catalytic EP300 inhibitor A-485 (10 μM). Histone H3 serves as protein loading control.


F . qRT - PCR showing increased expression levels of genes with BAZ2A - bound promoter and downregulated in metastatic tumors ( AOX1 , MANB2 , DMPK , and DDB1 in PC3 cells treated with siRNA - EP300 . Data were from three independent experiments . Values were normalized to GAPDH mRNA . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * < 0 . 05 , * * * * < 0 . 0001 ) . Error bars represent SD .

F. qRT-PCR showing increased expression levels of genes with BAZ2A-bound promoter and downregulated in metastatic tumors (AOX1, MANB2, DMPK, and DDB1 in PC3 cells treated with siRNA-EP300. Data were from three independent experiments. Values were normalized to GAPDH mRNA. Statistical significance (P-values) was calculated using two-tailed t-test (*<0.05, ****<0.0001). Error bars represent SD.


B . Inactive enhancers ( C2 ) are enriched in H3K14ac and BAZ2A levels . Read coverage at enhancer classes C1 and C2 for BAZ2A , H3K14ac , H3K27ac , H3K4me1 and H3K27me3 at ± 1Kb from annotated enhancer regions . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * * * < 0 . 001 ) . Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1 . 5 times the interquartile range .

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B. Inactive enhancers (C2) are enriched in H3K14ac and BAZ2A levels. Read coverage at enhancer classes C1 and C2 for BAZ2A, H3K14ac, H3K27ac, H3K4me1 and H3K27me3 at ±1Kb from annotated enhancer regions. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001). Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.


H . qRT - PCR showing increased expression levels of BAZ2A - bound C2 - enhancer genes in PC3 cells treated with siRNA - EP300 . Data were from three independent experiments . Values were normalized to GAPDH mRNA . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * < 0 . 05 , * * < 0 . 001 ) . Error bars represent SD .

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H. qRT-PCR showing increased expression levels of BAZ2A-bound C2-enhancer genes in PC3 cells treated with siRNA-EP300. Data were from three independent experiments. Values were normalized to GAPDH mRNA. Statistical significance (P-values) was calculated using two-tailed t-test (*<0.05, **<0.001). Error bars represent SD.


C . qRT - PCR of genes differentially expressed in PC3 - CSCs compared to PC3 cells showing the upregulation of CSC markers ( ALDH1A2 , LGR5 , CD24 ) and downregulation of genes related to developmental processes ( HOXA1 , HOXA4 , SOX7 ) . Data are from two independent experiments . mRNA levels were normalized to GAPDH .

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C. qRT-PCR of genes differentially expressed in PC3-CSCs compared to PC3 cells showing the upregulation of CSC markers (ALDH1A2, LGR5, CD24) and downregulation of genes related to developmental processes (HOXA1, HOXA4, SOX7). Data are from two independent experiments. mRNA levels were normalized to GAPDH.


D . BAZ2A is required for the dedifferentiation of PC3 into PC3 - CSCs . Left panel : Representative images from two independent experiments showing the impairment of PC3 cells to form tumorspheres upon stable depletion of BAZ2A . Right panel : BAZ2A mRNA levels in three independent PC3 cell lines stably expressing shRNA - BAZ2A were measured by qRT - PCR and normalized to GAPDH mRNA from 2 independent experiments .

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D. BAZ2A is required for the dedifferentiation of PC3 into PC3-CSCs. Left panel: Representative images from two independent experiments showing the impairment of PC3 cells to form tumorspheres upon stable depletion of BAZ2A. Right panel: BAZ2A mRNA levels in three independent PC3 cell lines stably expressing shRNA-BAZ2A were measured by qRT-PCR and normalized to GAPDH mRNA from 2 independent experiments.


E . Left panel : qRT - PCR showing BAZ2A mRNA levels in PC3 cells depleted of endogenous BAZ2A with siRNA specifically targeting human BAZ2A sequences and expressing mouse BAZ2Awt and BAZ2A - BRD mutant ( BAZ2AY1775F ) . Values were normalized to MRPS7 mRNA . Data are from 3 independent experiments . Right panel : Quantification of tumorspheres from 3 independent experiments . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * < 0 . 05 ; * * < 0 . 01 ) . Error bars represent SD .

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E. Left panel: qRT-PCR showing BAZ2A mRNA levels in PC3 cells depleted of endogenous BAZ2A with siRNA specifically targeting human BAZ2A sequences and expressing mouse BAZ2Awt and BAZ2A-BRD mutant (BAZ2AY1775F). Values were normalized to MRPS7 mRNA. Data are from 3 independent experiments. Right panel: Quantification of tumorspheres from 3 independent experiments. Statistical significance (P-values) was calculated using two-tailed t-test (* <0.05; **< 0.01). Error bars represent SD.


B . Cell proliferation curves of PC3 cells treated with siRNA - BAZ2A . Error bars represent the standard deviation from three independent experiments . C . Cell proliferation curves of PC3 cells treated with 5μM BAZ2 - ICR and GSK2801 . Error bars represent the standard deviation from three independent experiments .

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B. Cell proliferation curves of PC3 cells treated with siRNA-BAZ2A. Error bars represent the standard deviation from three independent experiments. C. Cell proliferation curves of PC3 cells treated with 5μM BAZ2-ICR and GSK2801. Error bars represent the standard deviation from three independent experiments.


D . Representative images of tumorspheres generated from PC3 cells treated with BAZ2 - ICR ( 1μM ) or GSK2801 ( 1μM ) .

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D. Representative images of tumorspheres generated from PC3 cells treated with BAZ2-ICR (1μM) or GSK2801 (1μM).


E . Half - maximal effective concentration ( EC50 ) of BAZ2 - ICR . Measurements were performed 5 days after treatment of PC3 cells and initiation of tumorspheres using the indicated BAZ2 - ICR concentration . The amount of tumorspheres was assessed by measurement of cell viability . Error bars represent SD . Values are from three independent experiments .

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E. Half-maximal effective concentration (EC50) of BAZ2-ICR. Measurements were performed 5 days after treatment of PC3 cells and initiation of tumorspheres using the indicated BAZ2-ICR concentration. The amount of tumorspheres was assessed by measurement of cell viability. Error bars represent SD. Values are from three independent experiments.


A . Representative bright field images of organoids derived from mouse prostate cells treated with DMSO , BAZ2 - ICR ( 5μM ) , GSK2801 ( 5μM ) or GSK126 ( 5μM ) inhibitors followed by transduction with shRNA - Control or shRNA - Pten .

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A. Representative bright field images of organoids derived from mouse prostate cells treated with DMSO, BAZ2-ICR (5μM), GSK2801 (5μM) or GSK126 (5μM) inhibitors followed by transduction with shRNA-Control or shRNA-Pten.


C . Representative images of Ki67 - immunostained mouse prostate organoid sections expressing shRNA - control and shRNA - Pten and treated with DMSO or BAZ2 - ICR ( 5μM ) . D . Quantification of Ki67 + cells in mouse prostate organoids shown in C . * * < 0 . 01 , * * * * P < 0 . 0001 , two - tailed Student ' s t test ; ns , not significant . The median values are represented by a line .

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C. Representative images of Ki67-immunostained mouse prostate organoid sections expressing shRNA-control and shRNA-Pten and treated with DMSO or BAZ2-ICR (5μM). D. Quantification of Ki67+ cells in mouse prostate organoids shown in C. **< 0.01, ****P < 0.0001, two-tailed Student's t test; ns, not significant. The median values are represented by a line.


E . Quantification of mouse prostate organoids per field expressing shRNA - control and shRNA - Pten and treated with DMSO , GSK126 ( 5μM ) , BAZ2 - ICR ( 5μM ) , or GSK2801 ( 5μM ) . Values were normalized to shRNA - control samples . Average values of two independent experiments .

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E. Quantification of mouse prostate organoids per field expressing shRNA-control and shRNA-Pten and treated with DMSO, GSK126 (5μM), BAZ2-ICR (5μM), or GSK2801 (5μM). Values were normalized to shRNA-control samples. Average values of two independent experiments.


B ) Co - immunoprecipitation analysis to elucidate the interaction between Suv39h1 and HP1 . Whole - cell extracts of HEK293T cells expressing 3xFlag - Suv39h1 or 3xFlag - ΔN - Suv39h1 were subjected to immunoprecipitation with the anti - Flag tag antibody followed by immunoblotting with the indicated antibodies . IgG represents the heavy or light

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B) Co-immunoprecipitation analysis to elucidate the interaction between Suv39h1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Suv39h1 or 3xFlag-ΔN-Suv39h1 were subjected to immunoprecipitation with the anti-Flag tag antibody followed by immunoblotting with the indicated antibodies. IgG represents the heavy or light chain


F ) Co - immunoprecipitation analysis to elucidate the interaction between Setdb1 and HP1 . Whole - cell extracts of HEK293T cells expressing 3xFlag - Setdb1 or 3xFlag - Setdb1 mutants were subjected to immunoprecipitation with the anti - HP1β antibody The immunocomplex was analyzed by immunoblotting with the indicated antibodies .

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F) Co-immunoprecipitation analysis to elucidate the interaction between Setdb1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Setdb1 or 3xFlag-Setdb1 mutants were subjected to immunoprecipitation with the anti-HP1β antibody The immunocomplex was analyzed by immunoblotting with the indicated antibodies.


G ) Protein decay analysis of HP1 interaction - defective Setdb1 mutant . mESC lines stably expressing 3xFlag - Setdb1 or 3xFlag - Setdb1 - 3A were treated with CHX and MG132 for the indicated times . Stacked bar plots show the summarized protein amounts of Setdb1 ( right ) . Data represented as mean ± SD ( n = 4 , biological replicates ) . * * p < 0 . 01 ( Tukey ' s honestly significant difference test ) .

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G) Protein decay analysis of HP1 interaction-defective Setdb1 mutant. mESC lines stably expressing 3xFlag-Setdb1 or 3xFlag-Setdb1-3A were treated with CHX and MG132 for the indicated times. Stacked bar plots show the summarized protein amounts of Setdb1 (right). Data represented as mean ± SD (n = 4, biological replicates). **p < 0.01 (Tukey's honestly significant difference test).


B ) Protein expression levels of H3K9 MTs and histone modification levels in HP1 - cTKO cells were monitored by immunoblotting . The intensities were quantified using ImageJ software and summarized on the right . The thin gray line and bold colored line show individual data and means of the experimental replicates ( n ≥ 3 , biological replicates ) , respectively .

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B) Protein expression levels of H3K9 MTs and histone modification levels in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and means of the experimental replicates (n ≥ 3, biological replicates), respectively.


C ) Comparison of H3K9me3 levels between HP1 - deficient cells and Suv39h / Setdb1 - deficient cells . H3K9me levels were quantified using ImageJ software and are summarized at the bottom ; the horizontal line represents the mean intensity of each cell . More than 75 nuclei were counted for each staining . * * p < 0 . 01 ( Tukey ' s honestly significant difference test ) .

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C) Comparison of H3K9me3 levels between HP1-deficient cells and Suv39h/Setdb1-deficient cells. H3K9me levels were quantified using ImageJ software and are summarized at the bottom; the horizontal line represents the mean intensity of each cell. More than 75 nuclei were counted for each staining. **p < 0.01 (Tukey's honestly significant difference test).


D ) Electron microscopy images of HP1 - cTKO cells in the absence of 4OHT ( left ) and of cells treated with 4OHT for 4 days ( right ) . Boxes represent enlarged images .

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D) Electron microscopy images of HP1-cTKO cells in the absence of 4OHT (left) and of cells treated with 4OHT for 4 days (right). Boxes represent enlarged images.


B ) Subcellular distribution of H3K9 MTs in the HP1 - cTKO cells was examined by immunoblotting . α - Tubulin , Ezh2 , and histone H3 were used as representative markers for each fraction . Stacked bar plots show the abundance ratios of H3K9 MTs among the three fractions ( right ) .

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B) Subcellular distribution of H3K9 MTs in the HP1-cTKO cells was examined by immunoblotting. α-Tubulin, Ezh2, and histone H3 were used as representative markers for each fraction. Stacked bar plots show the abundance ratios of H3K9 MTs among the three fractions (right).


B , Elucidation of protein - protein interaction between HP1 and H3K9 MTs by co - immunoprecipitation analysis . Whole - cell extracts of HEK293T cells co - expressing HA - HP1β mutants and epitope - tagged proteins of interest were subjected to immunoprecipitation with the anti - HA tag antibody . The immunocomplex was analyzed by immunoblotting with the indicated antibodies . The intensities were quantified using ImageJ software , normalized , and shown on the top .

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B, Elucidation of protein-protein interaction between HP1 and H3K9 MTs by co-immunoprecipitation analysis. Whole-cell extracts of HEK293T cells co-expressing HA-HP1β mutants and epitope-tagged proteins of interest were subjected to immunoprecipitation with the anti-HA tag antibody. The immunocomplex was analyzed by immunoblotting with the indicated antibodies. The intensities were quantified using ImageJ software, normalized, and shown on the top.


E ) Evaluation of HP1β mutants for maintaining H3K9me2 / 3 and ensuring protein stability of H3K9 MTs . H3K9me2 / 3 levels and protein levels of H3K9 MTs in HP1 - cTKO lines expressing 3xFlag - hHP1β mutants were compared before and after 4OHT treatment . The intensities were quantified with ImageJ software and are summarized on the right . Data represented as mean ± SD ( n > 3 , biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 ( Welch ' s test ) .

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E) Evaluation of HP1β mutants for maintaining H3K9me2/3 and ensuring protein stability of H3K9 MTs. H3K9me2/3 levels and protein levels of H3K9 MTs in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).


B ) Protein expression levels of Jmjd1a / b in HP1 - cTKO cells were monitored by immunoblotting . The intensities were quantified using ImageJ software and summarized on the right . The thin gray line and bold colored line show individual data and the mean of experimental replicates , respectively .

B) Protein expression levels of Jmjd1a/b in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and the mean of experimental replicates, respectively.


C ) Evaluation of HP1β mutants for ensuring protein stability of Jmjd1a / b . Protein levels of Jmjd1a / b in HP1 - cTKO lines expressing 3xFlag - hHP1β mutants were compared before and after 4OHT treatment . The intensities were quantified with ImageJ software and are summarized on the right . Data represented as mean ± SD ( n > 3 , biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 ( Welch ' s test ) .

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C) Evaluation of HP1β mutants for ensuring protein stability of Jmjd1a/b. Protein levels of Jmjd1a/b in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).


C ) Impact of Suv39h / Setdb1 / G9a depletion on HP1 - mediated protein stability regulation of Jmjd1a / b . Jmjd1a / b were destabilized in mESCs lacking Suv39h / Setdb1 / G9a , even though HP1 was present .

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C) Impact of Suv39h/Setdb1/G9a depletion on HP1-mediated protein stability regulation of Jmjd1a/b. Jmjd1a/b were destabilized in mESCs lacking Suv39h/Setdb1/G9a, even though HP1 was present.


D ) Functional evaluation of H3K9me2 / 3 for confining HP1 to chromatin . Subcellular localizations of HP1α , β and γ in TT2 and mESCs lacking Suv39h / Setdb1 / G9a were compared . α - Tubulin , Ezh2 , and histone H3 were used as representative markers for each fraction .

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D) Functional evaluation of H3K9me2/3 for confining HP1 to chromatin. Subcellular localizations of HP1α, β and γ in TT2 and mESCs lacking Suv39h/Setdb1/G9a were compared. α-Tubulin, Ezh2, and histone H3 were used as representative markers for each fraction.


( A , B ) Representative images ( A ) and quantification ( B ) of calcium - stable k - fiber intensity levels by immunofluorescence analysis of n ≥ 41 tubulin - stained mitotic cells of neonatal ( N ) and elderly ( 87y ) human dermal fibroblasts ( HDF ) at prometaphase and metaphase stages . Intensity levels were normalized to neonatal samples . Scale bar , 5µm . Data information : All values shown are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by two tailed Mann - Whitney

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(A, B) Representative images (A) and quantification (B) of calcium-stable k-fiber intensity levels by immunofluorescence analysis of n≥41 tubulin-stained mitotic cells of neonatal (N) and elderly (87y) human dermal fibroblasts (HDF) at prometaphase and metaphase stages. Intensity levels were normalized to neonatal samples. Scale bar, 5µm. Data information: All values shown are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by two tailed Mann-Whitney test


( C ) Intra - kinetochore distance ( between Hec1 and ACA immunostainings of a kinetochore ) in n = 50 kinetochore pairs scored from 10 elderly vs . neonatal cells in prometaphase and metaphase . ( D ) Inter - kinetochore distance ( between Hec1 staining of sister kinetochores ) in n = 50 kinetochore pairs scored from 10 elderly vs . neonatal cells in prometaphase and metaphase . Data information : All values shown are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by two tailed Mann - Whitney

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(C) Intra-kinetochore distance (between Hec1 and ACA immunostainings of a kinetochore) in n=50 kinetochore pairs scored from 10 elderly vs. neonatal cells in prometaphase and metaphase. (D) Inter-kinetochore distance (between Hec1 staining of sister kinetochores) in n=50 kinetochore pairs scored from 10 elderly vs. neonatal cells in prometaphase and metaphase. Data information: All values shown are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by two tailed Mann-Whitney test


( E , Live cell imaging of neonatal ( N ) and elderly ( 87y ) fibroblasts expressing H2B - GFP / α - Tubulin - mCherry treated with kinesin - 5 inhibitor ( STLC ) to induce monopolar spindles , followed by washout ( WO ) into medium with DMSO ( - ) or ZM447439 ( + ; 500nM ) . ( E ) Quantification of anaphases with lagging chromosomes in n = cells

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(E, Live cell imaging of neonatal (N) and elderly (87y) fibroblasts expressing H2B-GFP/α-Tubulin-mCherry treated with kinesin-5 inhibitor (STLC) to induce monopolar spindles, followed by washout (WO) into medium with DMSO (-) or ZM447439 (+; 500nM). (E) Quantification of anaphases with lagging chromosomes in n=cells scored


F ) Live cell imaging of neonatal ( N ) and elderly ( 87y ) fibroblasts expressing H2B - GFP / α - Tubulin - mCherry treated with kinesin - 5 inhibitor ( STLC ) to induce monopolar spindles , followed by washout ( WO ) into medium with DMSO ( - ) or ZM447439 ( + ; 500nM ) . ( F ) representative movie frame series of a young and an elderly dividing cell that underwent correct ( Normal ) and incorrect ( Lagging ) chromosome segregation , respectively . Time , min : sec . Scale bars : 5µm ( images ) or 2µm ( insets ) .

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F) Live cell imaging of neonatal (N) and elderly (87y) fibroblasts expressing H2B-GFP/α-Tubulin-mCherry treated with kinesin-5 inhibitor (STLC) to induce monopolar spindles, followed by washout (WO) into medium with DMSO (-) or ZM447439 (+; 500nM). (F) representative movie frame series of a young and an elderly dividing cell that underwent correct (Normal) and incorrect (Lagging) chromosome segregation, respectively. Time, min:sec. Scale bars: 5µm (images) or 2µm (insets).


( G ) Representative images of anaphases without ( top ) and with ( bottom ) mis - segregation ( MS ) , FISH - stained for three chromosome pairs ( 7 , 12 and 18 ) . Arrowheads indicate micronuclei containing centromeric signal for chromosome 18 . Scale bar , 10µm . ( H ) Percentage of anaphases with MS in neonatal ( N ) vs . elderly ( 87y ) n = cells scored by FISH analysis . Data information : All values shown are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by ( H ) chi - square test .

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(G) Representative images of anaphases without (top) and with (bottom) mis-segregation (MS), FISH-stained for three chromosome pairs (7, 12 and 18). Arrowheads indicate micronuclei containing centromeric signal for chromosome 18. Scale bar, 10µm. (H) Percentage of anaphases with MS in neonatal (N) vs. elderly (87y) n=cells scored by FISH analysis. Data information: All values shown are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by (H) chi-square test.


( I ) Western blot analysis of total MCAK protein levels in neonatal ( HDF N ) and elderly ( HDF 87y ) fibroblasts . GAPDH is shown as loading control .

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(I) Western blot analysis of total MCAK protein levels in neonatal (HDF N) and elderly (HDF 87y) fibroblasts. GAPDH is shown as loading control.


( J ) Immunofluorescence analysis of MCAK levels in neonatal ( HDF N ) and elderly ( HDF 87y ) mitotic cells . Scale bar , 5µm .

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(J) Immunofluorescence analysis of MCAK levels in neonatal (HDF N) and elderly (HDF 87y) mitotic cells. Scale bar, 5µm.


( A ) GFP protein levels in FACS - sorted neonatal ( N ) and elderly ( 87y ) fibroblasts transduced with empty ( control , - ) , GFP - MCAK ( + ) and GFP - Kif2b ( + ) lentiviral plasmids . Tubulin is shown as loading control .

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(A) GFP protein levels in FACS-sorted neonatal (N) and elderly (87y) fibroblasts transduced with empty (control, -), GFP-MCAK (+) and GFP-Kif2b (+) lentiviral plasmids. Tubulin is shown as loading control.


( B ) Mitotic duration scored by time - lapse phase - contrast microscopy of n ≥ 50 cells per condition measured from nuclear envelope breakdown ( NEB ) to anaphase onset . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by two tailed ( B Mann - Whitney test .

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(B) Mitotic duration scored by time-lapse phase-contrast microscopy of n≥50 cells per condition measured from nuclear envelope breakdown (NEB) to anaphase onset. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by two tailed (B Mann-Whitney test.


( C , D ) Representative images ( C ) and quantification ( D ) of calcium - stable k - fiber intensity levels by immunofluorescence analysis of n ≥ 26 tubulin - stained metaphase cells transduced as indicated . Levels were normalized to the control neonatal sample . Scale bar , 5μm . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by two tailed D ) Mann - Whitney test .

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(C, D) Representative images (C) and quantification (D) of calcium-stable k-fiber intensity levels by immunofluorescence analysis of n≥26 tubulin-stained metaphase cells transduced as indicated. Levels were normalized to the control neonatal sample. Scale bar, 5μm. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by two tailed D) Mann-Whitney test.


( E ) Representative images of cytochalasin D - induced binucleated ( BN ) cells without ( top ) and with ( bottom ) mis - segregation ( MS ) , FISH - stained for three chromosome pairs ( 7 , 12 and 18 ) . Scale bar , 10µm . ( F ) Percentage of BN cells with MS in n = cells scored . ( G ) Percentage of micronuclei in n = cells analyzed . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by ( F , G ) chi - square 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 O 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) Representative images of cytochalasin D-induced binucleated (BN) cells without (top) and with (bottom) mis-segregation (MS), FISH-stained for three chromosome pairs (7, 12 and 18). Scale bar, 10µm. (F) Percentage of BN cells with MS in n=cells scored. (G) Percentage of micronuclei in n=cells analyzed. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by (F,G) chi-square test.


( A ) Representative images of elderly cells scored as negative ( - ) or positive ( + ) for Cdkn1a / p21 ( cell cycle inhibitor ) and 53BP1 ( ≥ 1 foci ; DNA damage ) senescence biomarkers . Scale bar 20µm . ( B ) Percentage of n = cells staining double - positive for Cdkn1a / p21 and 53BP1 in neonatal ( N / N ) and elderly ( 75 / 87y ) human dermal fibroblasts ( HDF ) transduced with empty , GFP - MCAK or GFP - Kif2b lentiviral plasmids . ( C ) Representative images of elderly cells scored as negative ( - ) or positive ( + ) for SA - β - galactosidase ( SA - β - gal ) activity . Scale bar 20µm . ( D ) Percentage of n = cells positive for SA - β - gal in neonatal and elderly HDFs transduced with empty , GFP - MCAK or GFP - Kif2b lentiviral plasmids . Data Information : Values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square test ( B , D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of elderly cells scored as negative (-) or positive (+) for Cdkn1a/p21 (cell cycle inhibitor) and 53BP1 (≥1 foci; DNA damage) senescence biomarkers. Scale bar 20µm. (B) Percentage of n=cells staining double-positive for Cdkn1a/p21 and 53BP1 in neonatal (N/N) and elderly (75/87y) human dermal fibroblasts (HDF) transduced with empty, GFP-MCAK or GFP-Kif2b lentiviral plasmids. (C) Representative images of elderly cells scored as negative (-) or positive (+) for SA-β-galactosidase (SA-β-gal) activity. Scale bar 20µm. (D) Percentage of n=cells positive for SA-β-gal in neonatal and elderly HDFs transduced with empty, GFP-MCAK or GFP-Kif2b lentiviral plasmids. Data Information: Values are mean ± s.d. of at least two independent experiments. ns p>0.05, *** p<0.001, **** p<0.0001 by two tailed chi-square test (B,D).


( F ) Total number of genes altered ( up or downregulated , as indicated ) for the comparisons shown between control and kinesin - 13 - overexpressing young ( HDF N ) and / or aged ( HDF 87y ) 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

(F) Total number of genes altered (up or downregulated, as indicated) for the comparisons shown between control and kinesin-13-overexpressing young (HDF N) and/or aged (HDF 87y) cells.


( G , H ) Heatmaps of differentially expressed ( G ) SASP and senescence genes , and ( H ) cGAS / STING / NF - κB - related genes of neonatal and 87y HDFs . Down and Up refer to the expected changes as reported for senescent cells Gene symbols highlighted in grey indicate genes that were not modulated by overexpression of Kinesin - 13 proteins . Z - score row color intensities indicate higher ( red ) to lower ( blue ) expression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmaps of differentially expressed (G) SASP and senescence genes, and (H) cGAS/STING/NF-κB-related genes of neonatal and 87y HDFs. Down and Up refer to the expected changes as reported for senescent cells Gene symbols highlighted in grey indicate genes that were not modulated by overexpression of Kinesin-13 proteins. Z-score row color intensities indicate higher (red) to lower (blue) expression.


( A ) Mitotic duration of neonatal ( N ) and elderly ( 87y ) human dermal fibroblasts ( HDF ) treated for 24 hrs with different concentrations of UMK57 ( MCAK agonist ) . n ≥ 58 cells were analyzed per condition . For all subsequent experiments UMK57 was used at 1µM for 24 hrs . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed ( A Mann - Whitney tests .

O O O O O 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 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

(A) Mitotic duration of neonatal (N) and elderly (87y) human dermal fibroblasts (HDF) treated for 24 hrs with different concentrations of UMK57 (MCAK agonist). n≥58 cells were analyzed per condition. For all subsequent experiments UMK57 was used at 1µM for 24 hrs. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed (A Mann-Whitney tests.


( B , Representative images ( B ) of calcium - stable k - fiber intensity levels in metaphase , scored by immunofluorescence analysis of n ≥ 34 tubulin - stained mitotic cells of neonatal and elderly samples treated with DMSO ( - ) and UMK57 ( + ) . Levels were normalized to neonatal DMSO - treated condition . Scale bar , 5µm .

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

(B, Representative images (B) of calcium-stable k-fiber intensity levels in metaphase, scored by immunofluorescence analysis of n≥34 tubulin-stained mitotic cells of neonatal and elderly samples treated with DMSO (-) and UMK57 (+). Levels were normalized to neonatal DMSO-treated condition. Scale bar, 5µm.


C ) quantification ( C ) of calcium - stable k - fiber intensity levels in metaphase , of neonatal and elderly samples treated with DMSO ( - ) and UMK57 ( + ) . Levels were normalized to neonatal DMSO - treated condition . Scale bar , 5µm . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed C ) Mann - Whitney tests .

O O O O O O 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

C) quantification (C) of calcium-stable k-fiber intensity levels in metaphase, of neonatal and elderly samples treated with DMSO (-) and UMK57 (+). Levels were normalized to neonatal DMSO-treated condition. Scale bar, 5µm. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed C) Mann-Whitney tests.


( D ) Aneusomy index of chromosomes 7 , 12 and 18 measured by interphase FISH analysis of n = cells . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Aneusomy index of chromosomes 7, 12 and 18 measured by interphase FISH analysis of n=cells. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( E ) Percentage of cytochalasin D - induced binucleated ( BN ) n = cells with chromosomes 7 , 12 , and 18 mis - segregation ( MS ) . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of cytochalasin D-induced binucleated (BN) n=cells with chromosomes 7, 12, and 18 mis-segregation (MS). Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( F ) Percentage of micronuclei in n = cells scored when treated with DMSO or UMK57 . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

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

(F) Percentage of micronuclei in n=cells scored when treated with DMSO or UMK57. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( G ) Percentage of n = cells staining positive for double immunostaining of Cdkn1a / p21 ( cell cycle inhibitor ) and 53BP1 ( ≥ 1 foci ; DNA damage ) senescence biomarkers . ( H ) Percentage of n = cells staining positive for SA - β - galactosidase ( SA - β - gal ) activity . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of n=cells staining positive for double immunostaining of Cdkn1a/p21 (cell cycle inhibitor) and 53BP1 (≥1 foci; DNA damage) senescence biomarkers. (H) Percentage of n=cells staining positive for SA-β-galactosidase (SA-β-gal) activity. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( A ) Mitotic duration of n = 100 neonatal ( N ) and elderly ( 87y ) human dermal fibroblasts ( HDF ) treated with DMSO ( - ) or UMK57 ( + ) for 96 hrs . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed ( A Mann - Whitney tests .

O O O O O O O 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

(A) Mitotic duration of n=100 neonatal (N) and elderly (87y) human dermal fibroblasts (HDF) treated with DMSO (-) or UMK57 (+) for 96 hrs. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed (A Mann-Whitney tests.


( B ) Relative calcium - stable k - fiber intensity levels scored by immunofluorescence analysis of n ≥ 29 tubulin - stained metaphase cells of neonatal and elderly samples treated with DMSO and UMK57 for 96 hrs . Levels were normalized to neonatal DMSO - treated condition . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed B , Mann - Whitney tests .

O O O O O O O O O O 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

(B) Relative calcium-stable k-fiber intensity levels scored by immunofluorescence analysis of n≥29 tubulin-stained metaphase cells of neonatal and elderly samples treated with DMSO and UMK57 for 96 hrs. Levels were normalized to neonatal DMSO-treated condition. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed B, Mann-Whitney tests.


( C ) Aneusomy index of chromosomes 7 , 12 and 18 measured by interphase FISH analysis of neonatal and elderly n = cells treated for 96 hrs . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Aneusomy index of chromosomes 7, 12 and 18 measured by interphase FISH analysis of neonatal and elderly n=cells treated for 96 hrs. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( D ) Cytochalasin D - induced binucleated ( BN ) n = cells with mis - segregation ( MS ) of chromosomes 7 , 12 and 18 in neonatal and elderly samples treated for 96 hrs . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cytochalasin D-induced binucleated (BN) n=cells with mis-segregation (MS) of chromosomes 7, 12 and 18 in neonatal and elderly samples treated for 96 hrs. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( E ) Percentage of n = cells staining positive for double immunostaining of Cdkn1a / p21 ( cell cycle inhibitor ) and 53BP1 ( ≥ 1 foci ; DNA damage ) senescence biomarkers after 96hrs of treatment . ( F ) Percentage of n = cells staining positive for SA - β - gal activity when treated for 96 hrs with DMSO or UMK57 . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

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

(E) Percentage of n=cells staining positive for double immunostaining of Cdkn1a/p21 (cell cycle inhibitor) and 53BP1 (≥1 foci; DNA damage) senescence biomarkers after 96hrs of treatment. (F) Percentage of n=cells staining positive for SA-β-gal activity when treated for 96 hrs with DMSO or UMK57. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( G , Representative images of phospho - Aurora B Thr232 ( pAuroraB T232 ) levels at kinetochores / centromeres in n ≥ 75 neonatal and elderly metaphase cells treated with UMK57 for 24 hrs or 96 hrs . Intensity levels were normalized to ACA . Scale bar , 5µm .

O O O O O O O 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

(G, Representative images of phospho-Aurora B Thr232 (pAuroraB T232) levels at kinetochores/centromeres in n≥75 neonatal and elderly metaphase cells treated with UMK57 for 24 hrs or 96 hrs. Intensity levels were normalized to ACA. Scale bar, 5µm.


H ) quantification ( H ) of phospho - Aurora B Thr232 ( pAuroraB T232 ) levels at kinetochores / centromeres in n ≥ 75 neonatal and elderly metaphase cells treated with UMK57 for 24 hrs or 96 hrs . Intensity levels were normalized to ACA . Scale bar , 5µm . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed H ) Mann - Whitney tests .

O O O O O O O O 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

H) quantification (H) of phospho-Aurora B Thr232 (pAuroraB T232) levels at kinetochores/centromeres in n≥75 neonatal and elderly metaphase cells treated with UMK57 for 24 hrs or 96 hrs. Intensity levels were normalized to ACA. Scale bar, 5µm. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed H) Mann-Whitney tests.


A . Body weight gain of male mice with the specified genotypes under normal ( ND ) or high - fat diet ( HFD ) .

O O 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. Body weight gain of male mice with the specified genotypes under normal (ND) or high-fat diet (HFD).


E . Representative pictures of H & E staining of SubWAT and EpiWAT of indicated male mice fed HFD .

E. Representative pictures of H&E staining of SubWAT and EpiWAT of indicated male mice fed HFD.


lipids content ( F ) per gram of feces .

lipids content (F) per gram of feces.


D . Western blot analysis and quantification of phosphorylated PKD2 ( S876 ) and phosphorylated PKD ( S916 / S876 ) in small intestine of Pkd2wt / wt and Pkd2ki / ki male mice . n = 3 .

O O O O O O O O O O O O O O O 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. Western blot analysis and quantification of phosphorylated PKD2 (S876) and phosphorylated PKD (S916/S876) in small intestine of Pkd2wt/wt and Pkd2ki/ki male mice. n=3.


A . Western blot and quantification of phosphorylated PKD ( S916 / S876 ) in small intestine of Pkd2flox / flox and Pkd2gutΔ / Δ male mice . n = 3 . B . Western blot and quantification of phosphorylated PKD ( S744 - 748 ) in small intestine of Pkd2flox / flox and Pkd2gutΔ / Δ male mice . n = 3 . 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 O O O O O O O O O O O O O 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 and quantification of phosphorylated PKD (S916/S876) in small intestine of Pkd2flox/flox and Pkd2gutΔ/Δ male mice. n=3. B. Western blot and quantification of phosphorylated PKD (S744-748) in small intestine of Pkd2flox/flox and Pkd2gutΔ/Δ male mice. n=3. C


G . Free - fatty acids and H . Triglycerides content in serum of male mice after 17 weeks in HFD . n = 10 .

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

G. Free-fatty acids and H. Triglycerides content in serum of male mice after 17 weeks in HFD. n=10.


A . Western blot ( WB ) analysis of specified proteins in small intestine of Pkd2wt / wt and Pkd2ki / ki male mice after one week of HFD . Quantification of the bands for each protein normalized to loading control and relative to Pkd2wt / wt . n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (WB) analysis of specified proteins in small intestine of Pkd2wt/wt and Pkd2ki/ki male mice after one week of HFD. Quantification of the bands for each protein normalized to loading control and relative to Pkd2wt/wt. n=3.


H . WB with specified antibodies after an in - vitro kinase assay .

H. WB with specified antibodies after an in-vitro kinase assay.


C . Western blot analysis of phosphorylated PKD2 in jejunum of male C57BL / 6 mice in HFD and treated with CRT0066101 or control ( water ) for 13 weeks . n = 3 .

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

C. Western blot analysis of phosphorylated PKD2 in jejunum of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. n=3.


F . WB of Apolipoprotein A4 ( APOA4 ) in jejunum of male C57BL / 6 mice in HFD and treated with CRT0066101 or control ( water ) for 13 weeks . Quantification of bands normalized to GAPDH and relative to control ( water ) n = 6 .

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

F. WB of Apolipoprotein A4 (APOA4) in jejunum of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. Quantification of bands normalized to GAPDH and relative to control (water) n=6.


G . Body weight of male C57BL / 6 mice in HFD and treated with CRT0066101 or control ( water ) for 13 weeks . n = 6 for control and n = 5 for CRT0066101 .

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 B-CONTROLLED_VAR O

G. Body weight of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. n=6 for control and n=5 for CRT0066101.


A . Body weight gain of male C57BL / 6 mice that , after 7 weeks in HFD , started receiving treatment with CRT0066101 or water . HFD continued along with treatment . n = 8 .

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

A. Body weight gain of male C57BL/6 mice that, after 7 weeks in HFD, started receiving treatment with CRT0066101 or water. HFD continued along with treatment. n=8.


F . Western blot analysis of PKD phosphorylation in proximal jejunum of morbidly obese female patients . Samples are loaded according to fasting triglyceride levels ( lower to higher ) . n = 7 .

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F. Western blot analysis of PKD phosphorylation in proximal jejunum of morbidly obese female patients. Samples are loaded according to fasting triglyceride levels (lower to higher). n=7.


( B ) Interaction between PLD1 and ARL11 / 14 is stronger than with ARF1 / 6 , RhoA . Flag - tagged PLD1 was co - expressed with HA - tagged GTP - bound mutants of small GTPases in HEK293A cells as indicated . PLD1 was immunoprecipitated with Flag beads , and the co - precipitated small GTPases were detected with an HA antibody . The result is a representative of three biologically independent experiments .

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(B) Interaction between PLD1 and ARL11/14 is stronger than with ARF1/6, RhoA. Flag-tagged PLD1 was co-expressed with HA-tagged GTP-bound mutants of small GTPases in HEK293A cells as indicated. PLD1 was immunoprecipitated with Flag beads, and the co-precipitated small GTPases were detected with an HA antibody. The result is a representative of three biologically independent experiments.


( C ) ARL11 / 14 co - localize with PLD1 . Flag - tagged PLD1 was co - expressed with HA - tagged GTP - bound mutants of small GTPases in HEK293A cells as indicated . After 24 h , cells were fixed with 4 % paraformaldehyde , followed by incubation with anti - Flag and anti - HA primary antibodies . The Flag and HA - antibodies were detected by anti - mouse and anti - rabbit secondary antibodies , respectively . DAPI ( blue ) was used for DNA staining . Scale bars : 10 μm . The result is a representative of two biologically independent experiments .

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(C) ARL11/14 co-localize with PLD1. Flag-tagged PLD1 was co-expressed with HA-tagged GTP-bound mutants of small GTPases in HEK293A cells as indicated. After 24 h, cells were fixed with 4% paraformaldehyde, followed by incubation with anti-Flag and anti-HA primary antibodies. The Flag and HA-antibodies were detected by anti-mouse and anti-rabbit secondary antibodies, respectively. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. The result is a representative of two biologically independent experiments.


( D ) Mander ' s overlap coefficient of ( C ) . Mander ' s overlap coefficient calculated using JACoP plugin in ImageJ . Mean ± Standard Deviation ( SD ) ; n = 6 technically independent samples .

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(D) Mander's overlap coefficient of (C). Mander's overlap coefficient calculated using JACoP plugin in ImageJ. Mean± Standard Deviation (SD); n= 6 technically independent samples.


( E ) PLD1 preferentially binds to the GTP - form , not the GDP - form , of ARL11 . Flag - tagged PLD1 was co - expressed with HA - tagged ARL11Q67L ( GTP - form ) or ARL11T26N ( GDP - form ) mutant in HEK293A cells . The result is a representative of two biologically independent experiments .

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(E) PLD1 preferentially binds to the GTP-form, not the GDP-form, of ARL11. Flag-tagged PLD1 was co-expressed with HA-tagged ARL11Q67L (GTP-form) or ARL11T26N (GDP-form) mutant in HEK293A cells. The result is a representative of two biologically independent experiments.


( F ) The subcellular co - localization of ARL11 with PLD1 is GTP - dependent . HA - tagged GTP - bound ARL11Q67L , wild - type ARL11 or GDP - bound ARL11T26N was co - expressed with Flag - tagged PLD1 . After 24 h , cells were fixed , followed by incubation with anti - Flag and anti - HA primary antibodies . DAPI ( blue ) was used for DNA staining . Scale bars : 10 μm . The result is a representative of three biologically independent experiments .

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(F) The subcellular co-localization of ARL11 with PLD1 is GTP-dependent. HA-tagged GTP-bound ARL11Q67L, wild-type ARL11 or GDP-bound ARL11T26N was co-expressed with Flag-tagged PLD1. After 24 h, cells were fixed, followed by incubation with anti-Flag and anti-HA primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. The result is a representative of three biologically independent experiments.


( G ) Mander ' s overlap coefficient of ( F ) .

(G) Mander's overlap coefficient of (F).


( B ) The loop region of PLD1 is required for the interaction between ARL11 and PLD1 . Flag - tagged full - length or truncations of PLD1 was co - expressed with HA - tagged ARL11Q67L , PLD1 was immunoprecipitated with Flag beads , and ARL11 that associated with it was detected with an HA antibody . The result is a representative of two biologically independent experiments .

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(B) The loop region of PLD1 is required for the interaction between ARL11 and PLD1. Flag-tagged full-length or truncations of PLD1 was co-expressed with HA-tagged ARL11Q67L, PLD1 was immunoprecipitated with Flag beads, and ARL11 that associated with it was detected with an HA antibody. The result is a representative of two biologically independent experiments.


( C ) Deletion of the loop region disrupts the PLD1 and ARL11 co - localization . Flag - tagged full - length or ∆ Loop truncation of PLD1 was co - expressed with HA - tagged ARL11Q67L . After 24 h , cells were fixed with 4 % paraformaldehyde , followed by incubation with anti - Flag and anti - HA primary antibodies . DAPI ( blue ) was used for DNA staining . Scale bars : 10 μm . The result is a representative of three biologically independent experiments . ( D ) Mander ' s overlap coefficient of ( J ) . Mander ' s overlap coefficient calculated using JACoP plugin in ImageJ .

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(C) Deletion of the loop region disrupts the PLD1 and ARL11 co-localization. Flag-tagged full-length or ∆Loop truncation of PLD1 was co-expressed with HA-tagged ARL11Q67L. After 24 h, cells were fixed with 4% paraformaldehyde, followed by incubation with anti-Flag and anti-HA primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. The result is a representative of three biologically independent experiments. (D) Mander's overlap coefficient of (J). Mander's overlap coefficient calculated using JACoP plugin in ImageJ.


( B ) ARL11 Knockout of THP - 1 stable cell pools . ARL11 knockout THP - 1 cell pools were generated by the CRISPR / Cas9 technology with three different guide RNAs . Cell lysates were immunoblotted with the indicated antibodies .

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(B) ARL11 Knockout of THP-1 stable cell pools. ARL11 knockout THP-1 cell pools were generated by the CRISPR/Cas9 technology with three different guide RNAs. Cell lysates were immunoblotted with the indicated antibodies.


( D ) THP - 1 cell pool stably expressing HA - tagged ARL11Q67L or ARL11T26N . Lysates were immunoblotted with indicated antibodies .

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(D) THP-1 cell pool stably expressing HA-tagged ARL11Q67L or ARL11T26N. Lysates were immunoblotted with indicated antibodies.


( E ) ARL11Q67L , but not ARL11T26N , promotes phagocytosis of macrophages . Macrophages were differentiated from indicated THP1 cell pool , followed by incubated with green zymosan particles for 1 hour . Scale bars : 50 μm . The result is a representative of two biologically independent experiments .

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(E) ARL11Q67L, but not ARL11T26N, promotes phagocytosis of macrophages. Macrophages were differentiated from indicated THP1 cell pool, followed by incubated with green zymosan particles for 1 hour. Scale bars: 50 μm. The result is a representative of two biologically independent experiments.


( F ) Quantification result of ( E ) . Data are presented as mean ± SD , n = 8 technically independent samples .

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(F) Quantification result of (E). Data are presented as mean± SD, n = 8 technically independent samples.


( G ) PLD1 knockout efficiency and HA - tagged ARL11Q67L expression of THP - 1 stable cell pools . Lysates were immunoblotted with indicated antibodies .

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(G) PLD1 knockout efficiency and HA-tagged ARL11Q67L expression of THP-1 stable cell pools. Lysates were immunoblotted with indicated antibodies.


( H ) PLD1 knockout blocks the ARL11Q67L - stimulated phagocytosis . Macrophages were differentiated from indicated THP1 cell groups and then incubated with green zymosan particles for 1 . 5 hours . Scale bars : 50 μm . The result is a representative of two biologically independent experiments . ( I ) PLD1 is required for ARL11 to stimulate phagocytosis . Data are quantification of panel ( H ) .

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(H) PLD1 knockout blocks the ARL11Q67L-stimulated phagocytosis. Macrophages were differentiated from indicated THP1 cell groups and then incubated with green zymosan particles for 1.5 hours. Scale bars: 50 μm. The result is a representative of two biologically independent experiments. (I) PLD1 is required for ARL11 to stimulate phagocytosis. Data are quantification of panel (H).


( B ) Interaction of PI4KB with ARL5A / 5B is stronger than ARF1 , RAB11A / 11B , and RAB14 . Flag - tagged PI4KB was co - expressed with HA - tagged GTP - bound mutant of GTPases in HEK293A cells as indicated . PI4KB was immunoprecipitated with Flag beads , and the co - precipitated GTPases were detected by HA Western .

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(B) Interaction of PI4KB with ARL5A/5B is stronger than ARF1, RAB11A/11B, and RAB14. Flag-tagged PI4KB was co-expressed with HA-tagged GTP-bound mutant of GTPases in HEK293A cells as indicated. PI4KB was immunoprecipitated with Flag beads, and the co-precipitated GTPases were detected by HA Western.


( C ) PI4KB preferentially binds to the GTP - form of ARL5B . Flag - tagged PI4KB was co - expressed with HA - tagged ARL5BQ70L ( GTP - form ) or ARL5BT30N ( GDP - form ) mutant in HEK293A cells . Interaction was measured similar to panel B . The result is a representative of two biologically independent experiments .

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(C) PI4KB preferentially binds to the GTP-form of ARL5B. Flag-tagged PI4KB was co-expressed with HA-tagged ARL5BQ70L (GTP-form) or ARL5BT30N (GDP-form) mutant in HEK293A cells. Interaction was measured similar to panel B. The result is a representative of two biologically independent experiments.


( D ) The ARL5A colocalization with PI4KB is GTP - dependent . HA - tagged GTP - bound ARL5AQ70L , wild - type ARL5A or GDP - bound ARL5AT30N was co - expressed with Flag - tagged PI4KB . After 24 h , cells were fixed , followed by incubation with anti - Flag and anti - HA primary antibodies . DAPI ( blue ) was used for DNA staining . Scale bars : 10 μm . Shown is a representative of two biologically independent experiments . The left panel is the quantification of overlap coefficient . Mean ± SD ; n = 6 technically independent samples .

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(D) The ARL5A colocalization with PI4KB is GTP-dependent. HA-tagged GTP-bound ARL5AQ70L, wild-type ARL5A or GDP-bound ARL5AT30N was co-expressed with Flag-tagged PI4KB. After 24 h, cells were fixed, followed by incubation with anti-Flag and anti-HA primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. Shown is a representative of two biologically independent experiments. The left panel is the quantification of overlap coefficient. Mean± SD; n= 6 technically independent samples.


( E ) ARL5A / 5B double knockout diminishes the colocalization of PI4KB and TGN46 . Flag - tagged PI4KB was expressed in HEK293A wild - type or two ARL5A / 5B double knockout clones as indicated . After 24 hours , cells were fixed , followed by incubation with anti - Flag and anti - TGN46 primary antibodies . DAPI ( blue ) was used for DNA staining . Scale bars : 5 μm . The result is a representative of two biologically independent experiments . The left panel is the quantification of overlap coefficient .

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(E) ARL5A/5B double knockout diminishes the colocalization of PI4KB and TGN46. Flag-tagged PI4KB was expressed in HEK293A wild-type or two ARL5A/5B double knockout clones as indicated. After 24 hours, cells were fixed, followed by incubation with anti-Flag and anti-TGN46 primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 5 μm. The result is a representative of two biologically independent experiments. The left panel is the quantification of overlap coefficient.


( G ) The N1 region of PI4KB is required for the interaction with ARL5B . Flag - tagged full - length or truncations of PI4KB were co - expressed with HA - tagged ARL5BQ70L . PI4KB was immunoprecipitated with Flag beads , and the co - precipitated ARL5B was detected by HA Western . The result is a representative of three biologically independent experiments .

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(G) The N1 region of PI4KB is required for the interaction with ARL5B. Flag-tagged full-length or truncations of PI4KB were co-expressed with HA-tagged ARL5BQ70L. PI4KB was immunoprecipitated with Flag beads, and the co-precipitated ARL5B was detected by HA Western. The result is a representative of three biologically independent experiments.


( H ) The N1 region of PI4KB is required for the co - localization of ARL5A / B and PI4P . P4Mx2 - mEGFP was co - expressed with HA - tagged ARL5AQ70L in HEK293A wild - type or PI4KB knockout cells ( clone # 2 - 5 ) . HEK293A cells were co - transfected with indicated constructs . After 24 h , cells were fixed with 4 % paraformaldehyde , followed by incubation with anti - HA primary antibodies . DAPI ( blue ) was used for nuclei staining . Scale bars : 10 μm . The result is a representative of two biologically independent experiments . ( I ) Mander ' s overlap coefficient of ( H ) .

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(H) The N1 region of PI4KB is required for the co-localization of ARL5A/B and PI4P. P4Mx2-mEGFP was co-expressed with HA-tagged ARL5AQ70L in HEK293A wild-type or PI4KB knockout cells (clone#2-5). HEK293A cells were co-transfected with indicated constructs. After 24 h, cells were fixed with 4% paraformaldehyde, followed by incubation with anti-HA primary antibodies. DAPI (blue) was used for nuclei staining. Scale bars: 10 μm. The result is a representative of two biologically independent experiments. (I) Mander's overlap coefficient of (H).


( B ) ARL5A / 5B recruitment to mitochondria induces PI4P accumulation at the mitochondria . Tom20 - CFP - RFB and P4Mx2 - mEGFP were co - expressed with RFP - FKBP vector or RFP - FKBP - ARL5A / 5BQ70L constructs in HEK293A cells . Cell samples were treated with or without 100nM rapamycin for 1 hour . Scale bars : 10 μm . The dashed line box indicates the merged signal of PI4P with ARL5A / 5B or Tom20 . The result is a representative of two biologically independent experiments . ( C ) PI4KB knockout blocks the PI4P accumulation induced by mitochondria anchored ARL5A / 5B . Tom20 - CFP - RFB and P4Mx2 - mEGFP were co - expressed with RFP - FKBP vector or RFP - FKBP - ARL5A / 5BQ70L constructs in HEK293A PI4KB knockout cells ( clone # 2 - 5 ) . Cell samples were treated with or without 100nM rapamycin for 1 hour before imaging . Scale bars : 10 μm . The dashed line box indicates the merged signal of PI4P with ARL5A / 5B or Tom20 . The result is a representative of two biologically independent experiments . (

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(B) ARL5A/5B recruitment to mitochondria induces PI4P accumulation at the mitochondria. Tom20-CFP-RFB and P4Mx2-mEGFP were co-expressed with RFP-FKBP vector or RFP-FKBP-ARL5A/5BQ70L constructs in HEK293A cells. Cell samples were treated with or without 100nM rapamycin for 1 hour. Scale bars: 10 μm. The dashed line box indicates the merged signal of PI4P with ARL5A/5B or Tom20. The result is a representative of two biologically independent experiments. (C) PI4KB knockout blocks the PI4P accumulation induced by mitochondria anchored ARL5A/5B. Tom20-CFP-RFB and P4Mx2-mEGFP were co-expressed with RFP-FKBP vector or RFP-FKBP-ARL5A/5BQ70L constructs in HEK293A PI4KB knockout cells (clone#2-5). Cell samples were treated with or without 100nM rapamycin for 1 hour before imaging. Scale bars: 10 μm. The dashed line box indicates the merged signal of PI4P with ARL5A/5B or Tom20. The result is a representative of two biologically independent experiments. (


( D ) Mander ' s overlap coefficient of ( B and C , result of rapamycin treatment ) . Mean ± SD ; n = 5 technically independent samples .

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(D) Mander's overlap coefficient of (B and C, result of rapamycin treatment). Mean± SD; n= 5 technically independent samples.


( E ) ARL5A / 5B double knockout decreases protein secretion . Gaussia - luciferase was expressed in HEK293A wild - type or three ARL5A / 5B double knockout clones as indicated . After 24 hours , the medium was replaced with the fresh medium . After 4 hours , medium was collected , and the secreted Gaussia - luciferase was detected by Western . Cells were also collected and probed with indicated

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(E) ARL5A/5B double knockout decreases protein secretion. Gaussia-luciferase was expressed in HEK293A wild-type or three ARL5A/5B double knockout clones as indicated. After 24 hours, the medium was replaced with the fresh medium. After 4 hours, medium was collected, and the secreted Gaussia-luciferase was detected by Western. Cells were also collected and probed with indicated antibodies


( H ) PI4KB knockout blocks protein secretion stimulated by ARL5A / 5BQ70L . Experiments were similar to panel ( G ) . Gaussia - luciferase in culture medium was detected by Western and the cell lysate was immunoblotted with indicated antibodies .

(H) PI4KB knockout blocks protein secretion stimulated by ARL5A/5BQ70L. Experiments were similar to panel (G). Gaussia-luciferase in culture medium was detected by Western and the cell lysate was immunoblotted with indicated antibodies.


( A - B ) Primary cilia are enriched at branch points and the greater curvature of the aorta of wild type mice , schematized in ( A ) and quantified in ( B ) . The color in ( A ) indicates which side of the aorta is quantified in ( B ) . Numbers indicate the region of the aorta ( A ) whose proportion of ciliated cells is quantified in ( B ) . Error bars are + / - 1 S . E . M and n = 7 mice . ( C ) Aortic endothelial cilia are ablated in Tek - Cre Ift88C / - mice . Endothelial cells and cilia are labeled by staining for Pecam1 and Arl13b , respectively . Scale bars are 10μm .

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(A-B) Primary cilia are enriched at branch points and the greater curvature of the aorta of wild type mice, schematized in (A) and quantified in (B). The color in (A) indicates which side of the aorta is quantified in (B). Numbers indicate the region of the aorta (A) whose proportion of ciliated cells is quantified in (B). Error bars are +/- 1 S.E.M and n=7 mice. (C) Aortic endothelial cilia are ablated in Tek-Cre Ift88C/- mice. Endothelial cells and cilia are labeled by staining for Pecam1 and Arl13b, respectively. Scale bars are 10μm.