( F ) Western blot showing the levels of MPM - 2 , phosphor - CDK substrate motif [ ( K / H ) pSP ] and β - Actin in whole cell extracts of RPE cells treated with DMSO ( Control ) , 0 . 5 μM Okadaic acid ( OA ) for 2 hrs , 10 nM Calyculin A for 2 hrs , 25 μM P22077 for 4 hrs or 25 μM BAY 11 - 7082 for 2 hrs . Calyculin A was added to this experiment to illustrate the gain in CDK activity induced by an additional PP2A inhibitor . Ponceau staining is shown as loading control .

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(F) Western blot showing the levels of MPM-2, phosphor-CDK substrate motif [(K/H)pSP] and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid (OA) for 2 hrs, 10 nM Calyculin A for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Calyculin A was added to this experiment to illustrate the gain in CDK activity induced by an additional PP2A inhibitor. Ponceau staining is shown as loading control.


( E ) Western blot showing the levels of USP7 and γH2AX in whole cell extracts of HCT - 116 cells treated with DMSO ( Control ) , 50 μM P22077 for 2 - 4 hrs ( USP7i ) , 10 μM RO3306 for 4 hrs ( CDK1i ) or 50 μM P22077 + 10 μM RO3306 for 2 - 4 hrs ( USP7i + CDK1i ) . Ponceau staining is shown as loading control .

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(E) Western blot showing the levels of USP7 and γH2AX in whole cell extracts of HCT-116 cells treated with DMSO (Control), 50 μM P22077 for 2-4 hrs (USP7i), 10 μM RO3306 for 4 hrs (CDK1i) or 50 μM P22077 + 10 μM RO3306 for 2-4 hrs (USP7i + CDK1i). Ponceau staining is shown as loading control.


( B ) Quantification of apoptotic induction was measured by flow cytometry in RPE cells after treatment with DMSO as a control , 25 μM P22077 ( USP7i ) , 10 μM RO3306 ( CDK1i ) or 10 μM DT - 061 ( PP2Aa ) , and their combinations for the indicated times . DNA content was followed with Hoechst , and the mitochondrial membrane potential was measured by TMRE . The percentage of apoptotic cells ( TMRE negative ) is indicated for each condition . This experiment was repeated 3 times . ( * p < 0 . 05 , * * p < 0 . 01 , t - test ) . Bars represent mean values ± SEM .

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(B) Quantification of apoptotic induction was measured by flow cytometry in RPE cells after treatment with DMSO as a control, 25 μM P22077 (USP7i), 10 μM RO3306 (CDK1i) or 10 μM DT-061 (PP2Aa), and their combinations for the indicated times. DNA content was followed with Hoechst, and the mitochondrial membrane potential was measured by TMRE. The percentage of apoptotic cells (TMRE negative) is indicated for each condition. This experiment was repeated 3 times. (* p<0.05, ** p<0.01, t-test). Bars represent mean values ± SEM.


( C ) GLO viability assay in cells treated with 3 independent USP7 inhibitors ( P22077 , BAY 11 - 7082 or HBX 19818 ) alone or in combination with a 24hr treatment of 10 μM RO3306 ( CDK1i ) . This experiment was repeated 3 times . Data represent mean values ± SEM . ( * * * p < 0 . 05 , ANOVA ) .

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(C) GLO viability assay in cells treated with 3 independent USP7 inhibitors (P22077, BAY 11-7082 or HBX 19818) alone or in combination with a 24hr treatment of 10 μM RO3306 (CDK1i). This experiment was repeated 3 times. Data represent mean values ± SEM. (*** p<0.05, ANOVA).


b , Carboxy - terminal V5immunoblot of full - length ( 68 kDa ) and cleaved ( 36 kDa ) ATG16L1β . Silver stain ( bottom gels ) depicts total protein . Data represent 3 independent experiments . IB , immunoblot .

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b, Carboxy-terminal V5immunoblot of full-length (68 kDa) and cleaved (36 kDa) ATG16L1β. Silver stain (bottom gels) depicts total protein. Data represent 3 independent experiments. IB, immunoblot.


c , TNF - α - mediated processing of humanATG16L1 in monocyte - derived macrophages . Scatter plot of immunoblot densitometry depicts a ratio of cleaved : full - length ATG16L1 ( Extended Data Fig . 2 ) . n = 7 donors .

c, TNF-α-mediated processing of humanATG16L1 in monocyte-derived macrophages. Scatter plot of immunoblot densitometry depicts a ratio of cleaved:full-length ATG16L1 (Extended Data Fig. 2). n = 7 donors.


d , TNF - α - mediated processing of murineATG16L1 in macrophages . Immunoblot represents 5 independent experiments . Scatterplot represents data pooled from 5 independent experiments . WT , wild type .

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d, TNF-α-mediated processing of murineATG16L1 in macrophages. Immunoblot represents 5 independent experiments. Scatterplot represents data pooled from 5 independent experiments. WT, wild type.


a , HeLa cells transfected with control siRNA ( siCTL ) or siCaspase 8 were stimulated with 2 . 0 μM staurosporine or 20 ng ml − 1 TNF + 10 μg ml − 1 CHX .

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a, HeLa cells transfected with control siRNA (siCTL) or siCaspase 8 were stimulated with 2.0 μM staurosporine or 20 ng ml−1 TNF + 10 μg ml−1 CHX.


b , HeLa cells transfected with control siRNA or siCaspase 9 were stimulated as in a .

b, HeLa cells transfected with control siRNA or siCaspase 9 were stimulated as in a.


c , HeLa cells transfected with control , caspase - 3 - or caspase - 7 - specific siRNAs were stimulated with 20 ng ml − 1 TNF + 10 & amp ; amp ; mgr ; g ml − 1 CHX .

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c, HeLa cells transfected with control, caspase-3- or caspase-7-specific siRNAs were stimulated with 20 ng ml−1 TNF + 10 &amp;mgr;g ml−1 CHX.


d , Wild - type or caspase - 3 - knockout macrophages were stimulated with 2 . 0 µM staurosporine or 20 ng ml − 1 TNF + 10 µg ml − 1 CHX . Data in a - c represent 2 independent experiments ; data in d represent 4 independent experiments .

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d, Wild-type or caspase-3-knockout macrophages were stimulated with 2.0 µM staurosporine or 20 ng ml−1 TNF + 10 µg ml−1 CHX. Data in a-c represent 2 independent experiments; data in d represent 4 independent experiments.


a , Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1 . White mask in LC3 channel illustrates punctate LC3 staining , representing LC3 - II . Histogram depicts mean fluorescence intensity of 10 , 000 cells per sample . b , Quantification of mean ATG16L1 fluorescence intensity in a , n = 4 mice . Glu . , glucose .

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a, Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1. White mask in LC3 channel illustrates punctate LC3 staining, representing LC3-II. Histogram depicts mean fluorescence intensity of 10,000 cells per sample. b, Quantification of mean ATG16L1 fluorescence intensity in a, n = 4 mice. Glu., glucose.


c , Immunoblot of phosphorylated ( p - ) AMPK & amp ; amp ; agr ; ( Thr 172 ) and lipidated LC3 ( LC3 - II ) .

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c, Immunoblot of phosphorylated (p-) AMPK&amp;agr; (Thr 172) and lipidated LC3 (LC3-II).


d , Quantification of mean LC3 - II area in murine macrophages , n = 4 mice .

d, Quantification of mean LC3-II area in murine macrophages, n = 4 mice.


e , Caspase 3 activity in murine macrophages following glucose starvation , n = 3 mice . RLU , relative light units .

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e, Caspase 3 activity in murine macrophages following glucose starvation, n = 3 mice. RLU, relative light units.


f , Quantification of mean LC3 - II area in wild - type ( Casp3 + / + ) or Casp3 - knockout ( Casp3 − / − ) macrophages , n = 3 mice . Data in a - f represent 2 independent experiments .

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f, Quantification of mean LC3-II area in wild-type (Casp3+/+) or Casp3-knockout (Casp3−/−) macrophages, n = 3 mice. Data in a-f represent 2 independent experiments.


a , Quantification of ATG16L1 mean fluorescence intensity by image - based flow cytometry of 10 , 000 cells , n = 4 ( wild - type , WT ) , 5 ( T300A ) . Data represent 3 independent experiments . b , Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells , n = 5 . Data represent 2 independent experiments . c , Quantification of mean LC3 - II area of samples in a . Data represent 3 independent experiments .

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a, Quantification of ATG16L1 mean fluorescence intensity by image-based flow cytometry of 10,000 cells, n = 4 (wild-type, WT), 5 (T300A). Data represent 3 independent experiments. b, Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells, n = 5. Data represent 2 independent experiments. c, Quantification of mean LC3-II area of samples in a. Data represent 3 independent experiments.


d , Visualization of early autophagosomes by transmission electron micrographs ( TEM ) . Red arrows , early autophagosomes , characterized by vesicular structures with a double - membrane . Blue arrows , multivesicular lysosomal structures , identified by degraded intravesicular cargo . Micrographs representative of 10 - 12 images per donor , n = 4 ( WT ) , n = 3 ( T300A ) . A total of 29 ( WT ) and 26 ( T300A ) images containing autophagosomes was analysed . Histogram quantifies autophagic vesicles as a ratio of autophagosome : cytosolic area .

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d, Visualization of early autophagosomes by transmission electron micrographs (TEM). Red arrows, early autophagosomes, characterized by vesicular structures with a double-membrane. Blue arrows, multivesicular lysosomal structures, identified by degraded intravesicular cargo. Micrographs representative of 10-12 images per donor, n = 4 (WT), n = 3 (T300A). A total of 29 (WT) and 26 (T300A) images containing autophagosomes was analysed. Histogram quantifies autophagic vesicles as a ratio of autophagosome:cytosolic area.


a - c , Y . enterocolitica colony forming units ( c . f . u . ) in human and murine macrophages infected for 6 h ( 20 m . o . i . ) . Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype , total n = 16 ( non - risk , WT ) , 15 ( T300A , risk ) . Data in b represent 3 independent experiments , n = 4 mice . Data in c represent 3 independent experiments , n = 6 .

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a-c, Y. enterocolitica colony forming units (c.f.u.) in human and murine macrophages infected for 6 h (20 m.o.i.). Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype, total n = 16 (non-risk, WT), 15 (T300A, risk). Data in b represent 3 independent experiments, n = 4 mice. Data in c represent 3 independent experiments, n = 6.


d , TNF - α , IL - 1β and IL - 6 levels in culture media of WT and T316A macrophages infected with 20 m . o . i . Y . enterocolitica . Data represent 2 independent experiments , n = 4 mice .

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d, TNF-α, IL-1β and IL-6 levels in culture media of WT and T316A macrophages infected with 20 m.o.i. Y. enterocolitica. Data represent 2 independent experiments, n = 4 mice.


e , Ileum and colon histology ( haematoxylin and eosin staining ) of WT mice . Images are representative of 3 ( PBS ) and 8 ( Y . enterocolitica ) treated male mice .

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e, Ileum and colon histology (haematoxylin and eosin staining) of WT mice. Images are representative of 3 (PBS) and 8 (Y. enterocolitica) treated male mice.


f , Measurement of cytokine mRNA transcripts in mesenteric lymph nodes . Data are pooled from 2 independent experiments of 3 mice ( PBS ) and 7 mice ( Y . enterocolitica ) per experiment . Transcripts are normalized to Gapdh ( 2 − Dgr ; Ct ) .

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f, Measurement of cytokine mRNA transcripts in mesenteric lymph nodes. Data are pooled from 2 independent experiments of 3 mice (PBS) and 7 mice (Y. enterocolitica) per experiment. Transcripts are normalized to Gapdh (2−Dgr;Ct).


g , Y . enterocolitica c . f . u . in mesenteric lymph nodes of WT and T316A knock - in mice 48 h following oral gavage . Data represent 3 independent experiments , n = 9 male mice .

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g, Y. enterocolitica c.f.u. in mesenteric lymph nodes of WT and T316A knock-in mice 48 h following oral gavage. Data represent 3 independent experiments, n = 9 male mice.


A . ATPase activity of ABCC3 wild - type ( WT ) and E1451Q in nanodiscs . The data points for WT and E1451Q are fitted with a Michaelis - Menten equation . All data points are the average of three independent experiments in nanodiscs , and error bars represent standard deviation .

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A. ATPase activity of ABCC3 wild-type (WT) and E1451Q in nanodiscs. The data points for WT and E1451Q are fitted with a Michaelis-Menten equation. All data points are the average of three independent experiments in nanodiscs, and error bars represent standard deviation.


B . Substrate - stimulated ATPase activities of ABCC3 WT and E1451Q upon addition of substrate E217βG or DHEAS . All data points are the average of three independent experiments in nanodiscs , and error bars represent standard deviation .

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B. Substrate-stimulated ATPase activities of ABCC3 WT and E1451Q upon addition of substrate E217βG or DHEAS. All data points are the average of three independent experiments in nanodiscs, and error bars represent standard deviation.


C . Relative ATPase activities of ABCC3 WT and mutants in the presence of 2 mM ATP upon addition of E217βG . The relative activity represents the substrate - stimulated activity of ABCC3 WT or its mutants harboring mutation of residues at the substrate - binding pocket . For ( C ) the activities of ABCC3 WT are set as 100 % . One - way analysis of variance ( ANOVA ) is used for the comparison of statistical significance of mutants and WT . The P values of < 0 . 05 , 0 . 01 , and 0 . 001 are indicated with * , * * and * * * , respectively .

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C. Relative ATPase activities of ABCC3 WT and mutants in the presence of 2 mM ATP upon addition of E217βG. The relative activity represents the substrate-stimulated activity of ABCC3 WT or its mutants harboring mutation of residues at the substrate-binding pocket. For (C) the activities of ABCC3 WT are set as 100%. One-way analysis of variance (ANOVA) is used for the comparison of statistical significance of mutants and WT. The P values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***, respectively.


D . Transport [ 3H ] - E217βG activity assays of ABCC3 WT and mutants . For ( D ) , the activities of ABCC3 WT are set as 100 % . One - way analysis of variance ( ANOVA ) is used for the comparison of statistical significance of mutants and WT . The P values of < 0 . 05 , 0 . 01 , and 0 . 001 are indicated with * , * * and * * * , respectively .

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D. Transport [3H]-E217βG activity assays of ABCC3 WT and mutants. For (D), the activities of ABCC3 WT are set as 100%. One-way analysis of variance (ANOVA) is used for the comparison of statistical significance of mutants and WT. The P values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***, respectively.


C . Relative ATPase activities of ABCC3 WT and mutants in nanodiscs upon addition of DHEAS and 2 mM ATP . The relative activity represents the substrate - stimulated activity of ABCC3 WT or mutants harboring mutation of residues at the substrate - binding pocket .

C. Relative ATPase activities of ABCC3 WT and mutants in nanodiscs upon addition of DHEAS and 2 mM ATP. The relative activity represents the substrate-stimulated activity of ABCC3 WT or mutants harboring mutation of residues at the substrate-binding pocket.


E . Relative ATPase activities of ABCC3 WT and mutants K318A , T535A upon addition of E217βG . For ( C ) and ( E ) , the activities of ABCC3 WT are set as 100 % . One - way ANOVA is used for the comparison of statistical significance of mutants and WT . The P values of < 0 . 05 , 0 . 01 , and 0 . 001 are indicated with * , * * and * * * , respectively .

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E. Relative ATPase activities of ABCC3 WT and mutants K318A, T535A upon addition of E217βG. For (C) and (E), the activities of ABCC3 WT are set as 100%. One-way ANOVA is used for the comparison of statistical significance of mutants and WT. The P values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***, respectively.


A Representative picture of plants carrying pASDOG1 : : LUC sprayed with mock solution , ABA or GA and imaged 24 h later . The full pictures are shown in Fig EV2 and Appendix Fig S1 . Scale bar , 1 cm .

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A Representative picture of plants carrying pASDOG1::LUC sprayed with mock solution, ABA or GA and imaged 24 h later. The full pictures are shown in Fig EV2 and Appendix Fig S1. Scale bar, 1 cm.


B Quantification of emitted light intensity per plant after treatment with ABA and GA , compared with mock treatment .

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B Quantification of emitted light intensity per plant after treatment with ABA and GA, compared with mock treatment.


C Antisense expression is reduced in response to drought conditions . The full pictures are shown in Fig EV3 . Scale bar , 1 cm .

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C Antisense expression is reduced in response to drought conditions. The full pictures are shown in Fig EV3. Scale bar, 1 cm.


D Quantification of emitted light intensity per plant for control and drought - treated plants .

D Quantification of emitted light intensity per plant for control and drought-treated plants.


E Strand - specific RT - qPCR asDOG1 expression analysis of 3 - day Col - 0 ( WT ) drought - treated vs . control plants .

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E Strand-specific RT-qPCR asDOG1 expression analysis of 3-day Col-0 (WT) drought-treated vs. control plants.


F The asDOG1 expression level ( black line ) is reduced while DOG1 sense expression ( grey line ) is increased after application of ABA . Leaves of mature Col - 0 ( WT ) 40 - day - old plants were sprayed with ABA and collected 0 , 6 and 10 h later for RNA extraction and RT - qPCR . Signals were normalized against the level of the UBC transcript . The data points are the averages for at least three biological replicates and are normalized against the mock - treated value . Error bars represent standard deviation . * and * * * represent t - test P - values of < 0 . 05 and < 0 . 001 , respectively .

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F The asDOG1 expression level (black line) is reduced while DOG1 sense expression (grey line) is increased after application of ABA. Leaves of mature Col-0 (WT) 40-day-old plants were sprayed with ABA and collected 0, 6 and 10 h later for RNA extraction and RT-qPCR. Signals were normalized against the level of the UBC transcript. The data points are the averages for at least three biological replicates and are normalized against the mock-treated value. Error bars represent standard deviation.* and *** represent t-test P-values of < 0.05 and < 0.001, respectively.


A Col - 0 ( WT ) and dog1 - 3 mutant plants were either watered normally or subjected to water withdrawal for 5 days and then watered again and allowed to recover for 2 days . A picture of a representative experiment is shown . Scale bar , 2 cm .

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A Col-0 (WT) and dog1-3 mutant plants were either watered normally or subjected to water withdrawal for 5 days and then watered again and allowed to recover for 2 days. A picture of a representative experiment is shown. Scale bar, 2 cm.


B Quantification of recovered and dead Col - 0 ( WT ) , null - 3 and dog1 - 4 plants ; plants were scored as dead when unable to grow and produced offspring after an extended recovery period .

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B Quantification of recovered and dead Col-0 (WT), null-3 and dog1-4 plants; plants were scored as dead when unable to grow and produced offspring after an extended recovery period.


C Analysis of DOG1 sense expression in drought - treated vs . control Col - 0 ( WT ) and dog1 - 3 mutant plants .

C Analysis of DOG1 sense expression in drought-treated vs. control Col-0 (WT) and dog1-3 mutant plants.


D dog1 - 3 mutant plants are defective in the regulation of the majority of the tested drought marker genes . Col - 0 ( WT ) and dog1 - 3 mutant plants were subjected to drought for 3 days and the expression of five marker genes was determined using RT - qPCR . Error bars represent standard deviation , and * , * * and * * * represent t - test P - values of < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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D dog1-3 mutant plants are defective in the regulation of the majority of the tested drought marker genes. Col-0 (WT) and dog1-3 mutant plants were subjected to drought for 3 days and the expression of five marker genes was determined using RT-qPCR. Error bars represent standard deviation, and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.


A Plants expressing LUC fused with full - length DOG1 ( pDOG1LUC : : DOG1 ) were sprayed with mock solution or ABA 10 , 20 and 40 days after germination and analysed 24 h after treatment . The graphs show mean emitted light intensity per plant .

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A Plants expressing LUC fused with full-length DOG1 (pDOG1LUC::DOG1) were sprayed with mock solution or ABA 10, 20 and 40 days after germination and analysed 24 h after treatment. The graphs show mean emitted light intensity per plant.


B Representative picture of mock - and ABA - treated pDOG1LUC : : DOG1 plants . The full picture is shown in Fig EV2C and D . Scale bar , 1 cm .

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B Representative picture of mock- and ABA-treated pDOG1LUC::DOG1 plants. The full picture is shown in Fig EV2C and D. Scale bar, 1 cm.


C A truncated construct that lacks the DOG1 antisense promoter region ( pDOG1shDOG1 : : LUC ) is not induced by ABA and is highly expressed throughout development . The graphs show mean emitted light intensity per plant .

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C A truncated construct that lacks the DOG1 antisense promoter region (pDOG1shDOG1::LUC) is not induced by ABA and is highly expressed throughout development. The graphs show mean emitted light intensity per plant.


D Representative picture of mock - and ABA - treated pDOG1shDOG1 : : LUC plants . Scale bar : 1cm .

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D Representative picture of mock- and ABA-treated pDOG1shDOG1::LUC plants. Scale bar: 1cm.


E RT - qPCR analysis of DOG1 sense mRNA level .

E RT-qPCR analysis of DOG1 sense mRNA level.


F Strand - specific RT - qPCR analysis of antisense levels after 10 h of ABA treatment in 10 - , 20 - and 40 - day - old Col - 0 plants .

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F Strand-specific RT-qPCR analysis of antisense levels after 10 h of ABA treatment in 10-, 20- and 40-day-old Col-0 plants.


G The pASDOG1 : : LUC reporter is silenced by the application of ABA , while mutation of TATA elements in the asDOG1 promoter ( pASDOG1 ∆ TATA : : LUC ) leads to attenuation of expression in the presence and absence of ABA .

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G The pASDOG1::LUC reporter is silenced by the application of ABA, while mutation of TATA elements in the asDOG1 promoter (pASDOG1∆TATA::LUC) leads to attenuation of expression in the presence and absence of ABA.


H Mutation of TATA elements in the asDOG1 promoter leads to high - level DOG1 sense expression and non - responsiveness to ABA . Error bars represent standard deviation and * , * * and * * * represent t - test P - values of < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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H Mutation of TATA elements in the asDOG1 promoter leads to high-level DOG1 sense expression and non-responsiveness to ABA. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.


A Plants expressing the truncated asDOG1 promoter - deficient version pDOG1shDOG1 : : LUC are more resistant to drought than plants expressing the full - length genomic version pDOG1 : : LUC - DOG1 . Three independent lines are shown for each construct , following 10 days of water deprivation . Scale bar , 2 cm .

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A Plants expressing the truncated asDOG1 promoter-deficient version pDOG1shDOG1::LUC are more resistant to drought than plants expressing the full-length genomic version pDOG1::LUC-DOG1. Three independent lines are shown for each construct, following 10 days of water deprivation. Scale bar, 2 cm.


B RT - qPCR quantification of selected drought marker genes in mock - and ABA - treated Col - 0 ( WT ) and two independent pDOG1shDOG1 : : LUC transgenic lines . Error bars represent standard deviation and * , * * and * * * represent t - test P - values of < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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B RT-qPCR quantification of selected drought marker genes in mock- and ABA-treated Col-0 (WT) and two independent pDOG1shDOG1::LUC transgenic lines. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.


( D , E ) Representative immunofluorescence microscopy images of the distribution of septin 7 ( red ) or borg3 or borg4 ( green ) in young , aged and aged LT - HSCs treated with CASIN ( 5µM ) . Nuclei were stained with DAPI ( blue ) , scale bar = 5µm .

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(D,E) Representative immunofluorescence microscopy images of the distribution of septin 7 (red) or borg3 or borg4 (green) in young, aged and aged LT-HSCs treated with CASIN (5µM). Nuclei were stained with DAPI (blue), scale bar= 5µm.


( A ) Representative immunofluorescence confocal microscopy images of young , aged and aged + CASIN ( 5µM ) treated LT - HSCs for the proximity ligation assay ( red signal ) , testing the extent of close physical interaction of Cdc42 and borg4 . A red fluorescent signal indicates close proximity of the two proteins tested . Nuclei were stained with DAPI ( blue ) , scale bar = 5µm . ( B ) Quantification of the level of fluorescent signal in young , aged and aged + CASIN ( 5µM ) treated LT - HSCs to quantify the Cdc42 - borg4 proximity interaction . Data are normalized to the mean fluorescence signal of aged LT - HSCs . 3 biological repeats , n = at least 17 cells per condition , error bars indicate mean ± SEM , unpaired t - test , * * * * P < 0 . 0001 . ( C ) Representative immunofluorescence confocal microscopy images of young , aged and aged + CASIN ( 5µM ) treated LT - HSCs for the proximity ligation assay ( red signal ) , testing the extent of close physical interaction of borg4 and septin 7 . A red fluorescent signal indicates close proximity of the two proteins tested . Nuclei were stained with DAPI ( blue ) , scale bar = 5µm . ( D ) Quantification of the level of fluorescent signal in young , aged and aged + CASIN ( 5µM ) treated LT - HSCs to quantify the borg4 - septin 7 proximity interaction . Data are normalized to the mean fluorescence signal of aged LT - HSCs . 3 biological repeats , n = at least 17 cells per condition , error bars indicate mean ± SEM , unpaired t - test , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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(A) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of Cdc42 and borg4. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm. (B) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the Cdc42-borg4 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ****P<0.0001. (C) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of borg4 and septin 7. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm. (D) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the borg4-septin 7 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ***P<0.001, ****P<0.0001.


( A , B ) Representative immunofluorescence microscopy images of the distribution of septin 7 ( red ) , Cdc42 ( red ) and tubulin ( green ) in LT - HSCs from borg4fl / fl or borg4 ∆ / ∆ mice . Nuclei were stained with DAPI ( blue ) , scale bar = 5 µm . ( C ) Percentage of cells with a polar distribution of septin 7 , Cdc42 or tubulin in borg4fl / fl or borg4 ∆ / ∆ LT - HSCs . At least 3 biological repeats , at least 50 cells were scored per sample . Error bars indicate mean ± SEM , Two - way ANOVA analysis , * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 .

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(A,B) Representative immunofluorescence microscopy images of the distribution of septin 7 (red), Cdc42 (red) and tubulin (green) in LT-HSCs from borg4fl/fl or borg4∆/∆ mice. Nuclei were stained with DAPI (blue), scale bar= 5 µm. (C) Percentage of cells with a polar distribution of septin 7, Cdc42 or tubulin in borg4fl/fl or borg4∆/∆ LT-HSCs. At least 3 biological repeats, at least 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, ***P<0.001, **P<0.01, *P<0.05.


( D , E ) Representative immunofluorescence microscopy images of the distribution of borg4 ( red ) , Cdc42 ( red ) or tubulin ( green ) in LT - HSCs from septin 7fl / fl or septin 7 ∆ / ∆ mice . Nuclei were stained with DAPI ( blue ) . scale bar = 5 µm . ( F ) Percentage of cells with a polar distribution of borg4 , Cdc42 or tubulin in septin 7fl / fl or septin 7 ∆ / ∆ LT - HSCs . At least 3 biological repeats , at least 30 to 50 cells were scored per sample . Error bars indicate mean ± SEM , Two - way ANOVA analysis , * * P < 0 . 01 , * P < 0 . 05 .

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(D,E) Representative immunofluorescence microscopy images of the distribution of borg4 (red), Cdc42 (red) or tubulin (green) in LT-HSCs from septin 7fl/fl or septin 7∆/∆ mice. Nuclei were stained with DAPI (blue). scale bar= 5 µm. (F) Percentage of cells with a polar distribution of borg4, Cdc42 or tubulin in septin 7fl/fl or septin 7∆/∆ LT-HSCs. At least 3 biological repeats, at least 30 to 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, **P<0.01, *P<0.05.


( J - O ) Percentage of donor derived ( borg4fl / fl or borg4 ∆ / ∆ or septin 7fl / fl or septin 7 ∆ / ∆ ) B - cells ( B220 positive cells ) , T - cells ( CD3 positive cells ) and myeloid cells ( Mac - 1 + or Gr + positive cells ) among BM cells , percentage of donor derived LT - HSCs , ST - HSCs and LMPPs in LSK cells and percentage of donor derived CLPs in lin - Sca1C - kit low cells in animals at post - transplantation ( n = 12 ) . Error bars indicate mean ± SD , * * * * P < 0 . 0001 , * * P < 0 . 01 and * P < 0 . 05 , Two - way ANOVA analysis and 2 tailed unpaired t - test .

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(J-O) Percentage of donor derived (borg4fl/fl or borg4∆/∆ or septin 7fl/fl or septin 7∆/∆) B-cells (B220 positive cells), T-cells (CD3 positive cells) and myeloid cells (Mac-1+ or Gr+ positive cells) among BM cells, percentage of donor derived LT-HSCs, ST-HSCs and LMPPs in LSK cells and percentage of donor derived CLPs in lin-Sca1C-kit low cells in animals at post- transplantation (n=12). Error bars indicate mean±SD, ****P<0.0001, **P<0.01 and *P<0.05, Two-way ANOVA analysis and 2 tailed unpaired t-test.


( A ) Percentage of single LT - HSCs from septin 7fl / fl of septin 7 ∆ / ∆ mice that completed first and second division up to 48 hours post initiation of culture . ( 192 sorted HSCs cells were analysed , error bars indicate mean ± SD ) .

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(A) Percentage of single LT-HSCs from septin 7fl/fl of septin 7∆/∆ mice that completed first and second division up to 48 hours post initiation of culture. (192 sorted HSCs cells were analysed, error bars indicate mean±SD).


( A ) Picrosirius red ( PSR ) staining of collagens ( red ) in PDAC tissue following surgical resection . Scale bar represents 200 µm . ( B ) Quantification of PSR in low - grade ( 1 - 2 ) and high - grade ( 3 ) tumors indicated in panel A as percentage of area . n = 15 primary PDAC samples . Data information : data are represented as mean ± SD , Student ' s t - test .

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(A) Picrosirius red (PSR) staining of collagens (red) in PDAC tissue following surgical resection. Scale bar represents 200 µm. (B) Quantification of PSR in low-grade (1-2) and high-grade (3) tumors indicated in panel A as percentage of area. n=15 primary PDAC samples. Data information: data are represented as mean±SD, Student's t-test.


( C ) Quantification of tumor cell percentage as scored by pathologists in low - and high - grade PDAC indicated in panel A . n = 15 primary PDAC samples . Data information : data are represented as mean ± SD , Student ' s t - test .

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(C) Quantification of tumor cell percentage as scored by pathologists in low- and high-grade PDAC indicated in panel A. n=15 primary PDAC samples. Data information: data are represented as mean±SD, Student's t-test.


( D ) PSR and α - SMA staining of indicated PDAC cell lines which were injected in NSG mice . Scale bar represents 200 µm for PSR and 100 µm for α - SMA images . ( E ) Quantification of stainings indicated in panel D . Three tumor ROIs of a tumor derived from epithelial cell lines ( Capan - 2 and AsPC - 1 ) and mesenchymal cell lines ( MIA PaCa - 2 and PANC - 1 ) are displayed and averages were pooled for analysis . Data information : data are represented as mean ± SD , Student ' s t - test .

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(D) PSR and α-SMA staining of indicated PDAC cell lines which were injected in NSG mice. Scale bar represents 200 µm for PSR and 100 µm for α-SMA images. (E) Quantification of stainings indicated in panel D. Three tumor ROIs of a tumor derived from epithelial cell lines (Capan-2 and AsPC-1) and mesenchymal cell lines (MIA PaCa-2 and PANC-1) are displayed and averages were pooled for analysis. Data information: data are represented as mean±SD, Student's t-test.


( F ) IHC staining of α - SMA in 3D organotypic co - cultures of PS - 1 cells and indicated PDAC cell lines . Scale bar represents 100 µm .

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(F) IHC staining of α-SMA in 3D organotypic co-cultures of PS-1 cells and indicated PDAC cell lines. Scale bar represents 100 µm.


( A ) Brightfield images of PS - 1 cells which were exposed for 72 hrs to CM from control ( CM of PS - 1 cells ) , epithelial ( blue ) or mesenchymal ( red ) cell lines . Scale bar represents 100 µm .

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(A) Brightfield images of PS-1 cells which were exposed for 72 hrs to CM from control (CM of PS-1 cells ), epithelial (blue) or mesenchymal (red) cell lines. Scale bar represents 100 µm.


( B ) Quantification of bead - calibrated cell count of PS - 1 cells after treatment using flowcytometry . n = 6 per group , 3 biological replicates for each cell line .

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(B) Quantification of bead-calibrated cell count of PS-1 cells after treatment using flowcytometry. n=6 per group, 3 biological replicates for each cell line.


( C ) EdU assay performed after treatment showing percentage of PS - 1 cells in G0 / G1 phase , G2 / M phase and S phase . n = 6 per group , 3 biological replicates for each cell line . .

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(C) EdU assay performed after treatment showing percentage of PS-1 cells in G0/G1 phase, G2/M phase and S phase. n=6 per group, 3 biological replicates for each cell line..


( A ) Gene expression level of stromal activation markers COL1A1 , ACTA2 , SPARC , FN1 and FAP in PS - 1 cells which were exposed for 72 hrs to CM from control ( CM of PS - 1 cells ) , epithelial ( blue ) or mesenchymal ( red ) PDAC cell lines using qPCR . Values were normalized to control and n = 6 per group , 3 biological replicates for each cell line . Data information : data are represented as mean ± SD , Student ' s t - test .

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(A) Gene expression level of stromal activation markers COL1A1, ACTA2, SPARC, FN1 and FAP in PS-1 cells which were exposed for 72 hrs to CM from control (CM of PS-1 cells ), epithelial (blue) or mesenchymal (red) PDAC cell lines using qPCR. Values were normalized to control and n=6 per group, 3 biological replicates for each cell line. Data information: data are represented as mean±SD, Student's t-test.


( B ) Heatmap representation of COL1A1 and ACTA2 relative gene expression levels measured with qPCR in PS - 1 cells after treatment using a large cohort of PDAC cell lines and primary cell cultures 67 and 53m . Values were Log2 transformed and normalized to Suit - 2 expression . Data information : data are represented as mean ± SD , Student ' s t - test .

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(B) Heatmap representation of COL1A1 and ACTA2 relative gene expression levels measured with qPCR in PS-1 cells after treatment using a large cohort of PDAC cell lines and primary cell cultures 67 and 53m. Values were Log2 transformed and normalized to Suit-2 expression. Data information: data are represented as mean±SD, Student's t-test.


( C ) Brightfield images of PS - 1 cells exposed to 10 days of treatment with 1 and 10 µM all - trans retinoic acid ( ATRA ) . Scale bar represents 100 µm .

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(C) Brightfield images of PS-1 cells exposed to 10 days of treatment with 1 and 10 µM all-trans retinoic acid (ATRA). Scale bar represents 100 µm.


( D ) Gene expression levels of COL1A1 and ACTA2 in PS - 1 cells after treatment using qPCR , values were normalized to control . n = 3 technical replicates per group . Data information : data are represented as mean ± SD , Student ' s t - test .

(D) Gene expression levels of COL1A1 and ACTA2 in PS-1 cells after treatment using qPCR, values were normalized to control. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.


( E ) Brightfield images of primary human CAFs exposed to 72 hrs of treatment with CM from control ( CM of primary CAFs ) or mesenchymal PDAC cell lines . Scale bar represents 100 µm .

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(E) Brightfield images of primary human CAFs exposed to 72 hrs of treatment with CM from control (CM of primary CAFs) or mesenchymal PDAC cell lines. Scale bar represents 100 µm.


( F ) Relative gene expression levels of COL1A1 and ACTA2 in primary CAFs after treatment using qPCR , values were normalized to control . n = 3 technical replicates per group . Data information : data are represented as mean ± SD , Student ' s t - test .

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(F) Relative gene expression levels of COL1A1 and ACTA2 in primary CAFs after treatment using qPCR, values were normalized to control. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.


( A ) Brightfield images of PS - 1 cells after 72 hrs of treatment with unfiltered , < 100 kDa or < 30 kDa filtered CM of indicated cell lines . Scale bar represents 100 µm .

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(A) Brightfield images of PS-1 cells after 72 hrs of treatment with unfiltered, <100 kDa or <30 kDa filtered CM of indicated cell lines. Scale bar represents 100 µm.


( B ) Gene expression levels of COL1A1 and ACTA2 in PS - 1 cells after treatment using qPCR . Data was normalized against control CM . Statistical significance was tested against unfiltered CM samples . Graph shows n = 3 technical replicates of an example experiment that was performed three times . Data information : data are represented as mean ± SD , Student ' s t - test .

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(B) Gene expression levels of COL1A1 and ACTA2 in PS-1 cells after treatment using qPCR. Data was normalized against control CM. Statistical significance was tested against unfiltered CM samples. Graph shows n=3 technical replicates of an example experiment that was performed three times. Data information: data are represented as mean±SD, Student's t-test.


( C ) Graphical representation of 64 cyto - and chemokines present in CM of epithelial and mesenchymal PDAC cell lines using forward - phase protein array . Significantly differential expression of proteins ( triangles ) and genes ( green triangles ) are identified between epithelial and mesenchymal cell lines . Dotted line indicates P = 0 . 05 . Data information : data are represented as mean ± SD , Student ' s t - test .

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(C) Graphical representation of 64 cyto- and chemokines present in CM of epithelial and mesenchymal PDAC cell lines using forward-phase protein array. Significantly differential expression of proteins (triangles) and genes (green triangles) are identified between epithelial and mesenchymal cell lines. Dotted line indicates P=0.05. Data information: data are represented as mean±SD, Student's t-test.


( D ) Brightfield images of PS - 1 cells after 72 hrs of treatment with CM of control ( CM of PS - 1s ) or epithelial PDAC cells supplemented with recombinant human CSF - 1 . Scale bar represents 100 µm .

(D) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of control (CM of PS-1s) or epithelial PDAC cells supplemented with recombinant human CSF-1. Scale bar represents 100 µm.


( E ) Gene expression levels of COL1A1 in PS - 1 cells after treatment using qPCR . n = 3 biological replicates per group . Data information : data are represented as mean ± SD , Student ' s t - test .

(E) Gene expression levels of COL1A1 in PS-1 cells after treatment using qPCR. n=3 biological replicates per group. Data information: data are represented as mean±SD, Student's t-test.


( F ) PS - 1 cells were starved and stimulated with 500 ng / ml rh - CSF - 1 at indicated time points . Activation of pathways involved in CSF1R signaling was analyzed using western blot . Samples were derived from the same experiment and blots were processed in parallel .

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(F) PS-1 cells were starved and stimulated with 500 ng/ml rh-CSF-1 at indicated time points. Activation of pathways involved in CSF1R signaling was analyzed using western blot. Samples were derived from the same experiment and blots were processed in parallel.


( A ) Brightfield images of PS - 1 cells after 72 hrs of treatment with CM of control ( CM of PS - 1s ) or mesenchymal PDAC cell lines supplemented with anti - CSF - 1 . Scale bar represents 100 µm .

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(A) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of control (CM of PS-1s) or mesenchymal PDAC cell lines supplemented with anti-CSF-1. Scale bar represents 100 µm.


( B ) Gene expression levels of COL1A1 in PS - 1 cells after treatment using qPCR . Graph shows n = 3 technical replicates of an example experiment that was performed three times . Data information : data are represented as mean ± SD , Student ' s t - test .

(B) Gene expression levels of COL1A1 in PS-1 cells after treatment using qPCR. Graph shows n=3 technical replicates of an example experiment that was performed three times. Data information: data are represented as mean±SD, Student's t-test.


( C ) Brightfield images of PS - 1 cells after 72 hrs of treatment with PANC - 1 CM of pLKOctrl or shCSF1 ( # 560 - 562 ) . Scale bar represents 100 µm .

(C) Brightfield images of PS-1 cells after 72 hrs of treatment with PANC-1 CM of pLKOctrl or shCSF1 (#560-562). Scale bar represents 100 µm.


( D ) Correlation graph of CSF1 expression in PANC - 1 pLKOctrl or shCSF1 ( # 560 - 562 ) and how these various CM affect COL1A1 expression in PS - 1 cells using qPCR , values were normalized for RPLPO expression . n = 3 biological replicates for all individual experiments , error bars indicated SD . R2 and P - value were analyzed with linear regression .

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(D) Correlation graph of CSF1 expression in PANC-1 pLKOctrl or shCSF1 (#560-562) and how these various CM affect COL1A1 expression in PS-1 cells using qPCR, values were normalized for RPLPO expression. n=3 biological replicates for all individual experiments, error bars indicated SD. R2 and P-value were analyzed with linear regression.


( E ) Brightfield images of PS - 1 cells after 72 hrs of treatment with CM of Capan - 2 cells with control vector or Capan - 2 cells overexpressing CSF1 . Scale bar represents 100 µm .

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(E) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of Capan-2 cells with control vector or Capan-2 cells overexpressing CSF1. Scale bar represents 100 µm.


( F ) Gene expression level of CSF1 in Capan - 2 cells after transduction with control vector or overexpressing CSF1 . n = 3 technical replicates per group . Data information : data are represented as mean ± SD , Student ' s t - test .

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(F) Gene expression level of CSF1 in Capan-2 cells after transduction with control vector or overexpressing CSF1. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.


( G ) Gene expression levels of COL1A1 in PS - 1 cells after treatment indicated using qPCR . Graph shows n = 3 technical replicates of an example experiment that was performed three times . Data information : data are represented as mean ± SD , Student ' s t - test .

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(G) Gene expression levels of COL1A1 in PS-1 cells after treatment indicated using qPCR. Graph shows n=3 technical replicates of an example experiment that was performed three times. Data information: data are represented as mean±SD, Student's t-test.


( A ) IHC staining of CSF - 1 in PDAC tissue following surgical resection representative image of CSF - 1 expression in grade 1 and grade 3 PDAC is depicted . Scale bar represents 100 µm . ( B ) For each tumor of panel A , CSF - 1 expression in ductal adenocarcinoma cells was quantified . Student ' s t - test . Shown is mean ± SD . n = 15 primary PDAC samples .

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(A) IHC staining of CSF-1 in PDAC tissue following surgical resection representative image of CSF-1 expression in grade 1 and grade 3 PDAC is depicted. Scale bar represents 100 µm. (B) For each tumor of panel A, CSF-1 expression in ductal adenocarcinoma cells was quantified. Student's t-test. Shown is mean±SD. n=15 primary PDAC samples.


( C ) A validation cohort of PDAC patients ( n = 21 ) that underwent direct resection was stained for CSF - 1 ( scale bar represents 100 µm ) , PSR ( scale bar represents 500 µm ) and α - SMA ( scale bar represents 200 µm ) . OK8 is low in CSF - 1 expression and high in PSR and α - SMA expression , while the opposite accounts for OK7 . ( D ) Correlation of % of PSR in the tumor and CSF - 1 expressing tumor cells , as indicated in panel C . R2 and P - value were analyzed with linear regression . n = 21 primary PDAC samples ( E ) Correlation of % of α - SMA in the tumor and CSF - 1 expressing tumor cells , as indicated in panel C , R2 and P - value were analyzed with linear regression . n = 21 primary PDAC samples

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(C) A validation cohort of PDAC patients (n=21) that underwent direct resection was stained for CSF-1 (scale bar represents 100 µm), PSR (scale bar represents 500 µm) and α-SMA (scale bar represents 200 µm). OK8 is low in CSF-1 expression and high in PSR and α-SMA expression, while the opposite accounts for OK7. (D) Correlation of % of PSR in the tumor and CSF-1 expressing tumor cells, as indicated in panel C. R2 and P-value were analyzed with linear regression. n=21 primary PDAC samples (E) Correlation of % of α-SMA in the tumor and CSF-1 expressing tumor cells, as indicated in panel C, R2 and P-value were analyzed with linear regression. n=21 primary PDAC samples


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( A ) Localization of Ape1 relative to Ykt6 and Sec63 during nitrogen starvation . Cells expressing GFP - tagged Ykt6 under the control of the GAL1 promoter and tdTomato - tagged Sec63 were transformed with a centromeric plasmid expressing BFP - Ape1 . Cells were grown in SGC - LEU , shifted to SG - N for the indicated time , and analyzed by fluorescence microscopy . Images are shown as single planes . Scale bar , 5 µm . Arrows indicate Ykt6 dots colocalizing with Ape1 . ( B ) Percentage of Ykt6 puncta simultaneously co - localizing with both Ape1 and Sec63 . The data were quantified from ( A ) . Error bars represent standard deviation of three independent experiments . Cells ( n ≥ 250 ) and Ape1 dots ( n ≥ 50 ) were quantified .

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(A) Localization of Ape1 relative to Ykt6 and Sec63 during nitrogen starvation. Cells expressing GFP-tagged Ykt6 under the control of the GAL1 promoter and tdTomato-tagged Sec63 were transformed with a centromeric plasmid expressing BFP-Ape1. Cells were grown in SGC-LEU, shifted to SG-N for the indicated time, and analyzed by fluorescence microscopy. Images are shown as single planes. Scale bar, 5 µm. Arrows indicate Ykt6 dots colocalizing with Ape1. (B) Percentage of Ykt6 puncta simultaneously co-localizing with both Ape1 and Sec63. The data were quantified from (A). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Ape1 dots (n≥50) were quantified.


( C ) Localization of Ykt6 relative to different organelle marker proteins during nitrogen starvation . Cells expressing GFP - tagged Ykt6 under the control of the GAL1 promoter and tdTomato - tagged Snx41 ( endosomes ) , Sec63 ( ER ) , Vac8 ( vacuole ) , or Mnn9 ( Golgi ) were were grown in SGC - LEU , then shifted to SG - N for 2 h , and analyzed by fluorescence microscopy . Single imaging planes are shown . Scale bar , 5 µm . Arrows indicate Ykt6 dots that colocalize with marker proteins .

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(C) Localization of Ykt6 relative to different organelle marker proteins during nitrogen starvation. Cells expressing GFP-tagged Ykt6 under the control of the GAL1 promoter and tdTomato-tagged Snx41 (endosomes), Sec63 (ER), Vac8 (vacuole), or Mnn9 (Golgi) were were grown in SGC-LEU, then shifted to SG-N for 2 h, and analyzed by fluorescence microscopy. Single imaging planes are shown. Scale bar, 5 µm. Arrows indicate Ykt6 dots that colocalize with marker proteins.


Localization of Ykt6 in multiple atg mutants . ( A ) Wild - type or the respective mutant cells expressing mCherry - tagged Ape1 and GFP - tagged Ykt6 were grown in SGC to allow for Ykt6 expression from the GAL1 - promoter and analyzed Arrows in the images indicate dots of Ykt6 colocalizing with Ape1 ( A )

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Localization of Ykt6 in multiple atg mutants. (A) Wild-type or the respective mutant cells expressing mCherry-tagged Ape1 and GFP-tagged Ykt6 were grown in SGC to allow for Ykt6 expression from the GAL1-promoter and analyzed Arrows in the images indicate dots of Ykt6 colocalizing with Ape1 (A)


( B ) Quantification of colocalization of Ykt6 relative to Ape1 as a PAS marker . Cells ( n ≥ 250 ) and Ape1 dots ( n ≥ 70 ) were quantified .

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(B) Quantification of colocalization of Ykt6 relative to Ape1 as a PAS marker. Cells (n≥250) and Ape1 dots (n≥70) were quantified.


( C ) Localization of Atg9 relative to Ykt6 . Cells expressing GFP - tagged Ykt6 and mScarlet - tagged Atg9 were grown and analyzed The left is the quantification of colocalization of Ykt6 relative to Atg9 . Cells ( n ≥ 200 ) and Atg9 dots ( n ≥ 80 ) were quantified . Arrows in the images indicate dots of Ykt6 colocalizing with Atg9 ( C )

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(C) Localization of Atg9 relative to Ykt6. Cells expressing GFP-tagged Ykt6 and mScarlet-tagged Atg9 were grown and analyzed The left is the quantification of colocalization of Ykt6 relative to Atg9. Cells (n≥200) and Atg9 dots (n≥80) were quantified. Arrows in the images indicate dots of Ykt6 colocalizing with Atg9 (C)


Ykt6 localizes to the isolation membrane during starvation . ( D ) Cells expressing mCherry - tagged Atg8 and GFP - Ykt6 carrying the Ape1 encoding plasmid pRS315 - CUP1pr - BFP - APE1 were grown in SGC medium containing CuSO4 and starved for 1 h , before to be analyzed by microscopy . Scale bar , 5 µm . ( E ) Volume rendering of LLSM image from a single time point after 3D deconvolution from 3D view . Cells expressing mCherry - tagged Atg8 and GFP - Ykt6 carrying plasmid pRS315 - CUP1pr - BFP - APE1 , cells , images were analyzed by Imaris . Scale bar , 5 µm . Arrows in the images indicate dots of Ykt6 colocalizing with Atg8 ( D , E ) .

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Ykt6 localizes to the isolation membrane during starvation. (D) Cells expressing mCherry-tagged Atg8 and GFP-Ykt6 carrying the Ape1 encoding plasmid pRS315-CUP1pr-BFP-APE1 were grown in SGC medium containing CuSO4 and starved for 1 h, before to be analyzed by microscopy. Scale bar, 5 µm. (E) Volume rendering of LLSM image from a single time point after 3D deconvolution from 3D view. Cells expressing mCherry-tagged Atg8 and GFP-Ykt6 carrying plasmid pRS315-CUP1pr-BFP-APE1, cells , images were analyzed by Imaris. Scale bar, 5 µm. Arrows in the images indicate dots of Ykt6 colocalizing with Atg8 (D,E).


( F ) The only partial time points ( 90 s to 600 s ) of images were assembled , and analyzed by Imaris and image J . Scale bar , 5 µm . 500 - 1000 cells were analyzed in each independent experiment . The 3D stacks were cropped by Imaris and different channels were split by Image J .

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(F) The only partial time points (90 s to 600 s) of images were assembled, and analyzed by Imaris and image J. Scale bar, 5 µm. 500-1000 cells were analyzed in each independent experiment. The 3D stacks were cropped by Imaris and different channels were split by Image J.


( A ) Effect of ykt6 and dsl1 complex ts mutants on autophagosome - vacuole fusion . Cells carrying a CEN plasmid expressing GFP - Atg8 were grown at 24 ° C in SGC and then shifted to SG - N for 2 h at 24 ° C or 37 ° C . Vacuoles were stained by FM4 - 64 and analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar of inset is 0 . 6 µm .

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(A) Effect of ykt6 and dsl1 complex ts mutants on autophagosome-vacuole fusion. Cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 24°C in SGC and then shifted to SG-N for 2 h at 24°C or 37°C. Vacuoles were stained by FM4-64 and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset is 0.6 µm.


( B ) Analysis of autophagy over time . Cells were grown at 24 ° C and shifted to starvation medium at 24 ° C or 37 ° C for the indicated time periods . The cells were harvested and proteins were extracted from cells by TCA precipitation . The extracted proteins were analyzed by SDS - PAGE and Western blotting against GFP .

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(B) Analysis of autophagy over time. Cells were grown at 24°C and shifted to starvation medium at 24°C or 37°C for the indicated time periods. The cells were harvested and proteins were extracted from cells by TCA precipitation. The extracted proteins were analyzed by SDS-PAGE and Western blotting against GFP.


( C ) Protease protection assay of ykt6 - 11 and dsl3 mutants . Lysates of atg1 ∆ , ypt7 ∆ , ykt6ts and dsl3ts cells carrying a CEN plasmid expressing GFP - Atg8 were grown at 37 ° C and subjected to the protease ( PK ) -

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(C) Protease protection assay of ykt6-11 and dsl3 mutants. Lysates of atg1∆, ypt7∆, ykt6ts and dsl3ts cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 37°C and subjected to the protease (PK)-protection


( D ) Localization of Atg8 relative to Atg1 during nitrogen starvation condition in sec12 - 1 ts mutant . Cells carrying a CEN plasmid expressing GFP - Atg8 and encoding 3xmCherry - tagged Atg1 were grown at 24 ° C in SDC and then shifted to SD - N for 2 h at 24 ° C or 37 ° C , and analyzed by fluorescence microscopy . Individual slices are shown . Scale bar , 5 µm . Scale bar of inset is 0 . 6 µm . Arrows in images indicate dots colocalization of Atg1 with Atg8 ( D )

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(D) Localization of Atg8 relative to Atg1 during nitrogen starvation condition in sec12-1 ts mutant. Cells carrying a CEN plasmid expressing GFP-Atg8 and encoding 3xmCherry-tagged Atg1 were grown at 24°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. Arrows in images indicate dots colocalization of Atg1 with Atg8 (D)


( E ) Localization of Ykt6 relative to Dsl1 during growth and nitrogen starvation conditions . Cells expressing GFP - tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry - tagged Dsl1 were grown in SDC or SD - N for 2 h , and analyzed by fluorescence microscopy . Individual slices are shown . Scale bar , 5 µm . ( F ) Percentage of Ykt6 puncta co - localizing with Dsl1 . The data was quantified from ( E ) . Error bars represent standard deviation of three independent experiments . Cells ( n ≥ 250 ) and Dsl1 dots ( n ≥ 50 ) were quantified . Arrows in images indicate dots colocalization of Ykt6 with Dsl1 ( E ) .

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(E) Localization of Ykt6 relative to Dsl1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Dsl1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. (F) Percentage of Ykt6 puncta co-localizing with Dsl1. The data was quantified from (E). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Dsl1 dots (n≥50) were quantified. Arrows in images indicate dots colocalization of Ykt6 with Dsl1 (E).


( G ) Localization of Dsl3 relative to Atg8 during growth and nitrogen starvation . Cells expressing GFP - tagged Dsl3 and mCherry - tagged Atg8 were grown in SDC or SD - N for 2 h , and analyzed by fluorescence microscopy . Scale bar , 5 µm .

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(G) Localization of Dsl3 relative to Atg8 during growth and nitrogen starvation. Cells expressing GFP-tagged Dsl3 and mCherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Scale bar, 5 µm.


( A ) Effect of Dsl and COPII ts mutants on Ykt6 localization to autophagosomes . Wild - type and selected ts strains ( sec12 - 1 , tip20 - 5 and dsl3 - 2 ) expressing GFP - tagged Ykt6 and mCherry - tagged Atg8 were grown at 24 ° C in SGC for 8 h and then shifted to SG - N for 2 h at 37 ° C . Vacuoles were stained by CMAC and analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar in inset , 0 . 7 µm . Arrows indicate dot colocalization of Ykt6 and Atg8 ( A )

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(A) Effect of Dsl and COPII ts mutants on Ykt6 localization to autophagosomes. Wild-type and selected ts strains (sec12-1, tip20-5 and dsl3-2) expressing GFP-tagged Ykt6 and mCherry-tagged Atg8 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 37°C. Vacuoles were stained by CMAC and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar in inset, 0.7 µm. Arrows indicate dot colocalization of Ykt6 and Atg8 (A)


Wild - type and selected ts strains ( sec12 - 1 , tip20 - 5 and dsl3 - 2 ) expressing GFP - tagged Ykt6 and mCherry - tagged Atg8 were grown at 24 ° C in SGC for 8 h and then shifted to SG - N for 2 h at 37 ° C . ( B ) Percentage of Ykt6 puncta co - localizing with Atg8 . Cells ( n ≥ 150 ) and Atg8 dots ( n ≥ 100 ) were quantified .

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Wild-type and selected ts strains (sec12-1, tip20-5 and dsl3-2) expressing GFP-tagged Ykt6 and mCherry-tagged Atg8 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 37°C. (B) Percentage of Ykt6 puncta co-localizing with Atg8. Cells (n≥150) and Atg8 dots (n≥100) were quantified.


( C ) Localization of Ykt6 relative to Sec63 and Sec13 during growth and nitrogen starvation conditions . tip20 - 5 cells expressing GFP - tagged Ykt6 and 3xmCherry - tagged Sec63 or Sec13 were grown at 24 ° C in SGC for 8 h and then shifted to SG - N for 2 h at 24 ° C or 37 ° C , and analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar of inset , 0 . 7 µm . Arrows indicate dot colocalization of Ykt6 with Sec63 ( C ) .

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(C) Localization of Ykt6 relative to Sec63 and Sec13 during growth and nitrogen starvation conditions. tip20-5 cells expressing GFP-tagged Ykt6 and 3xmCherry-tagged Sec63 or Sec13 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset, 0.7 µm. Arrows indicate dot colocalization of Ykt6 with Sec63 (C).


( D ) Effect of dsl mutation on autophagosome - vacuole fusion in vitro . The vam3 ∆ or dsl ts strain expressing Atg9 - 3xFLAG and GFP - Atg8 were starved for 3 h at 30 ° C before purifying autophagosomes , which was sufficient to induce the ts phenotype . Vacuoles were isolated from pep4 ∆ cells expressing Vac8 - 3xmCherry and then incubated with autophagosomes at 26 ° C for 1 h with or without ATP ( E ) Quantification of ( D ) . Error bars represent standard deviation of 3 independent experiments . Fluorescence intensity of GFP - Atg8 in the vacuolar lumen was quantified by image J using the ROI ( region of interest ) manager tool . Vacuoles ( n ≥ 40 ) for each experiment were quantified .

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(D) Effect of dsl mutation on autophagosome-vacuole fusion in vitro. The vam3∆ or dsl ts strain expressing Atg9-3xFLAG and GFP-Atg8 were starved for 3 h at 30°C before purifying autophagosomes, which was sufficient to induce the ts phenotype. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes at 26°C for 1 h with or without ATP (E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified.


( A - B ) Quantification of size and number of autophagosomes in the indicated ts mutant strains . Error bars represent standard deviation of 3 independent experiments . GFP - tagged Atg8 in the different mutants were grown at 23 ° C ( ts mutant ) or 30 ° C ( atg1 ∆ ) in SDC and then shifted to SD - N for 2 h at 30 ° C ( atg1 ∆ ) or 37 ° C ( ts mutant ) , and analyzed by fluorescence microscopy . Scale bar , 1 µm .

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(A-B) Quantification of size and number of autophagosomes in the indicated ts mutant strains. Error bars represent standard deviation of 3 independent experiments. GFP-tagged Atg8 in the different mutants were grown at 23°C (ts mutant) or 30°C (atg1∆) in SDC and then shifted to SD-N for 2 h at 30°C (atg1∆) or 37°C (ts mutant), and analyzed by fluorescence microscopy. Scale bar, 1 µm.


( C ) Localization of Atg8 relative to Atg9 during nitrogen starvation . dsl3 cells expressing mCherry - tagged Atg9 and carrying a CEN plasmid expressing GFP - Atg8 were grown at 23 ° C in SDC and then shifted to SD - N for 2 h at 24 ° C or 37 ° C . Cells were analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar for inset is 0 . 6 µm .

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(C) Localization of Atg8 relative to Atg9 during nitrogen starvation. dsl3 cells expressing mCherry-tagged Atg9 and carrying a CEN plasmid expressing GFP-Atg8 were grown at 23°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C. Cells were analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar for inset is 0.6 µm.


( D ) Atg9 localization in dsl3 cells during starvation . dsl3 ts cells expressing mCherry - tagged Atg9 , and carrying a CEN plasmid expressing GFP - Atg8 and plasmid pRS315 - CUP1pr - BFP - APE1 were grown in SDC medium containing CuSO4 to induce formation of the giant Ape1 structure , and starved for 1 h . Cells were analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar for inset is 0 . 6 µm .

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(D) Atg9 localization in dsl3 cells during starvation. dsl3 ts cells expressing mCherry-tagged Atg9, and carrying a CEN plasmid expressing GFP-Atg8 and plasmid pRS315-CUP1pr-BFP-APE1 were grown in SDC medium containing CuSO4 to induce formation of the giant Ape1 structure, and starved for 1 h. Cells were analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar for inset is 0.6 µm.


( B ) Localization of Ykt6 relative to Atg1 during growth and nitrogen starvation conditions . Cells expressing GFP - tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry - tagged Atg1 were grown in SDC or SD - N for 2 h , and analyzed by fluorescence microscopy . Individual slices are shown . Scale bar , 5 µm . Scale bar of inset is 0 . 6 µm . Right , percentage of Ykt6 puncta co - localizing with Atg1 . Error bars represent standard deviation of three independent experiments . Cells ( n ≥ 200 ) and Atg1 dots ( n ≥ 100 ) were quantified . Arrows indicate Ykt6 positive dots colocalizing with Atg1 ( B )

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(B) Localization of Ykt6 relative to Atg1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Atg1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. Right, percentage of Ykt6 puncta co-localizing with Atg1. Error bars represent standard deviation of three independent experiments. Cells (n≥200) and Atg1 dots (n≥100) were quantified. Arrows indicate Ykt6 positive dots colocalizing with Atg1 (B)


( C ) In vitro phosphorylation of purified Ykt6 by the Atg1 kinase complex . Purified Ykt6 was incubated with the Atg1 - Atg13 complex in the presence or absence of ATP , and Ykt6 band was cut out and analyzed by mass spectrometry .

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(C) In vitro phosphorylation of purified Ykt6 by the Atg1 kinase complex. Purified Ykt6 was incubated with the Atg1-Atg13 complex in the presence or absence of ATP, and Ykt6 band was cut out and analyzed by mass spectrometry.


( D ) Localization of Ykt6 relative to Atg8 under growth and nitrogen starvation conditions . Wild - type and atg1D211A cells expressing GFP - tagged Ykt6 under the control of the PHO5 promoter and genomically Cherry - tagged Atg8 were grown in SDC or SD - N for 2 h , and analyzed by fluorescence microscopy . Individual focal planes are shown . Scale bar , 5 µm . ( E ) Percentage of Ykt6 puncta co - localizing with Atg8 . Numbers in the column indicates the percentage . Cells ( n ≥ 300 ) and Atg8 dots ( n ≥ 60 ) were quantified . Arrows indicate Ykt6 positive dots colocalizing with Atg8 ( D ) .

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(D) Localization of Ykt6 relative to Atg8 under growth and nitrogen starvation conditions. Wild-type and atg1D211A cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically Cherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual focal planes are shown. Scale bar, 5 µm. (E) Percentage of Ykt6 puncta co-localizing with Atg8. Numbers in the column indicates the percentage. Cells (n≥300) and Atg8 dots (n≥60) were quantified. Arrows indicate Ykt6 positive dots colocalizing with Atg8 (D).


( A ) Growth test of Ykt6 wild - type and mutants . ykt6 ∆ cells carrying a URA3 plasmid coding for wild - type Ykt6 and pRS413 - GAL1pr - YKT6 - GFP ( wild - type or mutant ) were spotted as serial dilutions on SGC - HIS to select for the mutant plasmid or 5 - Fluoroorotic acid ( 5 - FOA ) to force the loss of the plasmid coding for wild - type Ykt6 .

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(A) Growth test of Ykt6 wild-type and mutants. ykt6∆ cells carrying a URA3 plasmid coding for wild-type Ykt6 and pRS413-GAL1pr-YKT6-GFP (wild-type or mutant) were spotted as serial dilutions on SGC-HIS to select for the mutant plasmid or 5-Fluoroorotic acid (5-FOA) to force the loss of the plasmid coding for wild-type Ykt6.