( A ) p53 small molecule activators Tenovin - 1 and CP - 31398 reduce the levels of misfolded SOD1 proteins , as determined by the SOD1G85R solubility assay in HEK293 cells . Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions .

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(A) p53 small molecule activators Tenovin-1 and CP-31398 reduce the levels of misfolded SOD1 proteins, as determined by the SOD1G85R solubility assay in HEK293 cells. Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions.


( B ) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1G85R but not WT SOD1 proteins in the SOD1G85R aggregation assay , as shown by western blots of both supernatant ( S ) ( n = 2 ) and pellet ( P ) ( n = 3 ) fractions .

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(B) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1G85R but not WT SOD1 proteins in the SOD1G85R aggregation assay, as shown by western blots of both supernatant (S) (n = 2) and pellet (P) (n = 3) fractions.


( C ) A complete absence of p53 increases the accumulation of SOD1G85R mutant proteins in p53 - / - HCT116 cells when compared with controls . Representative western blots ( left panels ) and quantification of SOD1G85R levels in the supernatant lysates are shown . The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1 . The right graph panel shows the same data as shown in the middle panel , but normalized to the average SOD1G85R level for each amount of the transfected plasmid . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(C) A complete absence of p53 increases the accumulation of SOD1G85R mutant proteins in p53-/- HCT116 cells when compared with controls. Representative western blots (left panels) and quantification of SOD1G85R levels in the supernatant lysates are shown. The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1. The right graph panel shows the same data as shown in the middle panel, but normalized to the average SOD1G85R level for each amount of the transfected plasmid. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Left : p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown . Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs . Right : quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control , double ( UBE4B / LSD1 ) , or triple ( UBE4B / LSD1 / p53 ) shRNAs ( n = 2 ) .

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(A) Left: p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown. Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs. Right: quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control, double (UBE4B/LSD1), or triple (UBE4B/LSD1/p53) shRNAs (n = 2).


( B ) The degenerative TDP - 43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 ( p53 RNAi ) , or by the overexpression of a dominant - negative p53 mutant ( p53 . R155H ) . Expression of p53 RNAi , p53 . R155H , and TDP - 43M337V are driven by GMR - Gal4 .

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(B) The degenerative TDP-43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53 RNAi), or by the overexpression of a dominant-negative p53 mutant (p53.R155H). Expression of p53 RNAi, p53.R155H, and TDP-43M337V are driven by GMR-Gal4.


( C ) The p53 - activating drug Tenovin - 1 ( TEN1 ) protects spinal cord motor neurons from SOD1G85R - induced proteotoxicity . Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0 . 8 μM TEN1 or vehicle ( VEH ) DMSO , followed by infection of HSV - SOD1G85R or the HSV - LacZ control after 24 h . At day 5 post - infection , cells were fixed and stained with a motor neuron - specific anti - neurofilament H ( NF - H ) antibody . Left : representative images of motor neurons in each condition . Scale bar = 50 μm . Right : quantification of motor neuron survival ( one - way ANOVA with multiple comparisons and Tukey correction ) . Data represent means ± SEM .

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(C) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1G85R-induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM.


( A ) Top hit selected candidates are listed . The plot shows relative S9 . 6 nuclear intensity values for cells transformed with each of the indicated pool of siRNAs .

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(A) Top hit selected candidates are listed. The plot shows relative S9.6 nuclear intensity values for cells transformed with each of the indicated pool of siRNAs.


( B ) Representative images of HeLa cells immunostained with S9 . 6 and nucleolin antibodies after transfection with at least a pool of two siRNAs of each original pool .

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(B) Representative images of HeLa cells immunostained with S9.6 and nucleolin antibodies after transfection with at least a pool of two siRNAs of each original pool.


( C ) Relative S9 . 6 signal intensity per nucleus after nucleolus signal removal in HeLa cells transfected with the indicated siRNAs . More than 250 total cells from three independent experiments were considered . The median of each population is shown . Boxes and whiskers indicate 25 - 75 and 10 - 90 percentiles , respectively . * * * p < 0 . 001 ( Mann - Whitney U test ) .

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(C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells transfected with the indicated siRNAs. More than 250 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. ***p<0.001 (Mann-Whitney U test).


( A ) Relative DRIP - qPCR signal values at RPL13A , APOE , MIB2 and RHOT2 genes in HeLa cells transfected with the indicated siRNAs and treated in vitro with RNase H pre - immunoprecipitation where indicated . The mean ± SEM from at least three independent experiments is shown . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - tailed paired t - test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) Relative DRIP-qPCR signal values at RPL13A, APOE, MIB2 and RHOT2 genes in HeLa cells transfected with the indicated siRNAs and treated in vitro with RNase H pre-immunoprecipitation where indicated. The mean ± SEM from at least three independent experiments is shown. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( B ) Representative images of immunostaining with S9 . 6 and anti - nucleolin antibodies in HeLa cells transfected with the indicated

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(B) Representative images of immunostaining with S9.6 and anti-nucleolin antibodies in HeLa cells transfected with the indicated siRNAs


( C ) Relative S9 . 6 signal intensity per nucleus after nucleolus signal removal in HeLa cells after cytoplasm pre - extraction ( CE ) and treated in vitro with RNaseIII and RNase H where indicated . More than 500 total cells from three independent experiments were considered . The median of each population is shown . Boxes and whiskers indicate 25 - 75 and 10 - 90 percentiles , respectively . * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U test ) .

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(C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells after cytoplasm pre-extraction (CE) and treated in vitro with RNaseIII and RNase H where indicated. More than 500 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. **p<0.01, ***p<0.001 (Mann-Whitney U test).


( A ) Representative images of single - cell alkaline gel electrophoresis ( comet assay ) of HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) .

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(A) Representative images of single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+).


( B ) Comet tail moment from single - cell alkaline gel electrophoresis ( comet assay ) of HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . More than 250 total cells were considered . The mean ± SEM of the median from at least three independent experiments is shown , except for RAD1 , RAD17 and UBE2B ( n = 2 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - tailed unpaired t - test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(B) Comet tail moment from single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 250 total cells were considered. The mean ± SEM of the median from at least three independent experiments is shown, except for RAD1, RAD17 and UBE2B (n=2). *p<0.05, **p<0.01, ***p<0.001 (one-tailed unpaired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( A ) Representative images and percentage of HeLa cells with more than 5 H3S10 - P foci after transfection with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . Mitotic cells were excluded for the analysis by DAPI staining . More than 300 total cells were considered . Data represent mean ± SEM from three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - tailed paired t - test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) Representative images and percentage of HeLa cells with more than 5 H3S10-P foci after transfection with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). Mitotic cells were excluded for the analysis by DAPI staining. More than 300 total cells were considered. Data represent mean ± SEM from three independent experiments. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( B ) Representative images and H3K9me2 nuclear signal intensity of HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . At least three experiments were performed . The median of each population in a representative experiment with at least 100 cells per condition is shown . Boxes and whiskers indicate 25 - 75 and 10 - 90 percentiles , respectively . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - tailed paired t - test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(B) Representative images and H3K9me2 nuclear signal intensity of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). At least three experiments were performed. The median of each population in a representative experiment with at least 100 cells per condition is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( A ) Fork velocity as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . More than 200 tracks were considered except for RAD1 + RNH1 ( n = 125 ) , ATR + RNH1 ( n = 182 ) and RAD18 + RNH1 ( n = 146 ) . Median values are indicated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) Fork velocity as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 200 tracks were considered except for RAD1 +RNH1 (n=125), ATR +RNH1 (n=182) and RAD18 +RNH1 (n=146). Median values are indicated. *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.


( B ) Fork asymmetry as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . From 40 to 200 total measurements were considered for each candidate . Median values are indicated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(B) Fork asymmetry as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). From 40 to 200 total measurements were considered for each candidate. Median values are indicated. *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.


( A ) Left column : Flow cytometry profiles showing DNA content of the indicated RNase III - treated cells . G1 ( red ) , S ( blue ) and G2 ( green ) phases were calculated from the profile . Right column : Flow cytometry histograms depicting intensity of S9 . 6 signals in each phase of the cell cycle for the indicated cells . ( B ) Quantification of panel B . The mean ± SD of the S9 . 6 mean intensity of five experiments is shown . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( repeated measures ANOVA test with Bonferroni ' s post - test ) . ( C ) Graph represents fold change of S9 . 6 mean signal in G2 with respect to G1 cells . Values for five independent experiments together with the mean are shown . * p < 0 . 05 ( one - tailed paired t - test ) .

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(A) Left column: Flow cytometry profiles showing DNA content of the indicated RNase III-treated cells. G1 (red), S (blue) and G2 (green) phases were calculated from the profile. Right column: Flow cytometry histograms depicting intensity of S9.6 signals in each phase of the cell cycle for the indicated cells. (B) Quantification of panel B. The mean ± SD of the S9.6 mean intensity of five experiments is shown. ***p<0.001, **p<0.01, *p<0.05 (repeated measures ANOVA test with Bonferroni's post-test). (C) Graph represents fold change of S9.6 mean signal in G2 with respect to G1 cells. Values for five independent experiments together with the mean are shown. *p<0.05 (one-tailed paired t-test).


( A ) DNA - RNA hybrids , DNA damage and fork stalling in 9 - 1 - 1 / ATR / CHK1 , ATM / CHK2 and PRR deficient cells . DNA - RNA hybrids represent the mean ± SEM of all the DRIP data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( Paired t - test ) . DNA damage represents the mean of medians ± SEM of all the comet tail moment data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( paired t - test ) . Fork stalling represents the median ± SEM of all the fork asymmetry data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( Mann - Whitney U test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) DNA-RNA hybrids, DNA damage and fork stalling in 9-1-1/ATR/CHK1, ATM/CHK2 and PRR deficient cells. DNA-RNA hybrids represent the mean ± SEM of all the DRIP data ***p<0.001, **p<0.01, *p<0.05 (Paired t-test). DNA damage represents the mean of medians ± SEM of all the comet tail moment data ***p<0.001, **p<0.01, *p<0.05 (paired t-test). Fork stalling represents the median ± SEM of all the fork asymmetry data ***p<0.001, **p<0.01, *p<0.05 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.


IF by confocal microscopy performed on KRAB - HA overexpressing cells . Staining was performed with anti - HA ( Alexa - 488 , green ) , anti - HSPA5 ( Alexa - 647 , red ) and DNA was stained with Dapi ( blue ) .

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IF by confocal microscopy performed on KRAB-HA overexpressing cells. Staining was performed with anti-HA (Alexa-488, green), anti-HSPA5 (Alexa-647, red) and DNA was stained with Dapi (blue).


HA immunoprecipitation of stably expressed ZNF282 - HA in cells previously transfected with ΔDUF3669 - ZNF398 - GFP and WT - ZNF398 - GFP followed by detection of ZNF398 constructs in the IPs through Western - Blot using an anti - GFP antibody . Bottom : Western - Blot using an anti - HA antibody on the IPs . Input = Cellular lysate , IP =

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HA immunoprecipitation of stably expressed ZNF282-HA in cells previously transfected with ΔDUF3669-ZNF398-GFP and WT-ZNF398-GFP followed by detection of ZNF398 constructs in the IPs through Western-Blot using an anti-GFP antibody. Bottom: Western-Blot using an anti-HA antibody on the IPs. Input = Cellular lysate, IP = Immunoprecipitate


Left : DUF3669 - only and KRAB domain constructs used . Right : HA immunoprecipitation in cells previously co - transfected with ZNF398 - DUF3669 - HA and either ZNF282 - KRAB - Flag or ZNF282 - DUF3669 - Flag followed by detection of either of these protein constructs in the IPs through Western - Blot using an anti - Flag antibody . Bottom : Western - Blot using an anti - HA antibody on the IPs .

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Left: DUF3669-only and KRAB domain constructs used. Right: HA immunoprecipitation in cells previously co-transfected with ZNF398-DUF3669-HA and either ZNF282-KRAB-Flag or ZNF282-DUF3669-Flag followed by detection of either of these protein constructs in the IPs through Western-Blot using an anti-Flag antibody. Bottom: Western-Blot using an anti-HA antibody on the IPs.


Histograms representing the percentage of KZFPs ( identified in Fig . 3B ) binding sites falling in TEs ( red ) or TSSs ( blue ) .

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Histograms representing the percentage of KZFPs (identified in Fig. 3B) binding sites falling in TEs (red) or TSSs (blue).


KZFPs binding sites on L1PA5 and L1PA6 . Top : boxes containing the interactors of KZFPs found enriched on L1PA5 , 6 . Middle : plot showing the average ChIP - exo signals ( scaled between 0 and 1 ) for each selected KZFP plotted on top of L1PA5 and L1PA6 multiple sequence alignment ( MSA ) . Bottom : schematic representation of L1PA5 , 6 different domains .

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KZFPs binding sites on L1PA5 and L1PA6. Top: boxes containing the interactors of KZFPs found enriched on L1PA5,6. Middle: plot showing the average ChIP-exo signals (scaled between 0 and 1) for each selected KZFP plotted on top of L1PA5 and L1PA6 multiple sequence alignment (MSA). Bottom: schematic representation of L1PA5,6 different domains.


Human KZFPs KRAB domain A - boxes protein phylogenetic tree associated with their corresponding amino acid sequences colored according to the Clustal ZAPPO color scheme ( residues sharing common physicochemical properties display the same color : http : / / www . jal - view . org / help / html / colourSchemes / zappo . html ) . This figure also displays a zoom - in on the variant KRAB domain cluster ( right ) . On this zoom - in , the tested vKZFPs , whose interactomes were defined in our study , were marked by an asterisk . D . Box plot representing KAP1FC values in function of KZFPs age . On a superimposed swarm plot , the individual vKZFPs corresponding KAP1FC values were represented by red dots and for their sKZFPs counterparts , by blue dots . Mann - Whitney two - sided rank test .

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Human KZFPs KRAB domain A-boxes protein phylogenetic tree associated with their corresponding amino acid sequences colored according to the Clustal ZAPPO color scheme (residues sharing common physicochemical properties display the same color: http://www.jal- view.org/ help/html/colourSchemes/zappo.html). This figure also displays a zoom-in on the variant KRAB domain cluster (right). On this zoom-in, the tested vKZFPs, whose interactomes were defined in our study, were marked by an asterisk. D. Box plot representing KAP1FC values in function of KZFPs age. On a superimposed swarm plot, the individual vKZFPs corresponding KAP1FC values were represented by red dots and for their sKZFPs counterparts, by blue dots. Mann-Whitney two-sided rank test.


Immunoprecipitations of HA - tagged KRAB domains in order to check interaction with cKAP1 . Co - transfection of Flag - tagged cKAP1 ( upper panel ) or Flag - tagged hKAP1 ( lower panel ) and HA - tagged ZNF398 DUF3669 domain negative control , H2ZYM4 and M3XKG1 cKRAB domains and ZNF93 hKRAB domain in KAP1 KO HAP1 cells followed by HA - immunoprecipitation . cKAP1 and hKAP1 presence was revealed by Western - Blot using an anti - Flag antibody . Input = Cellular lysate , IP = Immunoprecipitate . Western - Blot using an HA antibody on the IPs at the bottom .

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Immunoprecipitations of HA-tagged KRAB domains in order to check
 interaction with cKAP1. Co-transfection of Flag-tagged cKAP1 (upper panel) or Flag-tagged hKAP1 (lower panel) and HA-tagged ZNF398 DUF3669 domain negative control, H2ZYM4 and M3XKG1 cKRAB domains and ZNF93 hKRAB domain in KAP1 KO HAP1 cells followed by HA-immunoprecipitation. cKAP1 and hKAP1 presence was revealed by Western-Blot using an anti-Flag antibody. Input = Cellular lysate, IP = Immunoprecipitate. Western-Blot using an HA antibody on the IPs at the bottom.


Immunoblot analysis of mouse intestinal crypts treated with Wnt3a ( 60 ng / mL ) for indicated time . The relative protein level was quantified and shown under each band .

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Immunoblot analysis of mouse intestinal crypts treated with Wnt3a (60 ng/mL) for indicated time. The relative protein level was quantified and shown under each band.


Polysome profiles of mouse intestinal crypt treated with or without Wnt3a for 30 min . The right panels show the distributions of Ythdf1 and Actb in polysome fractions . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - test ) .

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Polysome profiles of mouse intestinal crypt treated with or without Wnt3a for 30 min. The right panels show the distributions of Ythdf1 and Actb in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).


Immunoblot analysis of SW620 cells with or without APC overexpression . The relative protein level was quantified and shown under each band .

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Immunoblot analysis of SW620 cells with or without APC overexpression. The relative protein level was quantified and shown under each band.


Polysome profiles of SW620 cells with or without APC overexpression . The right panels show the distributions of YTHDF1 and ACTB in polysome fractions . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - test ) .

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Polysome profiles of SW620 cells with or without APC overexpression. The right panels show the distributions of YTHDF1 and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).


Dual - Luciferase assay wih a construct bearing the 5 ' UTR of YTHDF1 in SW620 cells with or without APC overexpression . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - test ) .

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Dual-Luciferase assay wih a construct bearing the 5'UTR of YTHDF1 in SW620 cells with or without APC overexpression. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).


YTHDF1 protein expression in intestinal tissue from 4 pairs of Apc + / + and Apcmin / + mice . N , normal tissue . T , tumor tissue . A , tumor adjacent tissue .

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YTHDF1 protein expression in intestinal tissue from 4 pairs of Apc+/+ and Apcmin/+ mice. N, normal tissue. T, tumor tissue. A, tumor adjacent tissue.


YTHDF1 staining in matched samples of human CRC tissue and adjacent non - tumor tissue . Scale bar , 450 μm . The right panel shows the statistics of IHC scores . Chi - square test ; n = 75 for each group .

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YTHDF1 staining in matched samples of human CRC tissue and adjacent non-tumor tissue. Scale bar, 450 μm. The right panel shows the statistics of IHC scores. Chi-square test; n = 75 for each group.


The representative jejunum from wildtype ( Ythdf1CTL ) and Ythdf1cKO mice . Left : hematoxylin and eosin staining ( H & E ) ; middle : BrdU staining ; right : quantification of crypt height and BrdU + cells . Scale bar , 50 μm . Data are represented as mean ± SEM . ( 9 biological replicates , t - test ) .

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The representative jejunum from wildtype (Ythdf1CTL) and Ythdf1cKO mice. Left: hematoxylin and eosin staining (H&E); middle: BrdU staining; right: quantification of crypt height and BrdU+ cells. Scale bar, 50 μm. Data are represented as mean ± SEM. (9 biological replicates, t-test).


Immunoblot analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice after 12 Gy IR .

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Immunoblot analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice after 12 Gy IR.


YTHDF1 ( left ) and Ki67 ( right ) staining of small intestine from wildtype mice 72 hr after 12 Gy IR . Scale bar , 50 μm . Red arrows indicate the proliferating cells .

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YTHDF1 (left) and Ki67 (right) staining of small intestine from wildtype mice 72 hr after 12 Gy IR. Scale bar, 50 μm. Red arrows indicate the proliferating cells.


Small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR . Left : H & E staining ; middle : Ki67 staining ; right : quantification of crypt height and Ki67 + cells . Scale bar , 50 μm . Vertical bars indicate the length of the crypts . Data are represented as mean ± SEM . * * * * p < 0 . 0001 ( 9 biological replicates , t - test ) .

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Small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Left: H&E staining; middle: Ki67 staining; right: quantification of crypt height and Ki67+ cells. Scale bar, 50 μm. Vertical bars indicate the length of the crypts. Data are represented as mean ± SEM. ****p<0. 0001 (9 biological replicates, t-test).


RT - qPCR analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 5 biological replicates , t - test ) .

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RT-qPCR analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (5 biological replicates, t-test).


Small intestines from 24 - week - old Apcmin / + ; Ythdf1CTL and Apcmin / + ; Ythdf1cKO mice .

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Small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice.


H & E staining of small intestines from 24 - week - old Apcmin / + ; Ythdf1CTL and Apcmin / + ; Ythdf1cKO mice . Scale bar , 1 mm . The black arrows indicate the tumors .

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H&E staining of small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Scale bar, 1 mm. The black arrows indicate the tumors.


Tumor number ( C ) and size distribution ( D ) from the small intestines of 24 - week - old Apcmin / + ; Ythdf1CTL and Apcmin / + ; Ythdf1cKO mice . Data are shown as mean ± SEM ( 6 mice for each group ) . * * p < 0 . 01 , * * * p < 0 . 001 ( t - test ) .

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Tumor number (C) and size distribution (D) from the small intestines of 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Data are shown as mean ± SEM (6 mice for each group). **p<0. 01, ***p<0. 001 (t-test).


Colons from Ythdf1CTL and Ythdf1cKO mice on day 84 of AOM / DSS induction .

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Colons from Ythdf1CTL and Ythdf1cKO mice on day 84 of AOM/DSS induction.


H & E staining of colons from Apcmin / + ; Ythdf1CTL and Apcmin / + ; Ythdf1cKO mice after AOM / DSS induction . Scale bar , 200 μm .

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H&E staining of colons from Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice after AOM/DSS induction. Scale bar, 200 μm.


Colon tumor number ( H ) and size distribution ( I ) from Ythdf1CTL and Ythdf1cKO mice on day 84 of induction . Data are shown as mean ± SEM ( 6 mice for each group ) . * * p < 0 . 01 ( t - test ) .

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Colon tumor number (H) and size distribution (I) from Ythdf1CTL and Ythdf1cKO mice on day 84 of induction. Data are shown as mean ± SEM (6 mice for each group). **p<0. 01 (t-test).


Fluorescent in situ hybridization for Ythdf1 mRNA in Ythdf1CTL and Ythdf1cKO small intestines . Scale bar , 50 μm .

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Fluorescent in situ hybridization for Ythdf1 mRNA in Ythdf1CTL and Ythdf1cKO small intestines. Scale bar, 50 μm.


Immunoblot analysis of fractionated murine intestine enriched for villus cells and crypt cells .

Immunoblot analysis of fractionated murine intestine enriched for villus cells and crypt cells.


Flowcytometry analysis of YTHDF1 protein expression in GFPhigh / low / neg populations from Lgr5 - GFP - IRES - creERT2 mice .

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Flowcytometry analysis of YTHDF1 protein expression in GFPhigh/low/neg populations from Lgr5-GFP-IRES-creERT2 mice.


Morphology of organoids from Lgr5 - creERT2 : Ythdf1fl / fl mice in Wnt3a - conditioned medium without or with 4 - OHT induction and infected with lentivirus expressing YTHDF1 or YTHDF1 mutant . Scale bar , 250 μm .

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Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing YTHDF1 or YTHDF1 mutant. Scale bar, 250 μm.


Quantification of differentiated versus undifferentiated organoids from ( D ) . Data are represented as mean ± SEM . * * p < 0 . 01 ( 4 biological replicates , t - test ) .

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Quantification of differentiated versus undifferentiated organoids from (D). Data are represented as mean ± SEM. **p<0. 01 (4 biological replicates, t-test).


Relative mRNA levels of intestinal stem cell and differentiation marker genes in organoids described in ( D ) . Data are represented as mean ± SEM . 4 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - test ) .

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Relative mRNA levels of intestinal stem cell and differentiation marker genes in organoids described in (D). Data are represented as mean ± SEM. 4 biological replicates. *p<0. 05, **p<0. 01, ***p<0. 001 (t-test).


Scatterplot of mRNA expression and ribosome profiling data from control and YTHDF1 knockdown cells . The upregulated ( red ) and downregulated ( blue ) genes in translational efficiency ( TE ) are highlighted .

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Scatterplot of mRNA expression and ribosome profiling data from control and YTHDF1 knockdown cells. The upregulated (red) and downregulated (blue) genes in translational efficiency (TE) are highlighted.


The relative m6A peak coverage in control cells for transcripts grouped according to TE changes after YTHDF1 knockdown . POI : peak over input . Down , genes significantly downregulated in YTHDF1 knockdown cells ( < 0 . 5 fold ) . Unchanged , genes not significantly changed . Up , genes significantly upregulated ( > 2 fold ) . The upper and lower quartiles and the median are shown for each group . Mann - Whitney test . * * p < 0 . 01 . * * * p < 0 . 001 .

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The relative m6A peak coverage in control cells for transcripts grouped according to TE changes after YTHDF1 knockdown. POI: peak over input. Down, genes significantly downregulated in YTHDF1 knockdown cells (<0.5 fold). Unchanged, genes not significantly changed. Up, genes significantly upregulated (>2 fold). The upper and lower quartiles and the median are shown for each group. Mann-Whitney test. **p<0. 01. ***p<0. 001.


Violin plots showing TE change between control and YTHDF1 knockdown cells for not - methylated ( non - m6A ) and methylated ( m6A ) transcripts . The upper and lower quartiles and the median are indicated for each group . Mann - Whitney test .

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Violin plots showing TE change between control and YTHDF1 knockdown cells for not-methylated (non-m6A) and methylated (m6A) transcripts. The upper and lower quartiles and the median are indicated for each group. Mann-Whitney test.


Cumulative distributions of TE change for non - m6A and m6A transcripts as in ( C ) . Kolmogorov - Smirnov test .

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Cumulative distributions of TE change for non-m6A and m6A transcripts as in (C). Kolmogorov-Smirnov test.


Heatmap showing the TE of Wnt signaling components in control and YTHDF1 knockdown cells . The values show the fold change of the indicated color of the heatmap .

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Heatmap showing the TE of Wnt signaling components in control and YTHDF1 knockdown cells. The values show the fold change of the indicated color of the heatmap.


Immunoblot analysis of HCT116 cells with YTHDF1 knockdown . Two major bands were detected for human TCF7L2 due to alternative splicing .

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Immunoblot analysis of HCT116 cells with YTHDF1 knockdown. Two major bands were detected for human TCF7L2 due to alternative splicing.


Polysome profiles of HCT116 cells with or without YTHDF1 knockdown . The right panels show the distributions of TCF7L2 , and ACTB in polysome fractions . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - test ) .

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Polysome profiles of HCT116 cells with or without YTHDF1 knockdown. The right panels show the distributions of TCF7L2, and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).


Immunoblot analysis of crypts from Ythdf1CTL and Ythdf1cKO mice . Multiple bands were detected for mouse TCF7L2 representing different isoforms .

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Immunoblot analysis of crypts from Ythdf1CTL and Ythdf1cKO mice. Multiple bands were detected for mouse TCF7L2 representing different isoforms.


Immunoblot analysis of mouse intestinal crypts treated with Wnt3a for 60 min .

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Immunoblot analysis of mouse intestinal crypts treated with Wnt3a for 60 min.


MeRIP - qPCR analysis of m6A levels of TCF7L2 in crypts treated with or without Wnt3a . Data are represented as mean ± SEM . * p < 0 . 05 ( 3 biological replicates , t - test ) .

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MeRIP-qPCR analysis of m6A levels of TCF7L2 in crypts treated with or without Wnt3a. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).


RIP analysis of the interaction of YTHDF1 with TCF7L2 mRNA . The enrichment was measured by qPCR and normalized to input . Data are represented as mean ± SEM . * p < 0 . 05 ( 3 biological replicates , t - test ) .

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RIP analysis of the interaction of YTHDF1 with TCF7L2 mRNA. The enrichment was measured by qPCR and normalized to input. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).


β - catenin / TCF4 reporter activity in YTHDF1 knockdown cells infected with lentivirus expressing TCF7L2 . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( 3 biological replicates , t - test ) .

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β-catenin/TCF4 reporter activity in YTHDF1 knockdown cells infected with lentivirus expressing TCF7L2. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01, ***p<0.001 (3 biological replicates, t-test).


Morphology of organoids from Lgr5 - creERT2 : Ythdf1fl / fl mice in Wnt3a - conditioned medium without or with 4 - OHT induction and infected with lentivirus expressing TCF7L2 . Scale bar , 250 μm . Quantification of differentiated versus undifferentiated organoids from ( H ) . Data are represented as mean ± SEM . * * p < 0 . 01 , * * * p < 0 . 001 ( 3 biological replicates , t - test ) .

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Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing TCF7L2. Scale bar, 250 μm. Quantification of differentiated versus undifferentiated organoids from (H). Data are represented as mean ± SEM. **p<0. 01, ***p<0. 001 (3 biological replicates, t-test).


Colons from mice in ( A ) treated with or without TAM on day 80 of AOM / DSS induction .

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Colons from mice in (A) treated with or without TAM on day 80 of AOM/DSS induction.


Colon tumor number from mice treated with or without TAM on day 80 of AOM / DSS induction . Data are shown as mean ± SEM ( 7 mice for each group ) . * * * * p < 0 . 0001 ( t - test ) .

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Colon tumor number from mice treated with or without TAM on day 80 of AOM/DSS induction. Data are shown as mean ± SEM (7 mice for each group). ****p<0. 0001 (t-test).


Immunoblot analysis of 2 pairs of colon tumors from mice treated with or without TAM .

Immunoblot analysis of 2 pairs of colon tumors from mice treated with or without TAM.


Kaplan - Meier survival plots for Apcmin / + ; Lgr5 - creERT2 : Ythdf1fl / fl mice ( 8 mice for each group ) treated with or without TAM . Log - rank ( Mantel Cox ) test . * * p < 0 . 01 ( t - test ) .

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Kaplan-Meier survival plots for Apcmin/+; Lgr5-creERT2:Ythdf1fl/fl mice (8 mice for each group) treated with or without TAM. Log-rank (Mantel Cox) test. **p<0. 01 (t-test).


( A ) Motility assay of C . c . ΔpseI cells expressing predicted Leg or Pse synthases ( LegI or PseI , in yellow or red , respectively ) from Plac on pSRK - Gm . Overnight cultures were spotted on PYE soft ( 0 . 3 % ) agar plates with gentamycin and IPTG ( 0 . 5 mM ) and incubated for 3 days at 30 ° C . Only predicted PseI - like synthase coding sequences ( CDSs ) restore motility of the C . c . ΔpseI cells . Note that the predicted synthases labeled in white do not share similarities with LegI or PseI of C . jejuni . Motility assays are representative of three independent experiments .

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

(A) Motility assay of C.c. ΔpseI cells expressing predicted Leg or Pse synthases (LegI or PseI, in yellow or red, respectively) from Plac on pSRK-Gm. Overnight cultures were spotted on PYE soft (0.3%) agar plates with gentamycin and IPTG (0.5 mM) and incubated for 3 days at 30°C. Only predicted PseI-like synthase coding sequences (CDSs) restore motility of the C.c. ΔpseI cells. Note that the predicted synthases labeled in white do not share similarities with LegI or PseI of C. jejuni. Motility assays are representative of three independent experiments.


( B ) Immunoblot of cell extracts from cells in ( A ) probed with antibodies to C . crescentus FljK ( FljKCc ) . The migration of flagellins in ΔpseI cells is shifted towards lower molecular mass suggesting that post - translational modification of flagellin is defective in ΔpseI mutant . Molecular sizes are indicated by the blues lines ( in kDa ) . Bs : Brevundimonas subvibrioides , Bl : Brevundimonas lutea , Bv : Brevundimonas viscosa , Dv : Dermabacter vaginalis , Li : Leptospira interrogans , Tp : Treponema pallidum , Td : Treponema denticola , AbACICU : Acinetobacter baumannii ACICU , AbLUH : Acinetobacter baumannii LUH , AbLAC : Acinetobacter baumannii LAC - 4 , Lp : Legionella pneumophila , PaY82 : Pseudomonas aeruginosa Y82 , PaPAPS475 : Pseudomonas aeruginosa PAPS475 , Sj : Shewanella japonicum , Basu : Bacillus subtilis , Ks : Kurthia sibirica , Cb : Clostridium botulinum , Gk : Geobacillus kaustophilus , Mh : Moorella humiferrea , Myco : Mycobacterium sp . KS0706 , HrrPV6 : Halorubrum sp . PV6 , Ms : Methanobrevibacter smithii . All the genes come from synthetic fragments codon optimized for E . coli ( except the synthase CDS from Gk that is codon optimized for C . c . ) . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Yellow boxes indicate PseI functional orthologs . Immunoblots are representative of at least two independent experiments .

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

(B) Immunoblot of cell extracts from cells in (A) probed with antibodies to C. crescentus FljK (FljKCc). The migration of flagellins in ΔpseI cells is shifted towards lower molecular mass suggesting that post-translational modification of flagellin is defective in ΔpseI mutant. Molecular sizes are indicated by the blues lines (in kDa). Bs: Brevundimonas subvibrioides, Bl: Brevundimonas lutea, Bv: Brevundimonas viscosa, Dv: Dermabacter vaginalis, Li: Leptospira interrogans, Tp: Treponema pallidum, Td: Treponema denticola, AbACICU: Acinetobacter baumannii ACICU, AbLUH: Acinetobacter baumannii LUH, AbLAC: Acinetobacter baumannii LAC-4, Lp: Legionella pneumophila, PaY82: Pseudomonas aeruginosa Y82, PaPAPS475: Pseudomonas aeruginosa PAPS475, Sj: Shewanella japonicum, Basu: Bacillus subtilis, Ks: Kurthia sibirica, Cb: Clostridium botulinum, Gk: Geobacillus kaustophilus, Mh: Moorella humiferrea, Myco: Mycobacterium sp. KS0706, HrrPV6: Halorubrum sp. PV6, Ms: Methanobrevibacter smithii. All the genes come from synthetic fragments codon optimized for E. coli (except the synthase CDS from Gk that is codon optimized for C.c.). Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Yellow boxes indicate PseI functional orthologs. Immunoblots are representative of at least two independent experiments.


( A ) and ( B ) Motility assays of WT , ΔlegI ( A ) and ΔflmG ( B ) B . s . cells . Overnight cultures of WT and mutants harboring the empty pSRK - Gm vector ( + Plac ) or the corresponding complementing plasmid were spotted on PYE soft agar plates with gentamycin and IPTG ( 0 . 5 mM ) and incubated for 7 days at 30 ° C . Motility assays are representative of three independent experiments . The same original picture was used to generate Figs 3B and EV2C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) and (B) Motility assays of WT, ΔlegI (A) and ΔflmG (B) B.s. cells. Overnight cultures of WT and mutants harboring the empty pSRK-Gm vector (+Plac) or the corresponding complementing plasmid were spotted on PYE soft agar plates with gentamycin and IPTG (0.5 mM) and incubated for 7 days at 30°C. Motility assays are representative of three independent experiments. The same original picture was used to generate Figs 3B and EV2C.


( C ) and ( D ) Immunoblots of cell extracts ( cells ) and supernatants ( SN ) of cultures from WT and mutant cells probed with antibodies to FljKCc , that detect the B . s . flagellins . The estimated molecular mass ( in kDa ) are indicated by the blue lines on the left . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Immunoblots are representative of at least two independent experiments .

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

(C) and (D) Immunoblots of cell extracts (cells) and supernatants (SN) of cultures from WT and mutant cells probed with antibodies to FljKCc, that detect the B.s. flagellins. The estimated molecular mass (in kDa) are indicated by the blue lines on the left. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of at least two independent experiments.


( F ) Flagellum length measurements determined by TEM of WT and mutants B . s . cells : WT , ΔlegX ( ΔBresu _ 3267 ) , ΔlegB ( ΔBresu _ 3266 ) expressing legX in trans from Plac on a plasmid , ΔlegH ( ΔBresu _ 0506 ) , ΔlegI ( ΔBresu _ 0507 ) , ΔflmG ( ΔBresu _ 2406 ) and ΔlegI ΔflmG . Box plots represent the distribution of the flagellum lengths . The cross indicates the average length , the central band indicates the median , the upper and lower sides of the box indicate the upper and lower quartiles , respectively , and the whiskers indicate the minimum and the maximum length measured . Twenty - five independent flagella were measured different cells ( biological replicates ) for each strain from TEM images .

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

(F) Flagellum length measurements determined by TEM of WT and mutants B.s. cells: WT, ΔlegX (ΔBresu_3267), ΔlegB (ΔBresu_3266) expressing legX in trans from Plac on a plasmid, ΔlegH (ΔBresu_0506), ΔlegI (ΔBresu_0507), ΔflmG (ΔBresu_2406) and ΔlegI ΔflmG. Box plots represent the distribution of the flagellum lengths. The cross indicates the average length, the central band indicates the median, the upper and lower sides of the box indicate the upper and lower quartiles, respectively, and the whiskers indicate the minimum and the maximum length measured. Twenty-five independent flagella were measured different cells (biological replicates) for each strain from TEM images.


( A ) Motility assays of B . s . ΔlegI cells complemented with LegI - type ( yellow ) or PseI - type ( red ) synthases expressed from Plac on pSRK - Gm . Motility assays are representative of three independent experiments .

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

(A) Motility assays of B.s. ΔlegI cells complemented with LegI-type (yellow) or PseI-type (red) synthases expressed from Plac on pSRK-Gm. Motility assays are representative of three independent experiments.


( B ) Immunoblots probed with antibodies to FljKCc , revealing the cell extracts levels of flagellin in B . s . ΔlegI derivatives shown in ( A ) . The blue lines represent the migration of the molecular size standards ( in kDa ) . Bl : Brevundimonas lutea , Bv : Brevundimonas viscosa , Dv : Dermabacter vaginalis , Li : Leptospira interrogans , Tp : Treponema pallidum , Td : Treponema denticola , AbACICU : Acinetobacter baumannii ACICU , AbLUH : Acinetobacter baumannii LUH , AbLAC : Acinetobacter baumannii LAC - 4 , Lp : Legionella pneumophila , PaY82 : Pseudomonas aeruginosa Y82 , PaPAPS475 : Pseudomonas aeruginosa PAPS475 , Sj : Shewanella japonicum , Basu : Bacillus subtilis , Ks : Kurthia sibirica , Cb : Clostridium botulinum , Gk : Geobacillus kaustophilus , Mh : Moorella humiferrea , Myco : Mycobacterium sp . KS0706 , HrrPV6 : Halorubrum sp . PV6 , Ms : Methanobrevibacter smithii . All the genes come from synthetic fragments codon optimized for E . coli ( except the synthase CDS from Gk that is codon optimized for C . c . ) . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Red boxes indicate LegI functional orthologs . Immunoblots are representative of at least two independent experiments .

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

(B) Immunoblots probed with antibodies to FljKCc, revealing the cell extracts levels of flagellin in B.s. ΔlegI derivatives shown in (A). The blue lines represent the migration of the molecular size standards (in kDa). Bl: Brevundimonas lutea, Bv: Brevundimonas viscosa, Dv: Dermabacter vaginalis, Li: Leptospira interrogans, Tp: Treponema pallidum, Td: Treponema denticola, AbACICU: Acinetobacter baumannii ACICU, AbLUH: Acinetobacter baumannii LUH, AbLAC: Acinetobacter baumannii LAC-4, Lp: Legionella pneumophila, PaY82: Pseudomonas aeruginosa Y82, PaPAPS475: Pseudomonas aeruginosa PAPS475, Sj: Shewanella japonicum, Basu: Bacillus subtilis, Ks: Kurthia sibirica, Cb: Clostridium botulinum, Gk: Geobacillus kaustophilus, Mh: Moorella humiferrea, Myco: Mycobacterium sp. KS0706, HrrPV6: Halorubrum sp. PV6, Ms: Methanobrevibacter smithii. All the genes come from synthetic fragments codon optimized for E. coli (except the synthase CDS from Gk that is codon optimized for C.c.). Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Red boxes indicate LegI functional orthologs. Immunoblots are representative of at least two independent experiments.


( A ) Motility assays of WT B . s . and ΔBresu _ 3266 ( ΔlegB ) cells expressing Bresu _ 3266 ( LegB ) , Bresu _ 3267 ( LegX ) or Bresu _ 3266 - Bresu _ 3267 ( LegB - LegX , Bresu _ 3266 - 67 ) from Plac on plasmid pSRK - Gm . Motility assays are representative of three independent experiments . ( D ) Motility assay of WT B . s . and ΔBresu _ 0506 ( ΔlegH ) cells complemented with a plasmid expressing Bresu _ 0506 ( LegH ) from Plac on pSRK - Gm . Motility assays are representative of three independent experiments .

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

(A) Motility assays of WT B.s. and ΔBresu_3266 (ΔlegB) cells expressing Bresu_3266 (LegB), Bresu_3267 (LegX) or Bresu_3266-Bresu_3267 (LegB-LegX, Bresu_3266-67) from Plac on plasmid pSRK-Gm. Motility assays are representative of three independent experiments. (D) Motility assay of WT B.s. and ΔBresu_0506 (ΔlegH) cells complemented with a plasmid expressing Bresu_0506 (LegH) from Plac on pSRK-Gm. Motility assays are representative of three independent experiments.


( B ) Immunoblots probed with anti - FljKCc antibodies to reveal flagellins in cell extracts ( cells ) and supernatants ( SN ) of the strains described in ( A ) . ( E ) Immunoblots probed with anti - FljKCc antibodies to reveal flagellins in cell extracts ( cells ) and supernatants ( SN ) of the strains described in ( D ) . Immunoblots are representative of two independent experiments .

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

(B) Immunoblots probed with anti-FljKCc antibodies to reveal flagellins in cell extracts (cells) and supernatants (SN) of the strains described in (A). (E) Immunoblots probed with anti-FljKCc antibodies to reveal flagellins in cell extracts (cells) and supernatants (SN) of the strains described in (D). Immunoblots are representative of two independent experiments.


( A ) Motility assay of ΔBresu _ 3267 ( ΔlegX ) B . s . cells compared to WT B . s . cells harbouring the empty pSRK - Gm vector ( + Plac ) or a complementing derivative with PtmE / LegX orthologs from C . jejuni ( Cj ) , Tanerella forsythia ( Tf ) or P . aeruginosa NMI1897 ( PaNMI ) . Motility assays are representative of three independent experiments .

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

(A) Motility assay of ΔBresu_3267 (ΔlegX) B.s. cells compared to WT B.s. cells harbouring the empty pSRK-Gm vector (+Plac) or a complementing derivative with PtmE/LegX orthologs from C. jejuni (Cj), Tanerella forsythia (Tf) or P. aeruginosa NMI1897 (PaNMI). Motility assays are representative of three independent experiments.


( B ) Immunoblots probed with anti - FljKCc antibodies from cell lysates ( cells ) and supernatants ( SN ) of WT B . s . and ΔBresu _ 3267 ( ΔlegX ) cultures harbouring either the empty pSRK - Gm vector ( + Plac ) or a complementing plasmid expressing different PtmE / LegX orthologs from C . jejuni ( Cj ) , Tanerella forsythia ( Tf ) or P . aeruginosa NMI1897 ( PaNMI ) . Molecular size standards are indicated by the blue lines with the corresponding value in kDa . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Immunoblots are representative of two independent experiments .

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

(B) Immunoblots probed with anti-FljKCc antibodies from cell lysates (cells) and supernatants (SN) of WT B.s. and ΔBresu_3267 (ΔlegX) cultures harbouring either the empty pSRK-Gm vector (+Plac) or a complementing plasmid expressing different PtmE/LegX orthologs from C. jejuni (Cj), Tanerella forsythia (Tf) or P. aeruginosa NMI1897 (PaNMI). Molecular size standards are indicated by the blue lines with the corresponding value in kDa. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of two independent experiments.


( B ) Anti - FljKCc immunoblot analyses of whole cell lysates ( cells ) from C . crescentus ( C . c . ) mutant cultures expressing fljKBssyn ( codon optimized for E . coli ) and flmGBs from the replicative pMT463 plasmid , in the presence or absence of a compatible integrative plasmid carrying a legBs synthetic operon ( pXGFP4 - legBs ) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu _ 3267 ( pMT375 ) . In the presence of pXGFP4 - legBs ( integrated at the xylX locus ) and pMT375 - Bresu _ 3267 , FljKBssyn migration is shifted toward higher molecular mass , indicative of glycosylation . Note that in the C . c . Δfljx6 background , the six flagellin - encoding genes have been deleted and the protein detected by the antibodies only corresponds to FljKBssyn . The legBs synthetic operon ( in red ) is composed of Bresu _ 3266 , Bresu _ 0765 , Bresu _ 0506 , Bresu _ 3264 , Bresu _ 0507 ( legI ) and Bresu _ 3265 . Molecular masses are indicated in kDa by the blue lines . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Asterisk indicates a modification of flagellin by FlmGBs that does not require Pse or Leg . Immunoblots are representative of at least two independent experiments .

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

(B) Anti-FljKCc immunoblot analyses of whole cell lysates (cells) from C. crescentus (C.c.) mutant cultures expressing fljKBssyn (codon optimized for E. coli) and flmGBs from the replicative pMT463 plasmid, in the presence or absence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). In the presence of pXGFP4-legBs (integrated at the xylX locus) and pMT375-Bresu_3267, FljKBssyn migration is shifted toward higher molecular mass, indicative of glycosylation. Note that in the C.c. Δfljx6 background, the six flagellin-encoding genes have been deleted and the protein detected by the antibodies only corresponds to FljKBssyn. The legBs synthetic operon (in red) is composed of Bresu_3266, Bresu_0765, Bresu_0506, Bresu_3264, Bresu_0507 (legI) and Bresu_3265. Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Asterisk indicates a modification of flagellin by FlmGBs that does not require Pse or Leg. Immunoblots are representative of at least two independent experiments.


( C ) Anti - FljKCc immunoblot analyses of whole cell lysates from C . crescentus mutant cultures expressing FlmGCc , FlmGBs and FlmGCc - Bschim chimera from the replicative pSRK - Gm plasmid ( Plac ) in the presence of a compatible integrative plasmid carrying a legBs synthetic operon ( pXGFP4 - legBs ) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu _ 3267 ( pMT375 ) . Molecular masses are indicated in kDa by the blue lines . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Immunoblots are representative of at least two independent experiments .

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

(C) Anti-FljKCc immunoblot analyses of whole cell lysates from C. crescentus mutant cultures expressing FlmGCc, FlmGBs and FlmGCc-Bschim chimera from the replicative pSRK-Gm plasmid (Plac) in the presence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of at least two independent experiments.


Strep - Tactin pull down of GIDAnt ( strep - tagged at Gid8 C - terminus ) probing binding of Gid1057 - 292 and Gid4117 - 362 to the complex visualized with Coomassie - stained SDS - PAGE . The experiment was performed with WT and C - terminal deletion ( ∆ C ) of the substrate receptors ( ∆ 289 - 292 ( FEIA ) and ∆ 359 - 362 ( FEFA ) for Gid10 and Gid4 , respectively ) and WT and mutant ( mut , Gid5W606A / Y613A / Q649A ) GIDAnt .

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

Strep-Tactin pull down of GIDAnt (strep-tagged at Gid8 C-terminus) probing binding of Gid1057-292 and Gid4117-362 to the complex visualized with Coomassie-stained SDS-PAGE. The experiment was performed with WT and C-terminal deletion (∆C) of the substrate receptors (∆289-292 (FEIA) and ∆359-362 (FEFA) for Gid10 and Gid4, respectively) and WT and mutant (mut, Gid5W606A/Y613A/Q649A) GIDAnt.


Lysates from yeast strains expressing endogenously tagged 3xFLAG - Gid4 or 3x - FLAG - Gid10 that were grown in SD complete at 30 ° C ( no stress ) , SE complete for 19 hours ( EtOH ) , SD complete for 1 hour following 19 hour ethanol treatment ( EtOH rec . ) , 42 ° C for 1 hour ( HS ) , SD complete supplemented with 0 . 5M NaCl for 1 hour ( OS ) , or SD - N for 1 hour ( - N ) were run on an SDS - PAGE gel and immunoblotted with αFLAG ( two exposures from the same gel are shown ) and αPGK .

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

Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3x-FLAG-Gid10 that were grown in SD complete at 30°C (no stress), SE complete for 19 hours (EtOH), SD complete for 1 hour following 19 hour ethanol treatment (EtOH rec.), 42°C for 1 hour (HS), SD complete supplemented with 0.5M NaCl for 1 hour (OS), or SD-N for 1 hour (-N) were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK.


Lysates from yeast strains expressing endogenously tagged 3xFLAG - Gid4 or 3xFLAG - Gid10 that were grown at 42 ° C for one hour , and then returned to 30 ° C for the indicated timepoints were run on an SDS - PAGE gel and immunoblotted with αFLAG ( two exposures from the same gel are shown ) and αPGK .

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Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK.


Lysates from wildtype and ∆ Gid2 yeast strains expressing endogenously tagged 3xFLAG - Gid10 that were grown at 42 ° C for one hour , and then returned to 30 ° C for the indicated timepoints were run on an SDS - PAGE gel and immunoblotted with αFLAG and αPGK .

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Lysates from wildtype and ∆Gid2 yeast strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.


Lysates from wildtype , Gid5W606A , Y613A , Q649A , and Gid10 ∆ C strains expressing endogenously tagged 3xFLAG - Gid10 that were grown at 42 ° C for one hour , and then returned to 30 ° C for the indicated timepoints were run on an SDS - PAGE gel and immunoblotted with αFLAG and αPGK .

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

Lysates from wildtype, Gid5W606A,Y613A,Q649A, and Gid10∆C strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.


Lysates from wildtype , ∆ Gid4 , and Gid4 ∆ C strains expressing endogenously tagged 3xFLAG - Gid10 that were grown at 42 ° C for one hour , and then returned to 30 ° C for the indicated timepoints were run on an SDS - PAGE gel and immunoblotted with αFLAG and αPGK .

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

Lysates from wildtype, ∆Gid4, and Gid4∆C strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.


Wildtype and Gid10 overexpressing yeast strains expressing endogenously tagged Gid4 were grown for 19 hours in YPE , and transitioned to YPD for the indicated timepoints . Lysates were run on an SDS - PAGE gel and immunoblotted with αFLAG and αPGK . Points represent mean , error bars represent standard deviation ( n > 3 biological replicates ) .

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

Wildtype and Gid10 overexpressing yeast strains expressing endogenously tagged Gid4 were grown for 19 hours in YPE, and transitioned to YPD for the indicated timepoints. Lysates were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK. Points represent mean, error bars represent standard deviation (n>3 biological replicates).


In vitro competition assay probing the ability of N - terminally 2xStrep - tagged Gid4 ( 2xS - Gid4 ) to exchange with Gid10 in the GIDSR10 complex . Proteins after Strep - Tactin pull - down were visualized by Coomassie - stained SDS - PAGE .

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In vitro competition assay probing the ability of N-terminally 2xStrep-tagged Gid4 (2xS-Gid4) to exchange with Gid10 in the GIDSR10 complex. Proteins after Strep-Tactin pull-down were visualized by Coomassie-stained SDS-PAGE.


Art2 - 3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10 , GidSR4 , GidAnt , or Gid10 for the indicated timepoints . Progress of the reaction was followed by αFLAG immunoblot .

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Art2-3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10, GidSR4, GidAnt, or Gid10 for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.


Mdh2 - 3xFLAG was immunocaptured from yeast cells following growth in YPE for 19h and incubated with GidSR10 , GidSR4 , GidAnt , or Gid4 for the indicated timepoints . Progress of the reaction was followed by αFLAG immunoblot .

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Mdh2-3xFLAG was immunocaptured from yeast cells following growth in YPE for 19h and incubated with GidSR10, GidSR4, GidAnt, or Gid4 for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.


Art2 - 3xFLAG or Art2P2S - 3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10 , or GidSR10 ∆ C for the indicated timepoints . Progress of the reaction was followed by αFLAG immunoblot .

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Art2-3xFLAG or Art2P2S-3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10, or GidSR10∆C for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.


In vitro ubiquitination assay probing the ability of Gid10 and Gid4 to promote ubiquitination of Art22 - 29 N - terminus and its P2S mutant . Progress of the reaction was monitored by fluorescent scan of the gel visualizing the Art2 peptide with FAM appended to its C - terminus ( pep * ) .

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In vitro ubiquitination assay probing the ability of Gid10 and Gid4 to promote ubiquitination of Art22-29 N-terminus and its P2S mutant. Progress of the reaction was monitored by fluorescent scan of the gel visualizing the Art2 peptide with FAM appended to its C-terminus (pep*).


A . Unified cell cycle map of unperturbed ( gray ) and serum - starved cells ( 1d : light blue , 7d : dark blue , N = 3007 cells ) . The proliferative cell cycle ( dotted gray line ) and the hypomitogenic arrest trajectory ( black dotted line ) are indicated on the map . Inset : Each treatment condition is shown individually on the unified map ( other conditions are shown in lighter gray ) .

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A. Unified cell cycle map of unperturbed (gray) and serum-starved cells (1d: light blue, 7d: dark blue, N = 3007 cells). The proliferative cell cycle (dotted gray line) and the hypomitogenic arrest trajectory (black dotted line) are indicated on the map. Inset: Each treatment condition is shown individually on the unified map (other conditions are shown in lighter gray).


( B ) Diffusion pseudotime , ( C ) p21 , ( D ) cyclin D1 , ( E ) DNA content of unperturbed ( left panels ) or serum - starved cells ( right panels ) are plotted on the map .

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(B) Diffusion pseudotime, (C) p21, (D) cyclin D1, (E) DNA content of unperturbed (left panels) or serum-starved cells (right panels) are plotted on the map.


( F ) phospho / total RB of unperturbed ( left panels ) or serum - starved cells ( right panels ) are plotted on the map .

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(F) phospho/total RB of unperturbed (left panels) or serum-starved cells (right panels) are plotted on the map.


G . Time - lapse imaging of cyclinD1 - mVenus intensity in unperturbed ( control , green ) and serum - starved cells ( blue ) . Cells were serum - starved for at least 8h prior to imaging . The solid line represents the population median and the shaded area indicates the 95 % confidence interval . N = 105 control cells and N = 111 starved cells .

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G. Time-lapse imaging of cyclinD1-mVenus intensity in unperturbed (control, green) and serum-starved cells (blue). Cells were serum-starved for at least 8h prior to imaging. The solid line represents the population median and the shaded area indicates the 95% confidence interval. N = 105 control cells and N = 111 starved cells.


I . Median nuclear p27 abundance in serum - starved cells is plotted on the map .

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I. Median nuclear p27 abundance in serum-starved cells is plotted on the map.


Unified cell cycle map of unperturbed ( gray ) and etoposide - treated cells ( 1 μM ; 1d : light green , 2d : green , 3d : dark green , 4d : darker green - see inset , N = 4315 cells ) . The unperturbed cell cycle trajectory ( dotted gray line ) and two arrest trajectories ( into G02C and G04C ; black dotted lines ) are indicated on the map . Inset : Each condition is shown individually on the map ( other conditions are shown in lighter gray ) .

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Unified cell cycle map of unperturbed (gray) and etoposide-treated cells (1 μM; 1d: light green, 2d: green, 3d: dark green, 4d: darker green - see inset, N = 4315 cells). The unperturbed cell cycle trajectory (dotted gray line) and two arrest trajectories (into G02C and G04C; black dotted lines) are indicated on the map. Inset: Each condition is shown individually on the map (other conditions are shown in lighter gray).


B - E . ( B ) Diffusion pseudotime , ( C ) DNA content , ( D ) phospho / total RB and ( E ) p21 of unperturbed ( left panels ) or etoposide - treated cells ( right panels ) are plotted on the arrest architecture . Median nuclear values are shown .

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B-E. (B) Diffusion pseudotime, (C) DNA content, (D) phospho/total RB and (E) p21 of unperturbed (left panels) or etoposide-treated cells (right panels) are plotted on the arrest architecture. Median nuclear values are shown.


F . Time - lapse imaging of CDK2 activity ( DHB - mCherry , gray ) and p21 - YPet ( green ) intensity in etoposide - treated cells . Schematic shows the two arrest trajectories observed following etoposide treatment . Cells that successfully complete G2 ( " Mothers " , N = 32 cells ) but arrest following cell division ( " Daughters " , N = 45 cells ) are shown in the two leftmost panels . Cells that arrest in G2 ( N = 40 cells ) are shown in the rightmost panel . The solid lines represent population medians and the shaded area indicates the 95 % confidence interval .

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F. Time-lapse imaging of CDK2 activity (DHB-mCherry, gray) and p21-YPet (green) intensity in etoposide-treated cells. Schematic shows the two arrest trajectories observed following etoposide treatment. Cells that successfully complete G2 ("Mothers", N = 32 cells) but arrest following cell division ("Daughters", N = 45 cells) are shown in the two leftmost panels. Cells that arrest in G2 (N = 40 cells) are shown in the rightmost panel. The solid lines represent population medians and the shaded area indicates the 95% confidence interval.


A . Arrest architecture of replicative stress . 2C and 4C arrest trajectories are shown , the regions that correspond to mitotic skipping and the senescent state are annotated and the arrest trajectory of 8C polyploid cells is indicated . N = 4315

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A. Arrest architecture of replicative stress. 2C and 4C arrest trajectories are shown, the regions that correspond to mitotic skipping and the senescent state are annotated and the arrest trajectory of 8C polyploid cells is indicated. N = 4315 cells


B . Progression of arrest states over time following etoposide treatment . Etoposide - treated cells ( upper panel : colored by condition , lower panel : colored by DNA content ) populating 2C , 4C , 8C and senescent arrest states at each day of treatment ( 1 - 4 d ) .

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B. Progression of arrest states over time following etoposide treatment. Etoposide-treated cells (upper panel: colored by condition, lower panel: colored by DNA content) populating 2C, 4C, 8C and senescent arrest states at each day of treatment (1-4 d).


C . Distribution of senescence - associated β - galactosidase ( SA - βgal ) activity in individual cells following etoposide treatment ( 1 μM , 0 - 4 d ) .

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C. Distribution of senescence-associated β-galactosidase (SA-βgal) activity in individual cells following etoposide treatment (1 μM, 0-4 d).


D - K . ( D ) GSK3β , ( E ) phospho ( Thr157 ) - p27 , ( F ) p27 , ( G ) CDK4 , ( H ) cyclin D1 , ( I ) cyclin E , ( J ) cell area and ( K ) DNA : cytoplasm ratio of etoposide - treated cells are plotted on the map . Median nuclear values are shown in D - I . Area indicated with a dotted line represents the senescent region of the map .

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D-K. (D) GSK3β, (E) phospho(Thr157)-p27, (F) p27, (G) CDK4, (H) cyclin D1, (I) cyclin E, (J) cell area and (K) DNA:cytoplasm ratio of etoposide-treated cells are plotted on the map. Median nuclear values are shown in D-I. Area indicated with a dotted line represents the senescent region of the map.


L - O . ( L ) Cyclin A , ( M ) cyclin B1 , ( N ) CDH1 and ( O ) SKP2 of etoposide - treated cells are plotted on the map . Median nuclear values are shown . Area indicated with a dotted line shows the trajectory of mitotic skipping and transition into senescence .

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L-O. (L) Cyclin A, (M) cyclin B1, (N) CDH1 and (O) SKP2 of etoposide-treated cells are plotted on the map. Median nuclear values are shown. Area indicated with a dotted line shows the trajectory of mitotic skipping and transition into senescence.


A . Distribution of senescence - associated β - galactosidase ( SA - βgal ) activity in individual cells following etoposide or palbociclib treatment ( 1 μM , 4 / 8 d ) . Control : N = 6050 cells , etop 4d : N = 409 cells , etop 8d : N = 732 cells , palbo 4d : N = 1837 cells , palbo 8d : N = 1677 cells . Statistical significance was determined using a one - way analysis of variance ( ANOVA ) with Sidak ' s post hoc test ( * * * p < 0 . 0001 ) .

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A. Distribution of senescence-associated β-galactosidase (SA-βgal) activity in individual cells following etoposide or palbociclib treatment (1 μM, 4/8 d). Control: N = 6050 cells, etop 4d: N = 409 cells, etop 8d: N = 732 cells, palbo 4d: N = 1837 cells, palbo 8d: N = 1677 cells. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.0001).


B . Unified cell cycle map of the unperturbed ( gray ) , palbociclib - ( palbo , 1 μM , 4 / 8 d , blue ) and etoposide - ( etop , 1 μM , 4 / 8 d , green ) treated cells . Control : N = 10499 cells , etop : N = 2692 cells , palbo : N = 4931

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B. Unified cell cycle map of the unperturbed (gray), palbociclib- (palbo, 1 μM, 4/8 d, blue) and etoposide- (etop,1 μM, 4/8 d, green) treated cells. Control: N = 10499 cells, etop: N = 2692 cells, palbo: N = 4931 cells


C - K . ( C ) DNA content , ( D ) phospho / total RB , ( E ) p16 , ( F ) TGFβ1 , ( G ) IL - 6 , ( H ) IL - 8 , ( I ) phospho - p38 , ( J ) phospho - STAT3 and ( K ) phospho - STAT5 are plotted on the map . Median nuclear values are shown .

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C-K. (C) DNA content, (D) phospho/total RB, (E) p16, (F) TGFβ1, (G) IL-6, (H) IL-8, (I) phospho-p38, (J) phospho-STAT3 and (K) phospho-STAT5 are plotted on the map. Median nuclear values are shown.


D . Representative single - cell trace demonstrating mitotic skipping and endoreduplication following etoposide treatment ( 1 μM ) by time - lapse imaging . CDK2 activity ( DHB - mCherry , blue ) , cell cycle phase ( PCNA - mTq2 , S phase shown as gray shaded area ) and p21 - YPet intensity ( green ) are plotted versus time of etoposide treatment .

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D. Representative single-cell trace demonstrating mitotic skipping and endoreduplication following etoposide treatment (1 μM) by time-lapse imaging. CDK2 activity (DHB-mCherry, blue), cell cycle phase (PCNA-mTq2, S phase shown as gray shaded area) and p21-YPet intensity (green) are plotted versus time of etoposide treatment.


E . Cyclin D1 abundance ( left ) and the proportion of polyploid cells ( right ) , as measured by immunofluorescence and Hoechst staining , respectively , following siRNA - mediated knockdown of cyclin D in control and etoposide - treated cells . Boxplots show data from four independent replicates ( gray circles ) . Data information : Statistical significance in right panels was determined using a two - way analysis of variance ( ANOVA ) with Sidak ' s post hoc test ( * * * p < 0 . 001 , * p = 0 . 02 ) . boxes show the interquartile range , the whiskers indicate the full distribution of points and the central band represents the population .

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E. Cyclin D1 abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following siRNA-mediated knockdown of cyclin D in control and etoposide-treated cells. Boxplots show data from four independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.


F . Cyclin D1 abundance ( left ) and the proportion of polyploid cells ( right ) , as measured by immunofluorescence and Hoechst staining , respectively , following doxycycline ( dox ) - induced upregulation of cyclin D1 in control and etoposide - treated cells . Boxplots show data from six independent replicates ( gray circles ) . Data information : Statistical significance in right panels was determined using a two - way analysis of variance ( ANOVA ) with Sidak ' s post hoc test ( * * * p < 0 . 001 , * p = 0 . 02 ) . boxes show the interquartile range , the whiskers indicate the full distribution of points and the central band represents the population .

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F. Cyclin D1 abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following doxycycline (dox)-induced upregulation of cyclin D1 in control and etoposide-treated cells. Boxplots show data from six independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.


G . Cyclin A abundance ( left ) and the proportion of polyploid cells ( right ) , as measured by immunofluorescence and Hoechst staining , respectively , following doxycycline ( dox ) - induced upregulation of cyclin A2 in control and etoposide - treated cells . Boxplots show data from six independent replicates ( gray circles ) . Data information : Statistical significance in right panels was determined using a two - way analysis of variance ( ANOVA ) with Sidak ' s post hoc test ( * * * p < 0 . 001 , * p = 0 . 02 ) . boxes show the interquartile range , the whiskers indicate the full distribution of points and the central band represents the population .

G. Cyclin A abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following doxycycline (dox)-induced upregulation of cyclin A2 in control and etoposide-treated cells. Boxplots show data from six independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.


( E ) Representative images of the pilot validation screen for the negative control ( no siRNA ) and for five sets of siRNA - treated cells with reduced TGN retrieval ratio . Different phenotypes that give rise to reduced TGN retrieval ratios are discussed in the text . Scale bar , 100 μm ( top two rows ) and 38 μm ( bottom row ) .

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(E) Representative images of the pilot validation screen for the negative control (no siRNA) and for five sets of siRNA-treated cells with reduced TGN retrieval ratio. Different phenotypes that give rise to reduced TGN retrieval ratios are discussed in the text. Scale bar, 100 μm (top two rows) and 38 μm (bottom row).


( A ) Primary screen anti - CD8 antibody - uptake images for control cells ( top ) and SFT2D2 KD cells ( bottom ) .

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(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and SFT2D2 KD cells (bottom).


( B ) Cells stably expressing SFT2D2 - Myc show localization of SFT2D2 to perinuclear membranes and endosomes , including VPS35 - positive endosomes ( arrowheads in inset ) . Treatment with nocodazole disperses the endosomes and even more clearly shows colocalization of SFT2D2 - Myc and VPS35 ( arrowheads in inset ) .

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(B) Cells stably expressing SFT2D2-Myc show localization of SFT2D2 to perinuclear membranes and endosomes, including VPS35-positive endosomes (arrowheads in inset). Treatment with nocodazole disperses the endosomes and even more clearly shows colocalization of SFT2D2-Myc and VPS35 (arrowheads in inset).