Figure 5B . 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 ) . 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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Figure 5B. 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).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.


Figure 6A . 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 . 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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Figure 6A. 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. 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.


Figure 1B ) 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 chainF ) 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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Figure 1B) 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 chainF) 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).


Figure 2B ) 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 ) . 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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Figure 2B) 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).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.


Figure 3B ) 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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Figure 3B) 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).


Figure 4B , 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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Figure 4B, 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).


Figure 5B ) 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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Figure 5B) 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).


Figure 6C ) 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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Figure 6C) 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.


Figure 1 ( 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 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 scoredF ) 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 . ( 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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Figure 1(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 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 scoredF) 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.(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.


Figure 2 ( 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 . ( 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 .

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Figure 2(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.(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.


Figure 3 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.(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.


Figure 4 ( 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 . 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 . ( 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 . ( 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 .

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Figure 4(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.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.(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.(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.


Figure 5 ( 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 . ( 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 . ( 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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.(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.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.


Figure 1A . 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 .

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Figure 1A. 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.


Figure 2lipids content ( F ) per gram of feces .

Figure 2lipids content (F) per gram of feces.


Figure 3D . 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 .

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Figure 3D. 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.


Figure 4A . 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 .

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Figure 4A. 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.


Figure 5A . 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 .

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Figure 5A. 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.


Figure 6C . 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 . 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 .

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Figure 6C. 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.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.


Figure 7A . 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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Figure 7A. 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.


Figure 3 ( 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 . ( 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O

Figure 3(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.(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.(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).


Figure 4 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.


Figure 5 ( 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 . ( 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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.(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).


Figure 6 ( 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 . ( 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.(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.(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).


Figure 7 ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.(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.


Figure 1 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.


Figure 2 ( A ) The vasculature in P5 retinas from ( A ) mTmG reporter mice with and without EC cilia was visualized by EGFP fluorescence . ( B ) The fractional distance migrated to the edge of the retina was quantified . There was no statistically significant difference ( p = 0 . 305 , Student ' s 2 - tailed t - test , n = 6 mice for each group ) . The average value of 8 measurements ( 4 retina leaflets measured per eye on both eyes ) was used for each mouse . ( C ) The number of vascular branchpoints was counted . There was no statistically significant difference ( p = 0 . 25 , Student ' s 2 - tailed t - test , n = 6 for control and n = 5 for mutant ) . ( D ) Control and mutant adult mouse aortas were stained for GM130 ( green ) to mark the Golgi , CD144 ( red ) to mark ECs and DAPI ( blue ) to label DNA . ( E ) The angle between the Golgi , nucleus and tissue axis was measured and binned as ' left ' , ' right ' , ' upstream ' or ' downstream ' . ( F ) Six areas in the aorta were analyzed . ( G ) There was no difference in Golgi distribution at any single region or overall , nor was there a difference in the variance of the individual un - binned angles . Error bars are + 1 S . E . M . Scale bars are 500μm . n = 3 control and mutant mice for each region except region 2 where n = 3 control and n = 2 mutant mice .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) The vasculature in P5 retinas from (A) mTmG reporter mice with and without EC cilia was visualized by EGFP fluorescence.(B) The fractional distance migrated to the edge of the retina was quantified. There was no statistically significant difference (p=0.305, Student's 2-tailed t-test, n=6 mice for each group). The average value of 8 measurements (4 retina leaflets measured per eye on both eyes) was used for each mouse.(C) The number of vascular branchpoints was counted. There was no statistically significant difference (p=0.25, Student's 2-tailed t-test, n=6 for control and n=5 for mutant).(D) Control and mutant adult mouse aortas were stained for GM130 (green) to mark the Golgi, CD144 (red) to mark ECs and DAPI (blue) to label DNA.(E) The angle between the Golgi, nucleus and tissue axis was measured and binned as 'left', 'right', 'upstream' or 'downstream'.(F) Six areas in the aorta were analyzed.(G) There was no difference in Golgi distribution at any single region or overall, nor was there a difference in the variance of the individual un-binned angles. Error bars are +1 S.E.M. Scale bars are 500μm. n=3 control and mutant mice for each region except region 2 where n=3 control and n=2 mutant mice.


Figure 3 ( B ) Loss of EC cilia by Tek - Cre increases atherosclerosis in the aortas of Apoe - / - mice on a high - fat high - cholesterol diet as visualized by Oil Red O ( ORO ) staining ( left panels ) whereas loss of cilia genes in blood by Mx1 - Cre has no effect ( right panels ) . ( C ) Cropped views of the boxed areas in ( B ) showing increased atherosclerosis in mice with Tek - Cre but not Mx1 - Cre mediated loss of Ift88 in the aortic arch . ( D - G ) Scatter plots of the percent ORO - positive area of the thoracic aorta in ( D - E ) Tek - Cre and ( F - G ) Mx1 - Cre mice , separated by ( D , F ) females and ( E , G ) males . Tek - Cre Apoe - / - Ift88C / - mice had a 58 . 9 % ( p = 0 . 0135 , Student ' s 2 - tailed t - test , n = 13 control and n = 10 mutant mice ) and 67 % ( p = 0 . 0106 , Student ' s 2 - tailed t - test , n = 17 control and n = 14 mutant ) increase in ORO - positive area versus control littermates in females and males , respectively . Mx1 - Cre Apoe - / - Ift88C / - mice had no significant difference in ORO - positive area in females ( p = 0 . 6643 , Student ' s 2 - tailed t - test , n = 7 control and mutant mice ) or males ( p = 0 . 9381 , Student ' s 2 - tailed t - test , n = 9 control and n = 7 mutant mice ) . Black bars represent the mean and error bars are + / - 1 S . E . M . Aortas depicted in ( B - C ) are red in the scatter plots .

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Figure 3(B) Loss of EC cilia by Tek-Cre increases atherosclerosis in the aortas of Apoe-/- mice on a high-fat high-cholesterol diet as visualized by Oil Red O (ORO) staining (left panels) whereas loss of cilia genes in blood by Mx1-Cre has no effect (right panels).(C) Cropped views of the boxed areas in (B) showing increased atherosclerosis in mice with Tek-Cre but not Mx1-Cre mediated loss of Ift88 in the aortic arch.(D-G) Scatter plots of the percent ORO-positive area of the thoracic aorta in (D-E) Tek-Cre and (F-G) Mx1-Cre mice, separated by (D, F) females and (E, G) males. Tek-Cre Apoe-/- Ift88C/- mice had a 58.9% (p=0.0135, Student's 2-tailed t-test, n=13 control and n=10 mutant mice) and 67% (p=0.0106, Student's 2-tailed t-test, n=17 control and n=14 mutant) increase in ORO-positive area versus control littermates in females and males, respectively. Mx1-Cre Apoe-/- Ift88C/- mice had no significant difference in ORO-positive area in females (p=0.6643, Student's 2-tailed t-test, n=7 control and mutant mice) or males (p=0.9381, Student's 2-tailed t-test, n=9 control and n=7 mutant mice). Black bars represent the mean and error bars are +/- 1 S.E.M. Aortas depicted in (B-C) are red in the scatter plots.


Figure 4 ( A ) Transcripts for the inflammatory cytokines Il1b , Il6 , Tnf1 and Tgfb1 , the adhesion molecules Vcam1 and Sele , the lymphocyte marker Nos2 and macrophage marker Cd68 , and the NFκB responsive genes Tnfaip3 and Hmox1 were upregulated in aortas from Apoe - / - mice lacking EC cilia by RT - qPCR , while transcripts of the Hh target genes Gli1 , Ptc1 , and Ptc2 were not . Error bars are + 1 S . E . M . n = 8 control and n = 10 mutant mice of mixed sex . P - values calculated by Student ' s 2 - tailed t - test . ( B ) Pkd2 localizes to EC cilia in vivo . Adult mouse aortas were stained for Pkd2 , acetylated α - Tubulin , and Hoechst to label DNA . Pkd2 localized strongly to primary cilia but also to other membranous structures in endothelial cells . Arrowheads point out cilia . Example cilium highlighted in inset . ( C ) Phosphorylation at Serine 1176 of eNOS was reduced in the aortas of mice lacking EC cilia . Aortas from control and mutant mice were stained for eNOS , p - eNOS - S1176 , acetylated α - Tubulin , and Hoechst to label DNA . ( D ) The ratio of the total fluorescence p - eNOS - S1176 to total eNOS in the upper thoracic aorta ( near the first intercostal artery ) was calculated for control and mutant mice . The relative p - eNOS - S1176 to eNOS ratio was reduced in the mutant to 72 . 7 % the level of control siblings ( p = 0 . 009 , paired 2 - tailed ratio t - test , n = 7 sets of mice ) . Error bar is + 1 S . E . M . Four sets consisted of one control mouse and one mutant mouse . Three sets consisted of two control or mutant mice and the average value of the two was used . Scale bars are 10μm .

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Figure 4(A) Transcripts for the inflammatory cytokines Il1b, Il6, Tnf1 and Tgfb1, the adhesion molecules Vcam1 and Sele, the lymphocyte marker Nos2 and macrophage marker Cd68, and the NFκB responsive genes Tnfaip3 and Hmox1 were upregulated in aortas from Apoe-/- mice lacking EC cilia by RT-qPCR, while transcripts of the Hh target genes Gli1, Ptc1, and Ptc2 were not. Error bars are +1 S.E.M. n=8 control and n=10 mutant mice of mixed sex. P-values calculated by Student's 2-tailed t-test.(B) Pkd2 localizes to EC cilia in vivo. Adult mouse aortas were stained for Pkd2, acetylated α-Tubulin, and Hoechst to label DNA. Pkd2 localized strongly to primary cilia but also to other membranous structures in endothelial cells. Arrowheads point out cilia. Example cilium highlighted in inset.(C) Phosphorylation at Serine 1176 of eNOS was reduced in the aortas of mice lacking EC cilia. Aortas from control and mutant mice were stained for eNOS, p-eNOS-S1176, acetylated α-Tubulin, and Hoechst to label DNA.(D) The ratio of the total fluorescence p-eNOS-S1176 to total eNOS in the upper thoracic aorta (near the first intercostal artery) was calculated for control and mutant mice. The relative p-eNOS-S1176 to eNOS ratio was reduced in the mutant to 72.7% the level of control siblings (p=0.009, paired 2-tailed ratio t-test, n=7 sets of mice). Error bar is +1 S.E.M. Four sets consisted of one control mouse and one mutant mouse. Three sets consisted of two control or mutant mice and the average value of the two was used. Scale bars are 10μm.


Figure 1C . Cell cycle fractions inferred from Cycleflow in IFN - β - treated NSCs . n = 3 biological replicates .

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Figure 1C. Cell cycle fractions inferred from Cycleflow in IFN-β-treated NSCs. n = 3 biological replicates.


Figure 2C , D . Ribo - Seq results depicting translation efficiency as the interaction of log2 fold changes ( LFC ) between footprints mapped to the CDS , referred to as " CDS counts " , and total RNA after 2h or 14h IFN - β treatment . FDR < 10 % , LR - Test in DESeq2 . Genes with a p value < 0 . 1 after FDR correction are highlighted . n = 4 biological replicates . I . Representative WB and phosphorylation levels quantification ( log2FC ) of the mTOR - related proteins ribosomal protein S6 kinase ( S6K ) , ribosomal protein S6 ( S6 ) and eIF4E - binding protein 1 ( 4E - BP1 ) from NSCs treated with IFN - β and normalized to control ( t = 0h ) . Bars represent the mean value . One - way ANOVA for the biphasic response test p - 4E - BP1S65 * ( p = 0 . 021 ) , p - S6S235 / 236 & p - S6S240 / 244 * * * ( p < 0 . 001 ) , p - S6KT389 * * ( p = 0 . 004 ) . n = biological replicates specified . p ≡ p - value .

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Figure 2C,D. Ribo-Seq results depicting translation efficiency as the interaction of log2 fold changes (LFC) between footprints mapped to the CDS, referred to as "CDS counts", and total RNA after 2h or 14h IFN-β treatment. FDR < 10%, LR-Test in DESeq2. Genes with a p value<0.1 after FDR correction are highlighted. n = 4 biological replicates.I. Representative WB and phosphorylation levels quantification (log2FC) of the mTOR-related proteins ribosomal protein S6 kinase (S6K), ribosomal protein S6 (S6) and eIF4E-binding protein 1 (4E-BP1) from NSCs treated with IFN-β and normalized to control (t=0h). Bars represent the mean value. One-way ANOVA for the biphasic response test p-4E-BP1S65 * (p=0.021), p-S6S235/236 & p-S6S240/244 ***(p<0.001), p-S6KT389 **(p=0.004). n = biological replicates specified. p ≡ p-value.


Figure 3A . Representative WB image and quantification ( log2FC ) of p - TSC2Thr1462 & p - AktThr308 from IFNβ - treated NSCs normalized to control ( t = 0h ) . Bars represent the mean value . One - way ANOVA for the biphasic response test p - TSC2T1462 * * * ( p < 0 . 001 ) , p - AktT308 * * ( p = 0 . 003 ) . n = 4 biological replicates . Data information : p ≡ p - value . C . Representative WB images and quantifications ( log2FC ) of p - eIF2αS51 in IFN - β - treated NSCs normalized to control ( t = 0h ) . Bars represent the mean value . Spearman ' s rank correlation test . p - eIF2αS51 * * ( p = 0 . 003724 ) . n = 4 biological replicates . Data information : p ≡ p - value . E . Representative WB image and quantifications ( log2FC ) of p - Rb1Ser780 from IFN - β - treated NSCs normalized to control ( t = 0h ) . Bars represent the mean value . Spearman ' s rank correlation test . p - Rb1S780 * * * ( p = 5 . 37e - 06 ) . n = 3 biological replicates . Data information : p ≡ p - value . I , J . Luciferase activity controlled by the upstream 5 ' UTR fragment from Sox2 ( WT and mutant ) , Rps21 ( WT and mutant ) and Actb . Treatments were applied for 12h . Data is normalized to vehicle and is represented as mean ± SD . n = 6 biological replicates . Two - way ANOVA with Tukey ' s multiple comparisons test ( p - value specified ) . Data information : p ≡ p - value .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Representative WB image and quantification (log2FC) of p-TSC2Thr1462 & p-AktThr308 from IFNβ-treated NSCs normalized to control (t=0h). Bars represent the mean value. One-way ANOVA for the biphasic response test p-TSC2T1462 ***(p<0.001), p-AktT308 **(p=0.003). n = 4 biological replicates. Data information: p ≡ p-value.C. Representative WB images and quantifications (log2FC) of p-eIF2αS51 in IFN-β-treated NSCs normalized to control (t=0h). Bars represent the mean value. Spearman's rank correlation test. p-eIF2αS51 **(p=0.003724). n = 4 biological replicates. Data information: p ≡ p-value.E. Representative WB image and quantifications (log2FC) of p-Rb1Ser780 from IFN-β-treated NSCs normalized to control (t=0h). Bars represent the mean value. Spearman's rank correlation test. p-Rb1S780 ***(p=5.37e-06). n = 3 biological replicates. Data information: p ≡ p-value.I,J. Luciferase activity controlled by the upstream 5'UTR fragment from Sox2 (WT and mutant), Rps21 (WT and mutant) and Actb. Treatments were applied for 12h. Data is normalized to vehicle and is represented as mean ± SD. n = 6 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test (p-value specified). Data information: p ≡ p-value.


Figure 4B . vSVZ niche FACS quantifications of total NSCs ( Total NSCs ) & immunofluorescence quantification of aNSCs ( Fraction of aNSCs ) in WT or IFNAGR KO mice at 2 , 7 and 22 months old ( MO ) . Data from individual mice is plotted . Inferred dynamics are depicted as a blue ( WT ) or red ( IFNAGR KO ) line over time . Sample sizes : WT total at 60 days : 8 mice , WT total at 210 days : 3 mice , WT total at 660 days : 3 mice , WT fraction aNSCs at 60 days : 3 mice , WT fraction aNSCs at 660 days : 4 mice , IFNAGRKO total at 60 days : 8 mice , IFNAGRKO total at 180 days : 3 mice , IFNAGRKO total at 660 days : 3 mice , IFNAGRKO fraction aNSCs at 60 days : 3 mice , IFNAGRKO fraction aNSCs at 660 days : 3 mice .

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Figure 4B. vSVZ niche FACS quantifications of total NSCs (Total NSCs) & immunofluorescence quantification of aNSCs (Fraction of aNSCs) in WT or IFNAGR KO mice at 2, 7 and 22 months old (MO). Data from individual mice is plotted. Inferred dynamics are depicted as a blue (WT) or red (IFNAGR KO) line over time. Sample sizes: WT total at 60 days: 8 mice, WT total at 210 days: 3 mice, WT total at 660 days: 3 mice, WT fraction aNSCs at 60 days: 3 mice, WT fraction aNSCs at 660 days: 4 mice, IFNAGRKO total at 60 days: 8 mice, IFNAGRKO total at 180 days: 3 mice, IFNAGRKO total at 660 days: 3 mice, IFNAGRKO fraction aNSCs at 60 days: 3 mice, IFNAGRKO fraction aNSCs at 660 days: 3 mice.


f1 ( a ) RT - PCR analysis of LC3 , IL - 8 , CHOP and GADD34 in U2OS cells subjected to glutamine deprivation ( − Q ) or exposed to TPG ( 1 μM ) . ( ( b ) Immunoblot analysis of ER stress - and autophagy - related proteins in U2OS cells subjected to glutamine deprivation or TPG treatment . Samples are run in triplicate . ( c ) Cytokine array analysis of conditioned media from U2OS and A549 cells grown in the presence ( + Q ) or absence ( − Q ) of glutamine . ( d ) IL - 8 enzyme - linked immunosorbent assay of conditioned media from U2OS and A549 cells grown in the presence ( + Q ) or absence ( − Q ) of glutamine . Error bars in all figures represent s . d . of three biological replicates .

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f1(a) RT-PCR analysis of LC3, IL-8, CHOP and GADD34 in U2OS cells subjected to glutamine deprivation (−Q) or exposed to TPG (1 μM). ((b) Immunoblot analysis of ER stress- and autophagy-related proteins in U2OS cells subjected to glutamine deprivation or TPG treatment. Samples are run in triplicate.(c) Cytokine array analysis of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine.(d) IL-8 enzyme-linked immunosorbent assay of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine. Error bars in all figures represent s.d. of three biological replicates.


f2 ( a ) Autophagosomes were visualized in U2OS cells stably expressing a GFP - LC3 reporter construct . GFP - labelled puncta were examined after 24 h in the presence ( + Q ) or absence ( − Q ) of glutamine , with or without co - addition of 100 nM CCI - 779 ( CCI ) and 400 nM BafA1 . Scale bar , 10 μm . ( b ) Graphical summary of experiments performed as described in a . Percentage of cells with > 10 puncta per cell from three independent experiments is depicted . Bars represent mean ± s . d . from three independent experiments ( > 50 cells per experiment ) . The statistical significance ( P value ) was determined by a two - tailed , paired Student ' s t - test . * P0 . 05 . ( c ) Immunoblot analysis of U2OS cells subjected to glutamine deprivation with or without 400 nM BafA1 . Cells were pretreated with BafA1 for 1 h before and during exposure to glutamine - deficient medium . Autophagic activity was monitored by detection of p62 and LC3 - II proteins . d ) U2OS mCherry - GFP - LC3 cells were cultured in the presence ( + Q ) or absence ( − Q ) of glutamine for 18 h . Red vesicles denote autolysosomes , whereas yellow vesicles represent autophagosomes . Bars indicate numbers of yellow vesicles ( autophagosomes ) or red vesicles ( autolysosomes ) per cell ± s . d . ( e ) Images of U2OS mCherry - GFP - LC3 cells cultured for 18 h in the presence ( + Q ) or absence ( − Q ) of glutamine . Scale bar , 10 μm . ( f ) Phagophore formation in mCherry - ULK1 . Scale bar , 10 μm . ( g ) mCherry - ATG5 - expressing U2OS cells after 24 h in the presence ( + Q ) or absence ( − Q ) of glutamine . Scale bar , 10 μm .

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f2(a) Autophagosomes were visualized in U2OS cells stably expressing a GFP-LC3 reporter construct. GFP-labelled puncta were examined after 24 h in the presence (+Q) or absence (−Q) of glutamine, with or without co-addition of 100 nM CCI-779 (CCI) and 400 nM BafA1. Scale bar, 10 μm. (b) Graphical summary of experiments performed as described in a. Percentage of cells with >10 puncta per cell from three independent experiments is depicted. Bars represent mean±s.d. from three independent experiments (>50 cells per experiment). The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. *P0.05.(c) Immunoblot analysis of U2OS cells subjected to glutamine deprivation with or without 400 nM BafA1. Cells were pretreated with BafA1 for 1 h before and during exposure to glutamine-deficient medium. Autophagic activity was monitored by detection of p62 and LC3-II proteins.d) U2OS mCherry-GFP-LC3 cells were cultured in the presence (+Q) or absence (−Q) of glutamine for 18 h. Red vesicles denote autolysosomes, whereas yellow vesicles represent autophagosomes. Bars indicate numbers of yellow vesicles (autophagosomes) or red vesicles (autolysosomes) per cell±s.d. (e) Images of U2OS mCherry-GFP-LC3 cells cultured for 18 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.(f) Phagophore formation in mCherry-ULK1. Scale bar, 10 μm. (g) mCherry-ATG5-expressing U2OS cells after 24 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.


f3 ( a ) Colocalization of lysosomes ( Lysotracker Green ) with phagophores ( mCherry - ULK1 and mCherry - glutamine puncta ) in glutamine - deprived U2OS cells . Scale bar , 25 μm . ( b ) Colocalization of LAMP1 - positive lysosomes with GFP - LC3 - labelled autophagosomes after glutamine deprivation in the absence or presence of 400 nM BafA1 in U2OS cells . Scale bar , 15 μm . ( c ) Immunofluorescence images of U2OS cells stained for p62 expression and LAMP1 following glutamine deprivation and BafA1 treatment . Nuclei were stained with DAPI . Scale bar , 15 μm . ( d ) Immunofluorescence images of cells stained with LAMP1 ( lysosomes ) and the Golgi marker , RCAS1 , following glutamine deprivation . Nuclei were stained with DAPI . Scale bar , 15 μm . ( e ) U2OS cells were treated for 24 h with cell culture medium containing the indicated amino acids . Cell lysates were immunoblotted to detect changes in S6K phosphorylation and p62 . ( ( f ) Immunofluorescence staining for LAMP1 ( green ) and mTOR ( red ) in U2OS cells subjected to glutamine deprivation ( 24 h ) , in the presence of 100 nM WYE - 125132 ( WYE - 132 ) . − AA denotes cells deprived of all amino acids . Scale bar , 15 μm . Error bars in all figures represent s . d . of three biological replicates . Ess . , essential .

O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3(a) Colocalization of lysosomes (Lysotracker Green) with phagophores (mCherry-ULK1 and mCherry-glutamine puncta) in glutamine-deprived U2OS cells. Scale bar, 25 μm.(b) Colocalization of LAMP1-positive lysosomes with GFP-LC3-labelled autophagosomes after glutamine deprivation in the absence or presence of 400 nM BafA1 in U2OS cells. Scale bar, 15 μm.(c) Immunofluorescence images of U2OS cells stained for p62 expression and LAMP1 following glutamine deprivation and BafA1 treatment. Nuclei were stained with DAPI. Scale bar, 15 μm.(d) Immunofluorescence images of cells stained with LAMP1 (lysosomes) and the Golgi marker, RCAS1, following glutamine deprivation. Nuclei were stained with DAPI. Scale bar, 15 μm.(e) U2OS cells were treated for 24 h with cell culture medium containing the indicated amino acids. Cell lysates were immunoblotted to detect changes in S6K phosphorylation and p62. ((f) Immunofluorescence staining for LAMP1 (green) and mTOR (red) in U2OS cells subjected to glutamine deprivation (24 h), in the presence of 100 nM WYE-125132 (WYE-132). −AA denotes cells deprived of all amino acids. Scale bar, 15 μm. Error bars in all figures represent s.d. of three biological replicates. Ess., essential.


f4 ( a ) Immunoblot of autophagy - related proteins in U2OS cells expressing a control TALEN ( U2OS - TAL2 - C1 ) or two independent subcloned TALEN cell lines targeting ATG7 ( U2OS - TAL2 - A7 , U2OS - TAL2 - A8 ) . p62 long and short refer to either long or short exposure times during film development . ( b ) U2OS cells expressing an ATG7 - TALEN ( TAL2 - ATG7 ) or control TALEN ( TAL2 - CTL ) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine - replete medium . Cells were then stained with SRB and absorbance was read at 540 nM . Graph shows average results from five clonal sub - lines for each condition . The statistical significance ( P value ) was determined by a two - tailed , paired Student ' s t - test . P0 . 05 . ( c ) Representative images of SRB - stained clones in bd ) IL - 8 enzyme - linked immunosorbent assay ( ELISA ) of U2OS - TAL2 - CTL - C1 ( control ) and U2OS - TAL2 - ATG7 - A8 ( ATG7 knockout ) following 24 h glutamine deprivation . ( e ) IL - 8 U2OS of U2OS parental cells starved of glutamine for the indicated times with or without BafA1 . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f4(a) Immunoblot of autophagy-related proteins in U2OS cells expressing a control TALEN (U2OS-TAL2-C1) or two independent subcloned TALEN cell lines targeting ATG7 (U2OS-TAL2-A7, U2OS-TAL2-A8). p62 long and short refer to either long or short exposure times during film development.(b) U2OS cells expressing an ATG7-TALEN (TAL2-ATG7) or control TALEN (TAL2-CTL) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine-replete medium. Cells were then stained with SRB and absorbance was read at 540 nM. Graph shows average results from five clonal sub-lines for each condition. The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. P0.05. (c) Representative images of SRB-stained clones in bd) IL-8 enzyme-linked immunosorbent assay (ELISA) of U2OS-TAL2-CTL-C1 (control) and U2OS-TAL2-ATG7-A8 (ATG7 knockout) following 24 h glutamine deprivation.(e) IL-8 U2OS of U2OS parental cells starved of glutamine for the indicated times with or without BafA1. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.