( A ) Fork velocity as measured by combing in HeLa transfected with indicated siRNAs either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . More than 200 tracks were considered except for RAD1 + RNH1 ( n = 125 ) , ATR + RNH1 ( n = 182 ) RAD18 + RNH1 ( n = 146 ) . Median values are indicated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U ) . 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 combing in HeLa transfected with indicated siRNAs 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 ) . 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 content of indicated RNase III - treated . G1 ( red ) , S ( blue ) G2 ( green ) phases were calculated from profile . Right column : Flow cytometry histograms depicting of S9 . 6 signals in each phase of cycle for indicated . ( B ) of panel B . mean ± SD of S9 . 6 mean of five experiments is shown . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( repeated measures ANOVA with Bonferroni ' s post - ) . ( C ) Graph represents fold change of S9 . 6 mean signal in G2 with respect to G1 . Values for five independent experiments together with mean are shown . * p < 0 . 05 ( one - paired t - ) .

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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 ) - hybrids , damage fork stalling in 9 - 1 - 1 / ATR / CHK1 , ATM / CHK2 PRR deficient . - hybrids represent mean ± of DRIP data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( Paired t - ) . damage represents mean of medians ± of comet tail moment data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( paired t - ) . Fork stalling represents median ± of fork asymmetry data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( Mann - Whitney U ) . 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 . was performed with anti - HA ( Alexa - 488 , green ) , anti - HSPA5 ( Alexa - 647 , red ) 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 previously transfected with ΔDUF3669 - ZNF398 - GFP WT - ZNF398 - GFP followed by detection of ZNF398 constructs in IPs through Western - Blot using an anti - GFP antibody . Bottom : Western - Blot using an anti - HA antibody on IPs . Input = 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 KRAB domain constructs used . Right : HA immunoprecipitation in previously co - transfected with ZNF398 - DUF3669 - HA either ZNF282 - KRAB - Flag or ZNF282 - DUF3669 - Flag followed by detection of either of these constructs in IPs through Western - Blot using an anti - Flag antibody . Bottom : Western - Blot using an anti - HA antibody on 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 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 L1PA6 . Top : boxes containing interactors of KZFPs found enriched on L1PA5 , 6 . Middle : plot showing average ChIP - exo signals ( scaled between 0 1 ) for each selected KZFP plotted on top of L1PA5 L1PA6 multiple 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 phylogenetic tree associated with their corresponding acid sequences colored according to Clustal ZAPPO color scheme ( residues sharing common physicochemical properties display same color : http : / / www . jal - view . org / help / html / colourSchemes / zappo . html ) . This figure also displays a zoom - in on variant KRAB domain cluster ( right ) . On this zoom - in , tested vKZFPs , whose interactomes were defined in our study , were marked by an asterisk . D . Box plot representing KAP1FC values in function of KZFPs . On a superimposed swarm plot , vKZFPs corresponding KAP1FC values were represented by red dots for their sKZFPs counterparts , by blue dots . Mann - Whitney two - sided rank .

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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 ) HA - tagged ZNF398 DUF3669 domain negative control , H2ZYM4 M3XKG1 cKRAB domains ZNF93 hKRAB domain in KAP1 KO HAP1 followed by HA - immunoprecipitation . cKAP1 hKAP1 presence was revealed by Western - Blot using an anti - Flag antibody . Input = lysate , IP = Immunoprecipitate . Western - Blot using an HA antibody on IPs at 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 of mouse intestinal crypts treated with Wnt3a ( 60 ng / mL ) for indicated time . relative level was quantified 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 . right panels show distributions of Ythdf1 Actb in polysome fractions . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - ) .

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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 of SW620 with or without APC overexpression . relative level was quantified 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 with or without APC overexpression . right panels show distributions of YTHDF1 ACTB in polysome fractions . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - ) .

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

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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 expression in intestinal tissue from 4 pairs of Apc + / + Apcmin / + mice . N , normal tissue . T , tissue . A , 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 in matched samples of human CRC tissue adjacent non - tissue . Scale bar , 450 μm . right panel shows statistics of scores . Chi - square ; 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.


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

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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 of small intestines from Ythdf1CTL 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 ) Ki67 ( right ) of small intestine from wildtype mice 72 hr after 12 Gy IR . Scale bar , 50 μm . Red arrows indicate proliferating .

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

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

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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 Apcmin / + ; Ythdf1cKO mice .

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


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


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

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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 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 of colons from Apcmin / + ; Ythdf1CTL 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 number ( H ) distribution ( I ) from Ythdf1CTL Ythdf1cKO mice on day 84 of induction . Data are shown as mean ± ( 6 mice for each group ) . * * p < 0 . 01 ( t - ) .

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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).


in situ hybridization for Ythdf1 mRNA in Ythdf1CTL 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 of fractionated murine intestine enriched for villus crypt .

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


Flowcytometry of YTHDF1 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.


of organoids from Lgr5 - creERT2 : Ythdf1fl / fl mice in Wnt3a - conditioned medium without or with 4 - OHT induction 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.


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

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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 of intestinal differentiation marker genes in organoids described in ( D ) . Data are represented as mean ± . 4 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - ) .

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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 ribosome profiling data from control YTHDF1 knockdown . upregulated ( red ) 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.


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

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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 m6A transcripts as in ( C ) . Kolmogorov - Smirnov .

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


showing TE of Wnt signaling components in control YTHDF1 knockdown . values show fold change of indicated color of .

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

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

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


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

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

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

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

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


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

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


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

O O O O O O B-MEASURED_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

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

O O O O B-MEASURED_VAR 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

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 infected with lentivirus expressing TCF7L2 . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( 3 biological replicates , t - ) .

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

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).


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

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

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 .

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


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

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

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 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 ) . * * p < 0 . 01 ( t - ) .

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

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 of C . c . ΔpseI 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 IPTG ( 0 . 5 mM ) incubated for 3 days at 30 ° C . Only predicted PseI - like synthase coding sequences ( CDSs ) restore motility of C . c . ΔpseI . Note that predicted synthases labeled in 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 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 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 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 extracts from in ( A ) probed with antibodies to C . crescentus FljK ( FljKCc ) . migration of flagellins in ΔpseI is shifted towards lower molecular mass suggesting that post - translational modification of flagellin is defective in ΔpseI mutant . Molecular sizes are indicated by 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 . genes come from synthetic fragments codon optimized for E . coli ( except synthase CDS from Gk that is codon optimized for C . c . ) . Empty carets indicate 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 B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR 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 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 ) ( B ) Motility assays of WT , ΔlegI ( A ) ΔflmG ( B ) B . s . . Overnight cultures of WT mutants harboring empty pSRK - Gm vector ( + Plac ) or corresponding complementing plasmid were spotted on PYE soft agar plates with gentamycin IPTG ( 0 . 5 mM ) incubated for 7 days at 30 ° C . Motility assays are representative of three independent experiments . same original picture was used to generate Figs 3B EV2C .

(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 ) ( D ) Immunoblots of extracts ( ) supernatants ( SN ) of cultures from WT mutant probed with antibodies to FljKCc , that detect B . s . flagellins . estimated molecular mass ( in kDa ) are indicated by blue lines on left . Empty carets indicate 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 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 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 measurements determined by of WT mutants B . s . : 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 ) ΔlegI ΔflmG . Box plots represent distribution of flagellum lengths . cross indicates average , central band indicates median , upper lower sides of box indicate upper lower quartiles , respectively , whiskers indicate minimum maximum measured . Twenty - five independent flagella were measured different ( biological replicates ) for each from .

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

(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 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 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 extracts of flagellin in B . s . ΔlegI derivatives shown in ( A ) . blue lines represent migration of molecular 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 . genes come from synthetic fragments codon optimized for E . coli ( except synthase CDS from Gk that is codon optimized for C . c . ) . Empty carets indicate 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 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 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 . ΔBresu _ 3266 ( ΔlegB ) 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 of WT B . s . ΔBresu _ 0506 ( ΔlegH ) 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 B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-CONTROLLED_VAR I-CONTROLLED_VAR 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 extracts ( ) supernatants ( SN ) of strains described in ( A ) . ( E ) Immunoblots probed with anti - FljKCc antibodies to reveal flagellins in extracts ( ) supernatants ( SN ) of 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 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

(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 of ΔBresu _ 3267 ( ΔlegX ) B . s . compared to WT B . s . harbouring 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 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 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 lysates ( ) supernatants ( SN ) of WT B . s . ΔBresu _ 3267 ( ΔlegX ) cultures harbouring either 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 standards are indicated by blue lines with corresponding value in kDa . Empty carets indicate 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 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 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 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 lysates ( ) from C . crescentus ( C . c . ) mutant cultures expressing fljKBssyn ( codon optimized for E . coli ) flmGBs from replicative pMT463 plasmid , in presence or absence of a compatible integrative plasmid carrying a legBs synthetic operon ( pXGFP4 - legBs ) in presence or absence of an additional compatible replicative plasmid carrying Bresu _ 3267 ( pMT375 ) . In presence of pXGFP4 - legBs ( integrated at xylX locus ) pMT375 - Bresu _ 3267 , FljKBssyn migration is shifted toward higher molecular mass , indicative of glycosylation . Note that in C . c . Δfljx6 background , six flagellin - encoding genes have been deleted detected by antibodies only corresponds to FljKBssyn . legBs synthetic operon ( in red ) is composed of Bresu _ 3266 , Bresu _ 0765 , Bresu _ 0506 , Bresu _ 3264 , Bresu _ 0507 ( legI ) Bresu _ 3265 . Molecular masses are indicated in kDa by blue lines . Empty carets indicate 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 B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-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 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 B-MEASURED_VAR 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 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 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 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 lysates from C . crescentus mutant cultures expressing FlmGCc , FlmGBs FlmGCc - Bschim chimera from replicative pSRK - Gm plasmid ( Plac ) in presence of a compatible integrative plasmid carrying a legBs synthetic operon ( pXGFP4 - legBs ) in presence or absence of an additional compatible replicative plasmid carrying Bresu _ 3267 ( pMT375 ) . Molecular masses are indicated in kDa by blue lines . Empty carets indicate 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 B-CONTROLLED_VAR 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 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 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 Gid4117 - 362 to visualized with Coomassie - stained SDS - PAGE . experiment was performed with WT C - terminal deletion ( ∆ C ) of substrate receptors ( ∆ 289 - 292 ( FEIA ) ∆ 359 - 362 ( FEFA ) for Gid10 Gid4 , respectively ) WT 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR 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 immunoblotted with αFLAG ( two exposures from same gel are shown ) 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

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 , then returned to 30 ° C for indicated timepoints were run on an SDS - PAGE gel immunoblotted with αFLAG ( two exposures from same gel are shown ) αPGK .

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

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

O O O O B-CONTROLLED_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

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

O O O O B-CONTROLLED_VAR 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

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

O O O O O B-CONTROLLED_VAR 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

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

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

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

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

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 grown in YPD incubated with GidSR10 , GidSR4 , GidAnt , or Gid10 for indicated timepoints . Progress of reaction was followed by αFLAG immunoblot .

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

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 following growth in YPE for 19h incubated with GidSR10 , GidSR4 , GidAnt , or Gid4 for indicated timepoints . Progress of reaction was followed by αFLAG immunoblot .

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

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 grown in YPD incubated with GidSR10 , or GidSR10 ∆ C for indicated timepoints . Progress of reaction was followed by αFLAG immunoblot .

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) content of unperturbed ( left panels ) or serum - starved ( right panels ) are plotted on map .

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

(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 ( right panels ) are plotted on map .

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

(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 in unperturbed ( control , green ) serum - starved ( blue ) . were serum - starved for at least 8h prior to imaging . solid represents population median shaded indicates 95 % confidence interval . N = 105 control N = 111 starved .

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

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 p27 abundance in serum - starved is plotted on map .

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

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


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

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

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 ) content , ( D ) phospho / total RB ( E ) p21 of unperturbed ( left panels ) or etoposide - treated ( right panels ) are plotted on arrest architecture . Median values are shown .

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

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 ) p21 - YPet ( green ) in etoposide - treated . Schematic shows two arrest trajectories observed following etoposide treatment . that successfully complete G2 ( " Mothers " , N = 32 ) but arrest following division ( " Daughters " , N = 45 ) are shown in two leftmost panels . that arrest in G2 ( N = 40 ) are shown in rightmost panel . solid lines represent population medians shaded indicates 95 % confidence interval .

O O O O O O O B-MEASURED_VAR 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

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 4C arrest trajectories are shown , regions that correspond to mitotic skipping senescent state are annotated arrest trajectory of 8C polyploid is indicated . N = 4315

O O O O O O O O O O O O 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. 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 ( upper panel : colored by condition , lower panel : colored by content ) populating 2C , 4C , 8C senescent arrest states at each day of treatment ( 1 - 4 d ) .

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

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 following etoposide treatment ( 1 μM , 0 - 4 d ) .

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

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 ) ( K ) : cytoplasm of etoposide - treated are plotted on map . Median values are shown in D - I . indicated with a dotted represents senescent region of map .

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

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 ( O ) SKP2 of etoposide - treated are plotted on map . Median values are shown . indicated with a dotted shows trajectory of mitotic skipping transition into senescence .

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

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 following etoposide or palbociclib treatment ( 1 μM , 4 / 8 d ) . Control : N = 6050 , etop 4d : N = 409 , etop 8d : N = 732 , palbo 4d : N = 1837 , palbo 8d : N = 1677 . Statistical significance was determined using a one - way of variance ( ANOVA ) with Sidak ' s post hoc ( * * * p < 0 . 0001 ) .

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

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

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

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

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 - trace demonstrating mitotic skipping endoreduplication following etoposide treatment ( 1 μM ) by time - lapse imaging . CDK2 activity ( DHB - mCherry , blue ) , cycle phase ( PCNA - mTq2 , S phase shown as gray shaded ) p21 - YPet ( 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 ) proportion of polyploid ( right ) , as measured by immunofluorescence Hoechst , respectively , following siRNA - mediated knockdown of cyclin D in control etoposide - treated . Boxplots show data from four independent replicates ( gray circles ) . Data information : Statistical significance in right panels was determined using a two - way of variance ( ANOVA ) with Sidak ' s post hoc ( * * * p < 0 . 001 , * p = 0 . 02 ) . boxes show interquartile range , whiskers indicate full distribution of points central band represents 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 ) proportion of polyploid ( right ) , as measured by immunofluorescence Hoechst , respectively , following doxycycline ( dox ) - induced upregulation of cyclin D1 in control etoposide - treated . Boxplots show data from six independent replicates ( gray circles ) . Data information : Statistical significance in right panels was determined using a two - way of variance ( ANOVA ) with Sidak ' s post hoc ( * * * p < 0 . 001 , * p = 0 . 02 ) . boxes show interquartile range , whiskers indicate full distribution of points central band represents 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 ) proportion of polyploid ( right ) , as measured by immunofluorescence Hoechst , respectively , following doxycycline ( dox ) - induced upregulation of cyclin A2 in control etoposide - treated . Boxplots show data from six independent replicates ( gray circles ) . Data information : Statistical significance in right panels was determined using a two - way of variance ( ANOVA ) with Sidak ' s post hoc ( * * * p < 0 . 001 , * p = 0 . 02 ) . boxes show interquartile range , whiskers indicate full distribution of points central band represents 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 of pilot validation screen for negative control ( no siRNA ) for five sets of siRNA - treated with reduced TGN retrieval . Different that give rise to reduced TGN retrieval ratios are discussed in text . Scale bar , 100 μm ( top two rows ) 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 ) screen anti - CD8 antibody - uptake for control ( top ) SFT2D2 KD ( bottom ) .

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


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


( C ) stably expressing SFT2D2 - Myc were costained with antibodies against various post - Golgi SNARE . Colocalization was quantified by Pearson ' s correlation coefficient ( right - graph ) indicates very extensive colocalization with STX6 , STX7 , VAMP8 . image panels illustrate colocalization of SFT2D2 - Myc STX5 at Golgi ( top , arrowhead ) but much more extensive colocalization of SFT2D2 - Myc STX6 ( bottom , arrowheads ) . In ( B ) ( C ) , dashed box delineates magnified in insets .

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(C) Cells stably expressing SFT2D2-Myc were costained with antibodies against various post-Golgi SNARE proteins. Colocalization was quantified by Pearson's correlation coefficient (right-hand graph) and indicates very extensive colocalization with STX6, STX7, and VAMP8. The image panels illustrate colocalization of SFT2D2-Myc and STX5 at the Golgi (top, arrowhead) but much more extensive colocalization of SFT2D2-Myc and STX6 (bottom, arrowheads). In (B) and (C), the white dashed box delineates the area magnified in the insets.


( D ) SNARE was compared for control SFT2D2 KD HeLa quantified . graph shows change in measured for each post - Golgi SNARE investigated . illustrate increased of VAMP3 in SFT2D2 KD .

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(D) SNARE protein staining was compared for control and SFT2D2 KD HeLa cells and quantified. The graph shows the change in cellular intensity measured for each post-Golgi SNARE investigated. Images illustrate the increased cellular intensity of VAMP3 in SFT2D2 KD cells.


( E ) Control SFT2D2 KD HeLa lysates were separated by LDS - PAGE blotted for indicated SNARE or actin .

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(E) Control and SFT2D2 KD HeLa cell lysates were separated by LDS-PAGE and blotted for the indicated SNARE proteins or actin.


( A ) screen anti - CD8 antibody - uptake for control ( top ) ZDHHC5 KD ( bottom ) .

(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and ZDHHC5 KD cells (bottom).


( B ) stably expressing ZDHHC5 - Myc show localization of ZDHHC5 to plasma to intracellular tubules vesicles . Some colocalization between ZDHHC5 retromer VPS35 is observed ( arrowheads in inset ) . Following nocodazole treatment endosomes are dispersed some are labeled with ZDHHC5 VPS35 ( arrowheads in inset ) .

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(B) Cells stably expressing ZDHHC5-Myc show localization of ZDHHC5 to the plasma membrane and to intracellular tubules and vesicles. Some colocalization between ZDHHC5 and retromer VPS35 is observed (arrowheads in inset). Following nocodazole treatment endosomes are dispersed and some are labeled with ZDHHC5 and VPS35 (arrowheads in inset).


( C ) Control ZDHHC5 siRNA - treated SFT2D2 - Myc were mixed stained for ZDHHC5 , Myc , VPS35 . KD are marked by an asterisk .

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(C) Control and ZDHHC5 siRNA-treated SFT2D2-Myc cells were mixed and stained for ZDHHC5, Myc, and VPS35. KD cells are marked by an asterisk.


( D ) Control ZDHHC5 siRNA - treated HeLa were mixed stained for ZDHHC5 , α5 - integrin , VPS35 . KD are indicated with an asterisk .

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(D) Control and ZDHHC5 siRNA-treated HeLa cells were mixed and stained for ZDHHC5, α5-integrin, and VPS35. KD cells are indicated with an asterisk.


( E ) Control ( top ) ZDHHC5 KD ( bottom ) HeLa were fixed stained for TGN46 β1 - integrin .

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(E) Control (top) and ZDHHC5 KD (bottom) HeLa cells were fixed and stained for TGN46 and β1-integrin.


( A ) screen anti - CD8 antibody uptake for control ( top ) GRINA KD ( bottom ) .

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


( B ) Control ( top ) GRINA KD ( bottom ) HeLa were stained for Golgi marker GM130 Golgi glycoprotein - 1 ( GLG1 ) .

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(B) Control (top) and GRINA KD (bottom) HeLa cells were stained for Golgi marker GM130 and Golgi glycoprotein-1 (GLG1).


( C ) stably expressing GFP - Rab6 were treated with GRINA siRNA ( bottom ) compared to control ( top ) upon for TGN46 VPS35 .

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(C) Cells stably expressing GFP-Rab6 were treated with GRINA siRNA (bottom) and compared to control cells (top) upon staining for TGN46 and VPS35.


( D ) Quantitation by western blotting of lysates from control GRINA - silenced HeLa GFP - Rab6 .

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(D) Quantitation by western blotting of lysates from control and GRINA-silenced HeLa cells and GFP-Rab6 cells.


( E - G ) HeLa were transiently transfected with GRINA - Myc for 24 hr before fixing . ( E F ) GRINA - Myc colocalizes with both TGN46 VPS35 . In this example , GRINA - Myc expression ( in marked by an asterisk in E ) reduced ' s TGN46 expression compared to surrounding untransfected caused enlargement of VPS35 - positive endosomes . in ( E ) magnified in ( F ) is indicated by a dashed . ( G ) GRINA - Myc expression also perturbs CIMPR localization . In this example , GRINA - Myc transfection ( in marked by an asterisk ) caused CIMPR to localize to round vesicular structures positive for GRINA - Myc , some of which appeared larger than regular endosomes , whereas TGN46 was almost absent in transfected .

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(E-G) HeLa cells were transiently transfected with GRINA-Myc for 24 hr before fixing and staining. (E and F) GRINA-Myc colocalizes with both TGN46 and VPS35. In this example, GRINA-Myc expression (in the cell marked by an asterisk in E) reduced the cell's TGN46 expression compared to surrounding untransfected cells and caused enlargement of VPS35-positive endosomes. The area in (E) magnified in (F) is indicated by a dashed line. (G) GRINA-Myc expression also perturbs CIMPR localization. In this example, GRINA-Myc transfection (in the cell marked by an asterisk) caused CIMPR to localize to round vesicular structures positive for GRINA-Myc, some of which appeared larger than regular endosomes, whereas TGN46 staining was almost absent in the transfected cell.


( A ) Control HeLa or transfected with indicated siRNAs were treated for 3 hr with cycloheximide , lysed , incubated with agarose - bound wheat germ agglutinin to capture glycosylated . Total lysates ( left ) lectin pull - down samples ( right ) were assayed by western blotting . experiment was repeated three times , representative data are shown .

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(A) Control HeLa cells or cells transfected with the indicated siRNAs were treated for 3 hr with cycloheximide, lysed, and incubated with agarose-bound wheat germ agglutinin to capture glycosylated membrane proteins. Total cell lysates (left) and lectin pull-down samples (right) were assayed by western blotting. The experiment was repeated three times, and representative data are shown.


( B - E ) Quantitative of SFT2D2 , ZDHHC5 , GRINA KD immunofluorescence using automated microscopy ( see Experimental Procedures ) . ( B ) Representative showing VPS35 , TGN46 , CIMPR . Scale bar , 50 μm . ( C ) Quantitative of CIMPR at Golgi indicates a significant increase in SFT2D2 , ZDHHC5 , or GRINA KD . ( D ) SFT2D2 , ZDHHC5 , GRINA KD increase Pearson ' s correlation coefficient for colocalization between CIMPR retromer VPS35 or SNX1 while decreasing correlation between CIMPR Golgi matrix GM130 . In some cases , identical correlation coefficients were measured in replicate experiments . ( E ) Quantitation of TGN46 GLG1 in three types of KD .

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(B-E) Quantitative analysis of SFT2D2, ZDHHC5, and GRINA KD cells immunofluorescence using automated microscopy (see Experimental Procedures). (B) Representative images showing VPS35, TGN46, and CIMPR staining. Scale bar, 50 μm. (C) Quantitative analysis of CIMPR intensity at the Golgi indicates a significant increase in SFT2D2, ZDHHC5, or GRINA KD cells. (D) SFT2D2, ZDHHC5, and GRINA KD increase the Pearson's correlation coefficient for colocalization between CIMPR and retromer proteins VPS35 or SNX1 while decreasing the correlation between CIMPR and Golgi matrix protein GM130. In some cases, identical correlation coefficients were measured in the replicate experiments. (E) Quantitation of the TGN46 and GLG1 intensity in the three types of KD cells.


D : Co - immunoprecipitation of TMBIM5 LETM1 in tandem in left 3 panels . input represents mitochondrial crudely isolated from HEK293 was used as input for co - IP , LETM1 was immunoprecipitated ( left panel , IP : LETM1 ) using a LETM1 monoclonal antibody G magnetic beads ( ProtG ) . ProtG beads alone were used as a negative control for binding , immunoprecipitates were immunoblotted ( IB ) for indicated to demonstrate interaction . 10 % of input was used for immunoblotting . Prohibitin ( PHB ) was used as a control to illustrate no nonspecific binding of inner mitochondrial complexes . middle right panel of coIPs illustrates converse experiment , in middle in TMBIM5WT right TMBIMKO , using TMBIM5 as bait ( right panel , IP : TMBIM5 ) . last two right panels show blots from BN - PAGE conducted in TMBIM5WT KO .

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D: Co-immunoprecipitation of TMBIM5 and LETM1 protein in tandem in the left 3 panels. The input represents the mitochondrial crudely isolated from HEK293 cells and was used as input for the co-IP, LETM1 was immunoprecipitated (left panel, IP: LETM1) using a LETM1 monoclonal antibody and Protein G magnetic beads (ProtG). ProtG beads alone were used as a negative control for binding, immunoprecipitates were immunoblotted (IB) for the indicated proteins to demonstrate interaction. 10% of the input was used for immunoblotting. Prohibitin (PHB) was used as a control to illustrate no nonspecific binding of inner mitochondrial membrane proteins complexes. The middle and right panel of the coIPs illustrates the converse experiment, in the middle in TMBIM5WT and right TMBIMKO, using TMBIM5 as bait (right panel, IP: TMBIM5). The last two right panels show blots from BN-PAGE conducted in TMBIM5WT and KO.


A : Western blot of TMBIM5 in control targeted HeLa HEK293 clones .

A: Western blot analysis of TMBIM5 in control and targeted HeLa and HEK293 clones.


E : Isolated mitochondria from three independent replicates of HEK293 TMBIM5WT TMBIM5KO1 KO2 were analyzed by immunoblotting using indicated antibodies , HSP60 TOM40 served as mitochondrial loading controls . F : Densitometric of bands in ( E ) normalized to loading control , bar graph of three experiments ( biological replicates ) , mean ± SD , One - way ANOVA with Bonferroni ' s multiple comparisons performed against TMBIM5WT * p < 0 . 05 , * * p < 0 . 008 , Two - way ANOVA with Bonferroni ' s multiple comparisons performed for OPA1 statistics against TMBIM5WT , * * * p = 0 . 0009 , * * * * p < 0 . 0001 .

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E: Isolated mitochondria from three independent replicates of HEK293 TMBIM5WT and TMBIM5KO1 and KO2 were analyzed by immunoblotting using the indicated antibodies, HSP60 and TOM40 served as mitochondrial loading controls. F: Densitometric analysis of the bands in (E) normalized to loading control, bar graph of three individual experiments (biological replicates), mean ± SD, One-way ANOVA with Bonferroni's multiple comparisons test performed against TMBIM5WT *p<0.05, **p<0.008, Two-way ANOVA with Bonferroni's multiple comparisons test performed for the OPA1 statistics against TMBIM5WT, ***p=0.0009, ****p<0.0001.


( A ) Western blot of purified reconstituted hTMBIM5 for evaluating incorporation of hTMBIM5 into proteoliposomes prepared as described in materials methods .

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(A) Western blot analysis of purified and reconstituted hTMBIM5 for evaluating the incorporation of hTMBIM5 into proteoliposomes prepared as described in materials and methods.


( C ) Acoustic startle reflex to compare hearing ability between Wt ( N = 7 ) Hpn - / - ( N = 4 ) mice .

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(C) Acoustic startle reflex test to compare hearing ability between Wt (N = 7) and Hpn-/- (N = 4) mice.


( F ) Immunoblot of phospho - Smad2 / 3 ( TGFβ pathway signaling marker ) total Smad2 / 3 in lysates from indicated tissues isolated from Wt , Hpn + / - Hpn - / - mice . GAPDH was used as loading control . histogram depicts of pSmad2 / 3 compared to Wt normalized to total Smad2 / 3 .

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(F) Immunoblot analysis of phospho-Smad2/3 (TGFβ pathway signaling marker) and total Smad2/3 in lysates from indicated tissues isolated from Wt, Hpn+/- and Hpn-/- mice. GAPDH was used as the loading control. The histogram depicts quantification of pSmad2/3 compared to Wt normalized to total Smad2/3.


( B ) Immunoblot of Wap - Myc mammary lysates for indicated TGFβ signaling markers hepsin ( T # denotes tumors ) . GAPDH was used as loading control . Lysates were derived from Wap - Myc mammary tumors from 6 mice with 6 mice without DOX - induced hepsin

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(B) Immunoblot analysis of Wap-Myc mammary tumor lysates for the indicated TGFβ signaling markers and hepsin (T# denotes individual tumors). GAPDH was used as the loading control. Lysates were derived from Wap-Myc mammary tumors from 6 mice with and 6 mice without DOX-induced hepsin overexpression


( D ) Immunoblot of phospho - Smad2 / 3 , total Smad2 / 3 , GAPDH ( loading control ) expression in Wt tumors transplanted into either Wt or Hpn - / - recipients ( n = 10 tumors each ) .

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(D) Immunoblot analysis of phospho-Smad2/3, total Smad2/3, and GAPDH (loading control) expression in Wt tumors transplanted into either Wt or Hpn-/- recipients (n = 10 tumors each).


( E ) Western blot of phospho - Smad2 / 3 , total Smad2 / 3 , hepsin , β - tubulin ( loading control ) in MCF10A - pIND20 - HPN treated with doxycycline ( DOX ; overexpression of hepsin ) TGFβR1 inhibitor Galunisertib ( TGFβR1i , 10µM ) as indicated .

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(E) Western blot analysis of phospho-Smad2/3, total Smad2/3, hepsin, and β-tubulin (loading control) in MCF10A-pIND20-HPN cells treated with doxycycline (DOX; overexpression of hepsin) and the TGFβR1 inhibitor Galunisertib (TGFβR1i, 10µM) as indicated.


( G ) TGFβ ELISA to detect active total TGFβ in conditioned medium from MCF10A - pIND20 - HPN pL6 - TGFβ1 with ( DOX + ) or without ( DOX - ) hepsin overexpressing . TGFβ ELISA detects active TGFβ ( first 2 columns ) , but heat treatment of conditioned medium activates TGFβ , thus effectively measuring total TGFβ ( right two columns ) .

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(G) TGFβ ELISA assay to detect active and total TGFβ levels in conditioned medium from MCF10A-pIND20-HPN pL6-TGFβ1 cells with (DOX+) or without (DOX-) hepsin overexpressing. The TGFβ ELISA assay detects active TGFβ levels (first 2 columns), but heat treatment of the conditioned medium activates all TGFβ, thus effectively measuring total TGFβ (right two columns).


( H ) Silver - stained gel with samples from an in vitro protease activity with recombinant SLC hepsin . SLC was incubated with increasing concentrations of recombinant hepsin ( numbers indicate of hepsin in nM ) .

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(H) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant SLC and hepsin. SLC was incubated with increasing concentrations of recombinant hepsin (numbers indicate the concentration of hepsin in nM).


( A ) Coomassie - stained gels with lysates concentrated culture supernatants of MCF10A - pIND20 - HPN , with ( + DOX ) or without ( - DOX ) hepsin overexpression . M indicates media only control . R NR above gels indicate reducing non - reducing conditions , respectively . Numbered arrowheads indicate areas of gel that were analyzed by mass spectrometry ; corresponding differently expressed in hepsin overexpressing are listed on left . image with two lanes on right is a copy of indicated with supernatant under reducing conditions , highlighting part from which fibronectin was identified . Red boxes indicate lanes that were analyzed by mass

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

(A) Coomassie-stained protein gels with cell lysates and concentrated culture supernatants of MCF10A-pIND20-HPN cells, with (+DOX) or without (-DOX) hepsin overexpression. M indicates the media only control. R and NR above the gels indicate reducing and non-reducing conditions, respectively. Numbered arrowheads indicate areas of the gel that were analyzed by mass spectrometry; corresponding proteins differently expressed in hepsin overexpressing cells are listed on the left. The image with two lanes on the right is a copy of the indicated area with supernatant under reducing conditions, highlighting the part from which fibronectin was identified. Red boxes indicate the lanes that were analyzed by mass specrtometry


( C ) Immunoblot of fibronectin , hepsin , β - tubulin ( loading control ) in lysates from MCF10A - pIND20 - HPN with ( DOX + ) or without ( DOX - ) hepsin overexpression . graph shows of fibronectin .

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

(C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) or without (DOX-) hepsin overexpression. The graph shows the quantification of fibronectin levels.