D ) In vitro organoids formation using crypts isolated from AhCre Eed + / + AhCre Eed - / - mice 15 days after first β - naphthoflavone injection . Organoids pictures were taken 5 days later at low higher magnification ( insets ) .

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D) In vitro organoids formation using crypts isolated from AhCre Eed+/+ and AhCre Eed-/- mice 15 days after the first β-naphthoflavone injection. Organoids pictures were taken 5 days later at low and higher magnification (insets).


A ) Volcano plot showing expression changes with a minimal fold change of 3 ( FC = 3 ) at genome - wide in AhCre Eed - / - crypts relative to AhCre Eed + / + crypts 15 days after β - naphthoflavone administration .

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A) Volcano plot showing the expression changes with a minimal fold change of 3 (FC=3) at genome-wide levels in AhCre Eed-/- crypts relative to AhCre Eed+/+ crypts 15 days after β-naphthoflavone administration.


B ) Bar plot showing of H3K27me3 - positive promoters among differentially regulated genes in Eed - / - crypts . gene promoters 5 independent random sampling of 167 gene promoters extracted mouse genome are presented as specificity controls .

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B) Bar plot showing the percentage of H3K27me3-positive promoters among the differentially regulated genes in Eed-/- crypts. All gene promoters and 5 independent random sampling of 167 gene promoters extracted the mouse genome are presented as specificity controls.


C ) profiles for H3K27me3 SUZ12 occupancy at promoters of up - regulated genes with respect to down - regulated genes in Eed - / - crypts .

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C) Intensity profiles for H3K27me3 and SUZ12 occupancy at the promoters of up-regulated genes with respect to down-regulated genes in Eed-/- crypts.


D ) Genomic snapshots of Cdkn2a locus showing SUZ12 occupancy H3K27me3 deposition at promoter regions of both p16 ARF isoforms .

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D) Genomic snapshots of the Cdkn2a locus showing SUZ12 occupancy and H3K27me3 deposition at the promoter regions of both the p16 and the ARF isoforms.


E ) Genomic snapshots of RNAseq profile of Cdkn2a locus in AhCre Eed + / + AhCre Eed - / - crypts 15 days after β - naphthoflavone administration .

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E) Genomic snapshots of the RNAseq profile of the Cdkn2a locus in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.


F ) Expression by qRT - PCR ( mean ± SD ) of both Cdkn2a isoforms ( p16Ink4A p19Arf ) in AhCre Eed + / + AhCre Eed - / - crypts 15 days after β - naphthoflavone administration .

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F) Expression analysis by qRT-PCR (mean ± SD) of both Cdkn2a isoforms (p16Ink4A and p19Arf) in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.


G ) Gene set enrichment ( GSEA ) using specific secretory enterocytic transcriptional signatures with respect to list of up - regulated genes in Eed - / - crypts .

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G) Gene set enrichment analysis (GSEA) using specific secretory and enterocytic transcriptional signatures with respect to the list of up-regulated genes in Eed-/- crypts.


A ) Haematoxylin eosin ( top panels ) PAS ( bottom panels ) of sections prepared from different intestinal tracts in AhCre Cdkn2a - / - Eed + / + AhCre Cdkn2a - / - Eedfl / fl mice 15 days after first β - naphthoflavone administration ( n = 5 ) .

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A) Haematoxylin and eosin (top panels) and PAS staining (bottom panels) of sections prepared from different intestinal tracts in AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration (n=5).


B ) ( mean ± SD ) of Goblet along crypt - to - villus axis from shown in A . More than 100 crypts villi were scored for each condition .

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B) Quantification (mean ± SD) of Goblet cells along the crypt-to-villus axis from the staining shown in A. More than 100 crypts and villi were scored for each condition.


C ) Immunhistochemistry using anti - KI67 antibody of intestinal sections prepared from AhCre Cdkn2a - / - Eed + / + AhCre Cdkn2a - / - Eedfl / fl mice 15 days after first β - naphthoflavone administration .

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C) Immunhistochemistry analysis using anti-KI67 antibody of intestinal sections prepared from AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration.


D ) Radiation - induced intestinal regeneration : AhCre Cdkn2a - / - Eed + / + AhCre Cdkn2a - / - Eedfl / fl mice ( n = 6 ) were injected with β - naphthoflavone , irradiated with 10 Gy 15 days later , sacrificed 8 days after irradiation . Sections prepared from different intestinal tracts were stained with haematoxylin eosin ( first panels ) Alcian Blue ( last panels ) . Sections were also stained for immunohistochemistry ( second third panels ) using H3K27me3 Ki67 - specific antibodies .

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D) Radiation-induced intestinal regeneration: AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice (n=6) were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (first panels) and Alcian Blue (last panels). Sections were also stained for immunohistochemistry analysis (second and third panels) using H3K27me3 and Ki67-specific antibodies.


A ) Expression by qRT - PCR ( mean ± SD ) of indicated intestinal epithelial markers regulated by Wnt Notch signaling pathways that are involved in homeostasis , regionalization , Goblet differentiation ( n = 3 ) .

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A) Expression analysis by qRT-PCR (mean ± SD) of the indicated intestinal epithelial cell markers regulated by the Wnt and Notch signaling pathways that are involved in stem cell homeostasis, regionalization, and Goblet cell differentiation (n=3).


B ) Genomic snapshots of indicated genomic loci for SUZ12 occupancy H3K27me3 deposition in intestinal crypts of wild type mice ( n = 3 ) .

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B) Genomic snapshots of the indicated genomic loci for SUZ12 occupancy and H3K27me3 deposition in intestinal crypts of wild type mice (n=3).


C ) Expression by qRT - PCR ( mean ± SD ) of Goblet markers ( Atoh1 , Gfi1 , Spdef ) , early secretory precursor ( Dll1 ) , NOTCH targets ( Hes1 , Olfm4 ) intestinal ( Lgr5 , Rnf43 ) in wild type intestinal crypts purified 38hrs after treatment with NOTCH inhibitor DBZ . DMSO served as vehicle negative control ( n = 4 ) .

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C) Expression analysis by qRT-PCR (mean ± SD) of the Goblet cell markers (Atoh1, Gfi1, Spdef), early secretory precursor cells (Dll1), NOTCH targets (Hes1, Olfm4) and intestinal stem cells (Lgr5, Rnf43) in wild type intestinal crypts purified 38hrs after treatment with the NOTCH inhibitor DBZ. DMSO served as vehicle negative control (n=4).


D ) ChIP of Atoh1 Gfi1 loci using H3K27me3 specific antibody . same crypts described in C were used . H3K27me3 were normalized on Histone H3 density ( of H3 signal ) . Graphs show mean ± SD .

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D) ChIP analysis of Atoh1 and Gfi1 loci using H3K27me3 specific antibody. The same crypts described in C were used. H3K27me3 levels were normalized on Histone H3 density (percentage of H3 signal). Graphs show mean ± SD.


( a ) PPARγ mRNA expression in sperm from control MSUS mice . Control n = 15 , MSUS n = 13 , two - Student ' s t - , P = 0 . 029 , t = 2 . 30 , df = 26 .

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(a) PPARγ mRNA expression in sperm from control and MSUS mice. Control n = 15, MSUS n = 13, two-tailed Student's t-test, P = 0.029, t = 2.30, df = 26.


( c ) Relative luciferase luminescence in transfected GC - 1 exposed to serum from control or MSUS mice . Numbers correspond to serum from applied to different culture wells . Control n = 17 , MSUS n = 22 , two - Student ' s t - , P = 0 . 042 , t = 2 . 1 , df = 37 .

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(c) Relative luciferase luminescence in transfected GC-1 cells exposed to serum from control or MSUS mice. Numbers correspond to serum from individual animals applied to different cell culture wells. Control n = 17, MSUS n = 22, two-tailed Student's t-test, P = 0.042, t = 2.1, df = 37.


( b ) Adult in offspring of tesaglitazar - injected ( Tesa - inj , n = 16 ) compared to offspring of vehicle - injected ( Vehicle - inj , n = 23 ) . Two - Student ' s t - , P = 0 . 019 , t = 2 . 44 , df = 37 .

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(b) Adult weight in the offspring of tesaglitazar-injected males (Tesa-inj, n = 16) compared to the offspring of vehicle-injected males (Vehicle-inj, n = 23). Two-tailed Student's t-test, P = 0.019, t = 2.44, df = 37.


( c ) Glucose level in offspring of Tesa - inj ( n = 14 ) Vehicle - inj ( n = 21 ) during a glucose tolerance . Repeated measures ANOVA , treatment effect P = 0 . 0083 , F ( 1 , 33 ) = 7 . 877 , time effect P < 0 . 0001 , F ( 4 , 132 ) = 347 . 7 , interaction P = 0 . 0776 , F ( 4 , 132 ) = 2 . 155 Conc . ; .

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(c) Glucose level in the offspring of Tesa-inj (n = 14) and Vehicle-inj (n = 21) males during a glucose tolerance test. Repeated measures ANOVA, treatment effect P = 0.0083, F (1, 33) = 7.877, time effect P < 0.0001, F (4, 132) = 347.7, interaction P = 0.0776, F (4, 132) = 2.155 Conc.; concentration.


( a Differentially expressed transposable elements in sperm from Tesa - inj MSUS . Data represent genes with P < 0 . 05 similar fold change . Fold change in heat map represents log2 ( fold change ) respective to corresponding control group . Total overlap is presented in Extended Data Fig . 11 .

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(a Differentially expressed transposable elements in sperm from Tesa-inj and MSUS males. Data represent genes with P < 0.05 and similar fold change. Fold change in heat map represents log2(fold change) respective to the corresponding control group. Total overlap is presented in Extended Data Fig. 11.


b ) mRNAs / lincRNAs in sperm from Tesa - inj MSUS . Data represent genes with P < 0 . 05 similar fold change . Fold change in heat map represents log2 ( fold change ) respective to corresponding control group . Total overlap is presented in Extended Data Fig . 11 .

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b) mRNAs/lincRNAs in sperm from Tesa-inj and MSUS males. Data represent genes with P < 0.05 and similar fold change. Fold change in heat map represents log2(fold change) respective to the corresponding control group. Total overlap is presented in Extended Data Fig. 11.


( c , d ) ( c ) Volcano plot ( d ) heat map of differentially expressed ( FDR < 0 . 05 ) mRNA / lincRNA in sperm from Tesa - inj . Dashed black lines in ( c ) represent y = 1 . 3 , equivalent to FDR = 0 . 05 , x = ± 0 . 03 , equivalent to FC = 1 . 23 0 . 81 . Black dots represent top candidates , which have FC > 2 or FC < 0 . 5 , FDR < 0 . 05 . Teal dots represent non - significant genes .

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(c,d) (c) Volcano plot and (d) heat map of differentially expressed (FDR < 0.05) mRNA/lincRNA in sperm from Tesa-inj males. Dashed black lines in (c) represent y = 1.3, equivalent to FDR = 0.05, and x = ±0.03, equivalent to FC = 1.23 and 0.81. Black dots represent top candidates, which all have FC > 2 or FC < 0.5, and FDR < 0.05. Teal dots represent non-significant genes.


( b ) in adult MSUS offspring offspring of injected with MSUS serum compared to respective control groups . MSUS offspring n = 22 , Control offspring n = 24 , one - Student ' s t - ( data reproduced ) P = 0 . 045 , t = 1 . 734 , df = 44 . MSUS serum - injected offspring n = 30 , Control serum - injected offspring n = 31 , two - Mann - Whitney U = 334 . 5 , P = 0 . 059 .

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(b) Weight in adult male MSUS offspring and the offspring of males injected with MSUS serum compared to respective control groups. MSUS offspring n = 22, Control offspring n = 24, one-tailed Student's t-test (data reproduced) P = 0.045, t = 1.734, df = 44. MSUS serum-injected offspring n = 30, Control serum-injected offspring n = 31, two-tailed Mann-Whitney U = 334.5, P = 0.059.


( c , d ) Blood glucose in MSUS offspring offspring of MSUS serum - injected following a 30 - min restraint challenge . MSUS offspring n = 13 , Control offspring n = 12 , repeat measures ANOVA , treatment effect P = 0 . 016 , F ( 1 , 23 ) = 6 . 704 , time effect P < 0 . 0001 , F ( 3 , 69 ) = 53 . 13 , interaction P = 0 . 027 , F ( 3 , 69 ) = 3 . 25 , at 15 - min adjusted P = 0 . 0015 , t = 3 . 693 , df = 92 . MSUS serum - injected offspring n = 17 , Control serum - injected offspring n = 14 , repeat measures ANOVA , treatment effect P = 0 . 023 , F ( 1 , 14 ) = 6 . 493 , time effect P < 0 . 0001 , F ( 3 , 42 ) = 48 . 4 , interaction P = 0 . 29 , F ( 3 , 42 ) = 1 . 29 . Conc . , .

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(c,d) Blood glucose levels in MSUS offspring and the offspring of MSUS serum-injected males following a 30-min restraint challenge. MSUS offspring n = 13, Control offspring n = 12, repeat measures ANOVA, treatment effect P = 0.016, F (1, 23) = 6.704, time effect P < 0.0001, F (3, 69) = 53.13, interaction P = 0.027, F (3, 69) = 3.25, at 15-min adjusted P = 0.0015, t = 3.693, df = 92. MSUS serum-injected offspring n = 17, and Control serum-injected offspring n = 14, repeat measures ANOVA, treatment effect P = 0.023, F (1,14) = 6.493, time effect P < 0.0001, F (3, 42) = 48.4, interaction P = 0.29, F (3, 42) = 1.29. Conc., concentration.


B , C , D . WT , ∆ cpt1 ∆ ept1 ( B ) , Δcho2 ( C ) or Δopi3 ( D ) expressing GFP - Atg8 were grown to logarithmic phase in SD - URA , shifted to SD - N for 4 h of starvation . were harvested at indicated time points subjected to western blotting ( left panel ) . Pgk1 was used as a loading control . Middle panel - Autophagic activity during starvation was quantified by calculating of free GFP to total GFP ( GFP - Atg8 + free GFP ) . Statistical was done by student ' s t - ( paired , two ) ( ns - not significant , * * * * , p ≤ 0 . 0001 ) , error bars represent of at least 3 independent experiments . Right panel - Ape1 maturation was quantified by measuring mApe1 level out of total Ape1 amount , during SD SD - N .

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B, C, D. WT, ∆cpt1∆ept1 (B), Δcho2 (C) or Δopi3 (D) cells expressing GFP-Atg8 were grown to logarithmic phase in SD-URA, and shifted to SD-N for 4 h of starvation. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was used as a loading control. Middle panel- Autophagic activity during starvation was quantified by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by student's t-test (paired, two tailed) (ns- not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount, during SD and SD-N.


E . WT , Δopi3 , Δcho2 as well as Δatg1 ( negative autophagy control ) expressing chromosomally - tagged Fba1 - GFP were grown to log phase in SD , starved in SD - N for 4 hours . were harvested at indicated starvation time points subjected to western blotting ( left panel ) . Pgk1 was monitored as a loading control . Right panel - Autophagic activity during starvation was quantified , by calculating of free GFP to total GFP ( Fba1 - GFP + free GFP ) .

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E. WT, Δopi3, and Δcho2 as well as Δatg1 (negative autophagy control) cells expressing chromosomally-tagged Fba1-GFP were grown to log phase in SD, and starved in SD-N for 4 hours. Cells were harvested at indicated starvation time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Right panel- Autophagic activity during starvation was quantified, by calculating the ratio of free GFP to total GFP (Fba1-GFP + free GFP).


F . WT , Δopi3 , Δcho2 Δatg1 on background of pho8Δ60 pho13Δ , were grown to log phase in SD , starved in SD - N for 4 hours . Pho8 activity was measured by alkaline phosphatase .

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F. WT, Δopi3, Δcho2 and Δatg1 cells on the background of pho8Δ60 pho13Δ strain, were grown to log phase in SD, and starved in SD-N for 4 hours. Pho8 activity was measured by the alkaline phosphatase assay.


A . Representative of GFP - Atg8 ( green ) vacuole ( red , stained with FM4 - 64 ) . WT , Δopi3 Δcho2 expressing GFP - Atg8 were grown to log phase in SD - URA medium shifted to SD - N for 3 h . were observed by confocal microscopy before ( SD ) during nitrogen starvation ( SD - N ) ( left panel ) . Scale bar 1 µm . Right panel - of of with or without GFP inside vacuoles . Statistical was done by Anova multiple comparisons - Sidak ' s , compared to WT ( * * * * , p ≤ 0 . 0001 ) ( ns - not significant ) , error bars represent of at least 3 independent experiments . Number of analyzed for each condition : SD ( WT ( n = 446 ) , Δcho2 ( n = 272 ) , Δopi3 ( n = 272 ) ) , SD - N ( WT ( n = 398 ) , Δcho2 ( n = 224 ) , Δopi3 ( n = 213 ) ) .

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A. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT, Δopi3 and Δcho2 cells expressing GFP-Atg8 were grown to log phase in SD-URA medium and shifted to SD-N for 3 h. Cells were observed by confocal microscopy before (SD) and during nitrogen starvation (SD-N) (left panel). Scale bar 1 µm. Right panel- Quantification of of cells with or without GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001) (ns- not significant), error bars represent SEM of at least 3 independent experiments. Number of cells analyzed for each strain and condition: SD (WT (n=446), Δcho2 (n=272), Δopi3 (n=272)), SD-N (WT (n=398), Δcho2 (n=224), Δopi3 (n=213)).


A , B . WT Δopi3 expressing GFP - Atg8 were grown to log phase in SD - URA , shifted to SD - N for 4 h , choline ( 1 mM ) was not supplemented ( - choline ) or supplemented to SD SD - N ( + choline SD , SD - N ) or only to SD - N ( + choline , SD - N ) as indicated . were harvested at indicated time points subjected to western blotting ( left panel ) . Pgk1 was monitored as a loading control . Middle panel - Autophagic activity was quantified during starvation by calculating of free GFP to total GFP ( GFP - Atg8 + free GFP ) . Statistical was done by Anova multiple comparisons - Sidak ' s , compared to WT ( ns - not significant , * * * * , p ≤ 0 . 0001 ) , error bars represent of at least 3 independent experiments . Right panel - Ape1 maturation was quantified by measuring mApe1 level out of total Ape1 amount in Δopi3 at growth ( SD ) or during starvation ( SD - N ) , with choline supplemented as indicated . Statistical was done by Sidak ' s multiple comparisons compared to WT ( ns - not significant , * , p ≤ 0 . 05 , * * , p ≤ 0 . 005 , * * * * , p ≤ 0 . 0001 ) , error bars represent of at least 3 independent experiments .

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A, B. WT and Δopi3 cells expressing GFP-Atg8 were grown to log phase in SD-URA, and shifted to SD-N for 4 h, choline (1 mM) was not supplemented (-choline) or supplemented to SD and SD-N (+choline SD, SD-N) or only to SD-N (+choline, SD-N) as indicated. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Middle panel- Autophagic activity was quantified during starvation by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (ns-not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount in Δopi3 cells at growth (SD) or during starvation (SD-N), with choline supplemented as indicated. Statistical analysis was done by Sidak's multiple comparisons test compared to WT (ns-not significant, *, p≤0.05, **, p≤0.005, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments.


C . Representative of GFP - Atg8 ( green ) vacuole ( red , stained with FM4 - 64 ) . WT Δopi3 expressing GFP - Atg8 were grown in SD - URA shifted to SD - N . Choline ( 1mM ) was either excluded ( - choline ) , added to growth starvation medium ( + choline SD , SD - N ) or added only to starvation medium ( + choline , SD - N ) . were observed using confocal microscopy during starvation ( left panel ) . Scale bar 1 µm . Right panel - of with GFP inside vacuoles . Statistical was done by Anova multiple comparisons - Sidak ' s , compared to WT ( * * * * , p ≤ 0 . 0001 , ns - not significant ) , error bars represent of 3 independent experiments . Number of analyzed for each condition ; - Choline : WT ( n = 487 ) , Δopi3 ( n = 316 ) , Choline SD , SD - N : WT ( n = 251 ) , Δopi3 ( n = 361 ) , Choline SD - N : WT ( n = 320 ) , Δopi3 ( n = 267 ) . D . PC determined by shotgun lipidomics for WT Δopi3 expressing GFP - Atg8 , with or without choline ( 1 mM ) supplementation to SD - N . Samples were taken after 4 h in SD - N medium .

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

C. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT and Δopi3 cells expressing GFP-Atg8 were grown in SD-URA and shifted to SD-N. Choline (1mM) was either excluded (-choline), added to growth and starvation medium (+choline SD, SD-N) or added only to starvation medium (+choline, SD-N). Cells were observed using confocal microscopy during starvation (left panel). Scale bar 1 µm. Right panel- Quantification of cells with GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001, ns- not significant), error bars represent SEM of 3 independent experiments. Number of cells analyzed for each strain condition; -Choline: WT (n=487), Δopi3 (n=316), Choline SD, SD-N: WT (n=251), Δopi3 (n=361), Choline SD-N: WT (n=320), Δopi3 (n=267). D. PC levels determined by shotgun lipidomics for WT and Δopi3 cells expressing GFP-Atg8, with or without choline (1 mM) supplementation to SD-N. Samples were taken after 4 h in SD-N medium.


A . Representative of Atg5 - mNG with sfTq2 - Atg8 during SD - N . Δatg3 Δatg3Δopi3 were grown to log phase in SD medium , shifted to SD - N for 4 h , observed by Widefield microscopy ( left panel ) , scale bar 1 µm . Right panel - of of with Atg5 during different time points . Statistical was done by Anova multiple comparisons - Sidak ' s ns - not significant ) , error bars represent of 2 independent experiments . Number of analyzed for each time point ( 0 hour : Δatg3 ( n = 296 ) , Δatg3Δopi3 ( n = 111 ) , 1 hour : Δatg3 ( n = 325 ) , Δatg3Δopi3 ( n = 110 ) , 2 hours : Δatg3 ( n = 338 ) , Δatg3Δopi3 ( n = 135 ) , 4 hours : Δatg3 ( n = 309 ) , Δatg3Δopi3 ( n = 167 ) .

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 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

A. Representative Images of Atg5-mNG with sfTq2-Atg8 during SD-N. Δatg3 and Δatg3Δopi3 cells were grown to log phase in SD medium, shifted to SD-N for 4 h, and observed by Widefield microscopy (left panel), scale bar 1 µm. Right panel- quantification of percentage of cells with Atg5 puncta during different time points. Statistical analysis was done by Anova multiple comparisons test- Sidak's ns- not significant), error bars represent SEM of 2 independent experiments. Number of cells analyzed for each strain and time point (0 hour: Δatg3 (n=296), Δatg3Δopi3 (n=111), 1 hour: Δatg3 (n=325), Δatg3Δopi3 (n=110), 2 hours: Δatg3 (n=338), Δatg3Δopi3 (n=135), 4 hours: Δatg3 (n=309), Δatg3Δopi3 (n=167).


C . Representative of WT Δopi3 expressing GFP - Atg8 . were grown to log phase in SD - URA medium , shifted to SD - N for 3 h observed by Airyscan microscopy ( left panel ) . dashes indicate boundaries , yellow dashes indicate phagophores . For each - Magnification of yellow dashed ( top right ) , schematic representation ( bottom right ) . Scale bar 1 µm . Right panel - of of with cup shaped GFP - Atg8 structures imaged by Airyscan microscopy . Statistical was done by student ' s t - ( unpaired ) ( * * p ≤ 0 . 05 ) , error bars represent of least 3 independent experiments . Number of analyzed for each , WT ( n = 45 ) , Δopi3 ( n = 110 ) .

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 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

C. Representative images of WT and Δopi3 cells expressing GFP-Atg8. Cells were grown to log phase in SD-URA medium, shifted to SD-N for 3 h and observed by Airyscan microscopy (left panel). White dashes indicate cell boundaries, yellow dashes indicate phagophores. For each cell - Magnification of yellow dashed area (top right), schematic representation (bottom right). Scale bar 1 µm. Right panel- quantification of the percentage of cells with cup shaped GFP-Atg8 structures imaged by Airyscan microscopy. Statistical analysis was done by student's t-test (unpaired) (** p≤0.05), error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain, WT (n=45), Δopi3 (n=110).


D . Representative CLEM of WT Δopi3 expressing GFP - Atg8 , grown to log phase in SD - URA medium shifted to SD - N for 3h . were harvested , deep frozen processed for CLEM ( left panel ) , V - vacuole . For Δopi3 - Magnification of yellow dashed of GFP - Atg8 positive phagophore ( top right ) , image only ( middle right ) , schematic representation of a phagophore ( bottom right ) . Scale bar 1 µm . Right panel - of of with cup shaped GFP - Atg8 positive cup shaped structures imaged by CLEM . Statistical was done by student ' s t - ( unpaired ) ( * * * p ≤ 0 . 005 ) , error bars represent of least 3 independent experiments . Number of analyzed for each , WT ( n = 20 ) , Δopi3 ( n = 21 ) .

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 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 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

D. Representative CLEM images of WT and Δopi3 cells expressing GFP-Atg8, grown to log phase in SD-URA medium and shifted to SD-N for 3h. Cells were harvested, deep frozen and processed for CLEM (left panel), V-vacuole. For Δopi3 cell - Magnification of yellow dashed area of GFP-Atg8 positive phagophore (top right), TEM image only (middle right), schematic representation of a phagophore (bottom right). Scale bar 1 µm. Right panel- quantification of the percentage of cells with cup shaped GFP-Atg8 positive cup shaped membrane structures imaged by CLEM. Statistical analysis was done by student's t-test (unpaired) (*** p≤0.005), error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain, WT (n=20), Δopi3 (n=21).


A . Representative of Δypt7 , Δopi3Δypt7 expressing GFP - Atg8 ( z - stack projection ) . were grown to log phase in SD - URA medium starved in SD - N , were taken during starvation in indicated time points by widefield microscopy ( left panel ) . Scale bar 1 µm . Right panel - of GFP - Atg8 per in Δypt7 , Δopi3Δypt7 at indicated time points . Statistical was done by Anova multiple comparisons - Sidak ' s ( * * * * , p ≤ 0 . 0001 , ns - not significant ) , compared to WT . Error bars represent of least 3 independent experiments . Number of analyzed for each time point , 0 hours : Δypt7 ( n = 322 ) , Δopi3Δypt7 ( n = 194 ) , 1 . 5 hours : Δypt7 ( n = 220 ) , Δopi3Δypt7 ( n = 155 ) , 3 . 5 hours Δypt7 ( n = 216 ) , Δopi3Δypt7 ( n = 307 ) . B . of cup - shaped phagophores in Δypt7 Δopi3Δypt7 expressing GFP - Atg8 during starvation ( 1 - 3h ) . Statistical was done by students t - ( * * * , p ≤ 0 . 001 ) , error bars represent from at least 3 independent experiments . Number of analyzed for each , Δypt7 ( n = 347 ) , Δopi3Δypt7 ( n = 474 ) .

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 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 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

A. Representative images of Δypt7, Δopi3Δypt7 expressing GFP-Atg8 (z-stack projection). Cells were grown to log phase in SD-URA medium and starved in SD-N, images were taken during starvation in indicated time points by widefield microscopy (left panel). Scale bar 1 µm. Right panel- quantification of GFP-Atg8 puncta per cell in Δypt7, Δopi3Δypt7 at indicated time points. Statistical analysis was done by Anova multiple comparisons test- Sidak's (****, p≤0.0001, ns- not significant), compared to WT. Error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain and time point, 0 hours: Δypt7 (n=322), Δopi3Δypt7 (n=194), 1.5 hours: Δypt7 (n=220), Δopi3Δypt7 (n=155), 3.5 hours Δypt7 (n=216), Δopi3Δypt7 (n=307). B. Quantification of cup-shaped phagophores in Δypt7 and Δopi3Δypt7 cells expressing GFP-Atg8 during starvation (1-3h). Statistical analysis was done by students t-test (***, p≤0.001), error bars represent SEM from at least 3 independent experiments. Number of cells analyzed for each strain, Δypt7 (n= 347), Δopi3Δypt7 (n= 474).


C . Representative of mNG - tagged Atg8 colocalized with mScarletI - PXVam7 . WT , Δopi3 , Δatg1 Δopi3Δatg1 on background of Δymr1 , expressing mScarletI - PXVam7 under CUP1 promoter from knocked - out VPS38 locus , were grown to log phase in SD medium in presence of 10 µM copper sulfate , shifted to SD - N in presence of 10 µM copper sulfate . were taken during SD - N ( 1 - 3 h ) by widefield microscopy . D . of autophagic structure diameters in WT Δopi3 on background of Δymr1Δvps38 ( C ) by ImageJ . Statistical was done by student ' s t - , unpaired , two sided ( ns - not significant ) , error bars represent of at least three independent experiments . Number of analyzed for each , WT ( n = 51 ) , Δopi3 ( n = 28 ) .

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

C. Representative images of mNG-tagged Atg8 colocalized with mScarletI-PXVam7. WT, Δopi3, Δatg1 and Δopi3Δatg1 cells on the background of Δymr1, expressing mScarletI-PXVam7 under the CUP1 promoter from the knocked-out VPS38 locus, were grown to log phase in SD medium in the presence of 10 µM copper sulfate, and shifted to SD-N in the presence of 10 µM copper sulfate. Images were taken during SD-N (1-3 h) by widefield microscopy. D. Quantification of autophagic structure diameters in WT and Δopi3 cells on the background of Δymr1Δvps38 (C) by ImageJ. Statistical analysis was done by student's t-test, unpaired, two sided (ns- not significant), error bars represent SEM of at least three independent experiments. Number of cells analyzed for each strain, WT (n=51), Δopi3 (n=28).


E . Representative of Δypt7 , Δopi3Δypt7 expressing GFP - Atg8 ( projection ) . were grown to log phase in SD - URA medium starved in SD - N , with supplementation of choline ( 1 mM ) to SD SD - N as indicated . were taken during growth starvation by confocal microscopy ( left panel ) . Scale bar 1 µm . Right panel - of with more than two GFP - Atg8 , in Δypt7 Δopi3Δypt7 at growth ( SD ) or starvation ( SD - N ) in presence ( + choline : SD , SD - N ) or absence ( - choline ) of choline ( 1 mM ) . Incidence were normalized to Δypt7 ( SD - N ) control . Statistical was done by Anova multiple comparisons - Sidak ' s ( * * * , p ≤ 0 . 001 ) . Error bars represent of least 3 independent experiments . Number of analyzed for each condition , SD : Δypt7 ( n = 209 ) , Δopi3Δypt7 ( n = 111 ) , SD - N : Δypt7 ( n = 118 ) , Δopi3Δypt7 ( n = 103 ) , SD + Choline : Δypt7 ( n = 107 ) , Δopi3Δypt7 ( n = 120 ) , Δypt7 ( n = 84 ) , Δopi3Δypt7 ( n = 74 ) .

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

E. Representative images of Δypt7, Δopi3Δypt7 cells expressing GFP-Atg8 (projection). Cells were grown to log phase in SD-URA medium and starved in SD-N, with supplementation of choline (1 mM) to SD and SD-N as indicated. Images were taken during growth and starvation by confocal microscopy (left panel). Scale bar 1 µm. Right panel- quantification of cell with more than two GFP-Atg8 puncta, in Δypt7 and Δopi3Δypt7 at growth (SD) or starvation (SD-N) in the presence (+choline: SD, SD-N) or absence (-choline) of choline (1 mM). Incidence were normalized to Δypt7 (SD-N) control. Statistical analysis was done by Anova multiple comparisons test- Sidak's (***, p≤0.001). Error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain and condition, SD: Δypt7 (n=209), Δopi3Δypt7 (n=111), SD-N: Δypt7 (n=118), Δopi3Δypt7 (n=103), SD+Choline: Δypt7 (n=107), Δopi3Δypt7 (n=120), Δypt7 (n=84), Δopi3Δypt7 (n=74).


Overexpression of USP36 promotes SUMOylation in . Whole lysates ( WCL ) from H1299 ( A ) , U2OS HeLa ( B ) transfected with control empty vector or Flag - USP36 were assayed by IB .

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

Overexpression of USP36 promotes SUMOylation in cells. Whole cell lysates (WCL) from H1299 (A), U2OS and HeLa (B) cells transfected with control empty vector or Flag-USP36 were assayed by IB.


H1299 transfected with WT USP36 or indicated mutants His - SUMO2 were subjected to Ni2 + - NTA agarose beads pull down ( PD ) followed by IB using anti - SUMO2 / 3 antibody .

O 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 B-MEASURED_VAR O O O O

H1299 cells transfected with WT USP36 or the indicated mutants and His-SUMO2 were subjected to Ni2+-NTA agarose beads pull down (PD) followed by IB using anti-SUMO2/3 antibody.


USP36 interacts with Ubc9 in . H1299 transfected with indicated plasmids were subjected to co - IP with anti - Flag antibody followed by IB .

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

USP36 interacts with Ubc9 in cells. H1299 cell transfected with the indicated plasmids were subjected to co-IP with anti-Flag antibody followed by IB.


USP36 SUMOylates PARP1 in vitro . Recombinant T7 - PARP1 ( 0 . 1 μM ) was incubated with SUMO E1 ( 30 nM ) , Ubc9 ( 50 nM ) , SUMO2 ( 4 μM ) in presence of USP361 - 800 ( 50 nM ) / or ATP ( 2 . 5 mM ) at 30ºC for 5 hours then assayed by IB .

B-MEASURED_VAR 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 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

USP36 SUMOylates PARP1 in vitro. Recombinant T7-PARP1 protein (0.1 μM) was incubated with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the presence of USP361-800 (50 nM) and/or ATP (2.5 mM) at 30ºC for 5 hours and then assayed by IB.


USP36 SUMOylates Nhp2 Nop58 in vitro . In vitro SUMOylation assays were performed by incubating recombinant Nhp2 ( F , G ) or Nop58 ( H ) ( 0 . 1 μM ) with SUMO E1 ( 30 nM ) , Ubc9 ( 50 nM ) , SUMO2 ( 4 μM ) in absence or presence of USP361 - 800 ( 50 nM , 1x ; 100 nM , 2x ) / or ATP ( 2 . 5 mM ) at 37ºC for two hours or indicated times . reactions were assayed by IB using anti - Nhp2 anti - Nop58 antibodies , respectively .

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

USP36 SUMOylates Nhp2 and Nop58 in vitro. In vitro SUMOylation assays were performed by incubating recombinant Nhp2 (F, G) or Nop58 (H) (0.1 μM) with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the absence or presence of USP361-800 (50 nM, 1x; 100 nM, 2x) and/or ATP (2.5 mM) at 37ºC for two hours or the indicated times. The reactions were assayed by IB using anti-Nhp2 and anti-Nop58 antibodies, respectively.


USP36 does not change of endogenous snoRNP . HeLa infected with scrambled or indicated USP36 shRNA lentiviruses were assayed by IB .

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

USP36 does not change the levels of endogenous snoRNP proteins. HeLa cells infected with scrambled or the indicated USP36 shRNA lentiviruses were assayed by IB.


USP36 promotes binding of Nhp2 to snoRNAs . H1299 transfected with Flag - Nhp2 V5 - USP36 individually or together were assayed by - IP with anti - Flag or control mouse IgG , followed by RT - qPCR detection of indicated box H / ACA snoRNAs ( C ) . Shown is one representative experiment of three independent experiments . Data were presented as mean ± SD , n = 3 technical replicates . * P < 0 . 05 ; * * P < 0 . 01 , compared to Nhp2 alone ( Student ' s t - ) .

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

USP36 promotes the binding of Nhp2 to snoRNAs. H1299 cells transfected with Flag-Nhp2 and V5-USP36 individually or together were assayed by RNA-IP with anti-Flag or control mouse IgG, followed by RT-qPCR detection of the indicated box H/ACA snoRNAs (C). Shown is one representative experiment of three independent experiments. Data were presented as mean ± SD, n=3 technical replicates. *P<0.05; **P<0.01, compared to Nhp2 alone (Student's t-test).


( B ) . USP36 is required for rRNA processing . HeLa transfected with scr or USP36 siRNA ( left panels ) or infected with scr or USP36 shRNA lentiviruses ( right panels ) were assayed for rRNA processing by Northern blot using 5 ' - ETS , ITS1 ITS2 probes as indicated .

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 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

(B). USP36 is required for rRNA processing. HeLa cells transfected with scr or USP36 siRNA (left panels) or infected with scr or USP36 shRNA lentiviruses (right panels) were assayed for rRNA processing by Northern blot using 5'- ETS, ITS1 and ITS2 probes as indicated.


Knockdown of USP36 inhibits translation . HeLa infected with scr or USP36 shRNA lentiviruses were incubated with O - propargyl - puromycin ( OPP ) followed by Click - iT OPP synthesis assays . Representative ( C ) from three independent experiments ( D ) . Data were presented as mean ± SD , n = 3 biological replicates . * * * P < 0 . 001 , compared to scr control ( Student ' s t - ) . Scale bar = 50 μM .

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Knockdown of USP36 inhibits translation. HeLa cells infected with scr or USP36 shRNA lentiviruses were incubated with O-propargyl-puromycin (OPP) followed by Click-iT OPP protein synthesis assays. Representative images (C) and quantification from three independent experiments (D). Data were presented as mean ± SD, n=3 biological replicates. ***P<0.001, compared to scr control (Student's t-test). Scale bar = 50 μM.


( A ) proliferation for PC3 transfected with siSCR or siCLU monitored in real time for 60h with SP Dynamic Xcelligence system ( Voltage 2 . 5 K ) . Error bars represent mean ± , n = 4 . Slopes compared by ANCOVA , p < 0 . 0001 .

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(A) Cell proliferation for PC3 cells transfected with siSCR or siCLU monitored in real time for 60h with SP Dynamic Xcelligence system (Voltage 2.5 K). Error bars represent mean ± SEM, n=4. Slopes compared by ANCOVA test, p<0.0001.


( B ) Left panel : cycle profile of PC3 transfected with siSCR or siCLU . Right panel : of variation in G1 , S G2 / M phases . Error bars represent mean ± , n = 3 , * * p < 0 . 01 by paired Student ' s t - . ( G2 / M population p = 0 . 0058 ; G1 p = 0 . 008 . ) . Inset : Western blot with CLU antibody in PC3 after CLU silencing . Vinculin was used as loading control .

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(B) Left panel: Cell cycle profile of PC3 cells transfected with siSCR or siCLU. Right panel: Percentage quantification of variation in G1, S and G2/M phases. Error bars represent mean ± SEM, n=3, **p<0.01 by paired Student's t-test. (G2/M population p= 0.0058; G1 p=0.008.). Inset: Western blot with CLU antibody in PC3 cells after CLU silencing. Vinculin was used as loading control.


( C ) Left panel : - Microarray comparing PC3 transfected with siCLU versus siSCR Kinexus phosphokinome microarray comparing LNCaP - transfected with siCLU versus siSCR . Target genes expression is shown as fold change relative to siSCR , p ≤ 0 . 05 by an unpaired t - for genes listed . Central panel : Venn diagram to graphically illustrate overlap of mRNA involved in regulation of mitosis in both transcriptome phosphokinome . comparison of these 2 experiments shows modification of 4 mitosis regulators . Right panel : Western blot validating expression of target .

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(C) Left panel: RNA-Microarray analysis comparing PC3 cells transfected with siCLU versus siSCR and Kinexus phosphokinome microarray analysis comparing LNCaP-transfected with siCLU versus siSCR. Target genes expression is shown as fold change relative to siSCR, p ≤0.05 by an unpaired t-test for all the genes listed. Central panel: Venn diagram to graphically illustrate the overlap of mRNA and proteins involved in the regulation of mitosis in both transcriptome and phosphokinome analysis. The comparison of these 2 experiments shows modification of 4 mitosis regulators. Right panel: Western blot validating the expression levels of target proteins.


( A ) Real - time PCR for CLU Cdc25C in three different prostate cancer lines . Target genes expression was calculated relative to GAPDH normalized to siSCR . Error bars represent mean ± SD , n = 3 .

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(A) Real-time PCR for CLU and Cdc25C in three different prostate cancer cell lines. Target genes expression was calculated relative to GAPDH and normalized to siSCR. Error bars represent mean ± SD, n=3.


( B ) Western blot for CLU Cdc25C after siSCR or si CLU transfection in same prostate cancer lines as in A . Vinculin was used as loading control .

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(B) Western blot for CLU and Cdc25C after siSCR or si CLU transfection in the same prostate cancer cell lines as in A. Vinculin was used as loading control.


( C ) Western blot for CLU Cdc25C in PC3 after transfection with siSCR or siCLU followed by transfection with CLU or empty expression plasmid for additional 48h . Vinculin was used as loading

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(C) Western blot for CLU and Cdc25C in PC3 cells after transfection with siSCR or siCLU followed by transfection with CLU or empty expression plasmid for additional 48h. Vinculin was used as loading control


( A ) Upper panel : Western blot for CLU Cdc25C from LNCaP xenografts harvested from mice treated with OGX - 011 ( n = 8 ) or SCR ASO control ( n = 6 ) . Vinculin was used as loading control . Lower panel : of , relative to loading control , reported as dot plots . * p < 0 . 05 by t - followed by Welch ' s correction ( CLU p = 0 . 049 , Cdc25C p = 0 . 024 ) .

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(A) Upper panel: Western blot for CLU and Cdc25C from LNCaP xenografts harvested from mice treated with OGX-011 (n=8) or SCR ASO control (n=6). Vinculin was used as loading control. Lower panel: Quantification of the protein levels, relative to the loading control, reported as dot plots. *p<0.05 by t-test followed by Welch's correction (CLU p= 0.049, Cdc25C p= 0.024).


( B ) Western blot with correspondent of ( left ) real time PCR ( right ) from untreated LNCaP xenografts harvested at endpoint . negative correlation between CLU - Cdc25C - ΔΔCT values was calculated using a spearman ( rho = - 0 . 86 ; p = 0 . 0107 ) .

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(B) Western blot with the correspondent quantification of protein levels (left) and real time PCR (right) from untreated LNCaP xenografts harvested at endpoint. The negative correlation between CLU-RNA and Cdc25C-RNA ΔΔCT values was calculated using a spearman test (rho=-0.86; p=0.0107).


( C ) Immunoistochemistry for CLU Cdc25C of radical prostatectomy samples from historical control specimens with no neoadjuvant therapy ( n = 5 ) or neoadjuvant hormone therapy , ( NHT ) ( n = 5 ) or combination of NHT OGX - 011 ( n = 9 ) for 3 months prior surgery . Average score from 3 groups ( left ) representative sections for CLU Cdc25C ( right ) . Error bars represent mean ± . CLU : * p < 0 . 05 ; untreated versus 3mNHT ( p = 0 . 0213 ) ; 3mNHT versus 3mNHT + OGX - 011 ( p = 0 . 012 ) by ANOVA followed by Bonferroni post hoc . cases were normalized by clinical stage , Gleason score serum PSA . For each , pathologist selected with highest Gleason score . Scale bar represents 100

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(C) Immunoistochemistry staining for CLU and Cdc25C of radical prostatectomy samples from historical control specimens with no neoadjuvant therapy (n=5) or neoadjuvant hormone therapy, (NHT) (n=5) or the combination of NHT and OGX-011 (n=9) for 3 months prior surgery. Average score staining from the 3 groups (left) and representative sections for CLU and Cdc25C (right). Error bars represent mean ± SEM. CLU: *p<0.05; untreated versus 3mNHT (p=0.0213); 3mNHT versus 3mNHT +OGX-011 (p=0.012) by ANOVA followed by Bonferroni post hoc analysis. All cases were normalized by clinical stage, Gleason score and serum PSA. For each patient, the pathologist selected the area with the highest Gleason score. Scale bar represents 100 µm


( A ) Co - immunoprecipitation from parental PC3 transfected with CLU Cdc25C plasmid expression vectors . were immunoprecipitated with specific anti - Cdc25C ( left ) CLU ( right ) antibodies analyzed by immunoblotting .

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(A) Co-immunoprecipitation from parental PC3 cells transfected with CLU and Cdc25C plasmid expression vectors. Proteins were immunoprecipitated with specific anti-Cdc25C (left) and CLU (right) antibodies and analyzed by immunoblotting.


( B ) Dual immunofluorescence with Cdc25C ( green ) CLU ( red ) antibodies in PC3 . Confocal microscopy was used to identify interaction ( arrows ) . was counterstained with ( DAPI ) ( blue ) . Scale bar represent 10 µm .

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(B) Dual immunofluorescence staining with Cdc25C (green) and CLU (red) antibodies in PC3 cells. Confocal microscopy was used to identify the interaction (white arrows). DNA was counterstained with (DAPI) (blue). Scale bar represent 10 µm.


( C ) Duolink proximity ligation between Cdc25C CLU in PC3 . Confocal microscopy was used to detect interaction ( red dots ) . was counterstained with DAPI ( blue ) . PC3 transfected with siCLU were used as a negative control . Scale bar represent 10 µm .

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(C) Duolink proximity ligation assay between Cdc25C and CLU in PC3 cells. Confocal microscopy was used to detect the interaction (red dots). DNA was counterstained with DAPI (blue). PC3 cells transfected with siCLU were used as a negative control. Scale bar represent 10 µm.


( A ) Left panel : Western blot for CLU , Cdc25C , Cdc25C - T48 , Cdc25C S216 Cdc25C S198 in PC - 3 after siSCR or siCLU transfection , synchronization nocodazole release at indicated time points . Vinculin was used as loading control . Right panel : Immunofluorescence microscopy for Cdc25C - T48 in PC3 after siSCR or siCLU transfection . was counterstained with ( DAPI ) ( blue ) . Scale bar represents 10 µm .

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(A) Left panel: Western blot for CLU, Cdc25C, Cdc25C-T48, Cdc25C S216 and Cdc25C S198 in PC-3 cells after siSCR or siCLU transfection, synchronization and nocodazole release at indicated time points. Vinculin was used as loading control. Right panel: Immunofluorescence microscopy for Cdc25C-T48 in PC3 cells after siSCR or siCLU transfection. DNA was counterstained with (DAPI) (blue). Scale bar represents 10 µm.


( B ) Western blot for CLU , Cdc25C - T48 , Cdk1 PP2A in PC3 after siSCR or siCLU transfection after synchronization followed by treatment with or without okadaic acid ( OA ) ( 100 nM ) for 12 hours . were extracted 15 min after nocodazole release . Vinculin was used as loading control .

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(B) Western blot for CLU, Cdc25C-T48, Cdk1 and PP2A in PC3 cells after siSCR or siCLU transfection and after synchronization followed by treatment with or without okadaic acid (OA) (100 nM) for 12 hours. Proteins were extracted 15 min after nocodazole release. Vinculin was used as loading control.


( C ) Western blot for PP2A , CLU , Cdc25C - T48 , Cdc25C , Wee1 Cdk1 in PC3 transfected with siSCR or siPP2A synchronized nocodazole released at indicated time points . Vinculin was used as loading control .

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(C) Western blot for PP2A, CLU, Cdc25C-T48, Cdc25C, Wee1 and Cdk1 in PC3 cells transfected with siSCR or siPP2A synchronized and nocodazole released at indicated time points. Vinculin was used as loading control.


( D ) PP2A phosphatase activity measurement in PC3 after siSCR or siCLU transfection , synchronization nocodazole release at indicated time points . Error bar represent mean ± , n = 3 , * * p < 0 . 01 ( p = 0 . 0025 ) by ANOVA followed by Bonferroni ' s post hoc comparisons . treated with no peptide were used as a negative control .

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(D) PP2A phosphatase activity measurement in PC3 cells after siSCR or siCLU transfection, synchronization and nocodazole release at indicated time points. Error bar represent mean ± SEM, n=3, ** p<0.01 (p=0.0025) by ANOVA followed by Bonferroni's post hoc comparisons. Cells treated with no peptide were used as a negative control.


( A ) Western blot for CLU , Cdk1 Y15 , Cdk1 Wee1 in PC - 3 after siSCR or siCLU transfection followed by synchronization nocodazole release at indicated time points . Vinculin was used as loading

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(A) Western blot for CLU, Cdk1 Y15, Cdk1 and Wee1 in PC-3 cells after siSCR or siCLU transfection followed by synchronization and nocodazole release at indicated time points. Vinculin was used as loading control


( B ) Wee1 expression level was evaluated in PC3 after CLU silencing by real - time PCR . Error bar represent mean ± , n = 3 , * * * p < 0 . 001 ( p = 0 . 0001 ) by unpaired t - followed by Welch ' s correction .

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(B) Wee1 expression level was evaluated in PC3 cells after CLU silencing by real-time PCR. Error bar represent mean ± SEM, n=3, *** p<0.001 (p=0.0001) by unpaired t-test followed by Welch's correction.


( C ) FACS showing of PC3 positive for phosphohistone H3 after transfection with siSCR or siCLU in presence absence of Wee1 inhibitor , MK - 1775 . Error bar represent mean ± , n = 3 , * * * * p < 0 . 0001 by Mann -

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(C) FACS analysis showing percentage of the PC3 cells positive for phosphohistone H3 after transfection with siSCR or siCLU in presence and absence of Wee1 inhibitor, MK-1775. Error bar represent mean ± SEM, n=3, ****p<0.0001 by Mann-Whitney


( D ) Western blot for CLU , Wee1 , Cdk1 Y15 , Cdk1 Cyclin B1 in PC3 after transfection with siSCR or siCLU followed by synchronization treatment with or without MK - 1775 , a specific Wee1 inhibitor , for 12 hours . were harvested at different time points after nocodazole release . Vinculin was used as loading control .

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(D) Western blot for CLU, Wee1, Cdk1 Y15, Cdk1 and Cyclin B1 in PC3 cells after transfection with siSCR or siCLU followed by synchronization and treatment with or without MK-1775, a specific Wee1 inhibitor, for 12 hours. Cells were harvested at different time points after nocodazole release. Vinculin was used as loading control.


( A ) Cabazitaxel IC50 calculation using WST1 in PC3 after transfection with siSCR or siCLU ( left ) , SCR or OGX - 011 ( right ) followed by 72h of cabazitaxel treatment . of surviving was calculated relative to control . Error bar represent mean ± , n = 3 . Inset : Western blot was performed to verify CLU knockdown in .

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(A) Cabazitaxel IC50 calculation using WST1 assay in PC3 cells after transfection with siSCR or siCLU (left), and SCR or OGX-011 (right) followed by 72h of cabazitaxel treatment. Percentage of surviving cells was calculated relative to control. Error bar represent mean ± SEM, n=3. Inset: Western blot was performed to verify CLU knockdown in cells.


( B ) Mitotic in mitotic catastrophe were counted using DAPI in PC - 3 after SCR or OGX - 011 transfection followed by different of cabazitaxel as indicated ( > 1000 ) Error bar represent mean ± . , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by Student ' s t - . ( caba 1 . 5 nM p = 0 . 00115 ; 3 nM p = 0 . 0105 ; 4 . 5 nM p = 1 . 93x10 - 7 , ; 6 . 25 nM p = 8 . 9x10 - 9 ; 12 . 5 nM p = 6 . 8x10 - 9 ; 25 nM p = 1 . 2x10 - 9 ) .

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(B) Mitotic cells and cells in mitotic catastrophe were counted using DAPI in PC-3 cells after SCR or OGX-011 transfection followed by different concentration of cabazitaxel as indicated (>1000 counts) Error bar represent mean ± SEM.,*p<0.05, ***p<0.001, ****p<0.0001 by Student's t-test.( caba 1.5 nM p=0.00115 ; 3 nM p=0.0105 ; 4.5 nM p=1.93x10-7, ; 6.25 nM p=8.9x10-9; 12.5 nM p=6.8x10-9; 25 nM p=1.2x10-9).


( C ) Clonogenic in PC - 3 after SCR or OGX - 011 transfection followed by treatment with different concentrations of cabazitaxel as indicated . number of survival clones was assessed after 15 days using crystal violet . Error bars represent mean ± n = 3 , * p < 0 . 05 , * * p < 0 . 01 by Mann - Whitney ( 0 nM caba p = 0 . 039 ; 0 . 5 nM p = 0 . 0079 ; 2 nM p = 0 . 0079 )

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(C) Clonogenic assay in PC-3 cells after SCR or OGX-011 transfection followed by treatment with different concentrations of cabazitaxel as indicated. The number of survival clones was assessed after 15 days using crystal violet assay. Error bars represent mean ± SEM n=3,*p<0.05, **p<0.01 by Mann-Whitney (0 nM caba p=0.039; 0.5 nM p=0.0079; 2 nM p=0.0079)


( A ) cycle distribution of syncronized PC3 PC3 - R - caba 60 min after nocodazole release at indicated time points .

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(A) Cell cycle distribution of syncronized PC3 and PC3-R-caba cells 60 min after nocodazole release at indicated time points.


( B ) Western blot for CLU , Cdc25C , Cdc25C - T48 , Cdk1 , Cdk1 - Y15 Wee1 in IGRCaP - 1 PC3 parental ( WT ) or cabazitaxel - resistant ( R - caba ) as well as for PC3 - R - caba transfected with siSCR or siCLU . Vinculin was used as loading control .

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(B) Western blot for CLU, Cdc25C, Cdc25C-T48, Cdk1, Cdk1-Y15 and Wee1 in IGRCaP-1 and PC3 parental (WT) or cabazitaxel-resistant (R-caba) cells as well as for PC3-R-caba cells transfected with siSCR or siCLU. Vinculin was used as loading control.


( C ) survival measured using WST1 in PC3 - R - caba after SCR or OGX - 011 transfection treatment with 10 nM cabazitaxel for 48h . Error bar represent mean ± , n = 3 , * * * p < 0 . 001 by one way ANOVA followed by Bonferroni ' s post hoc correction .

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(C) Cell survival measured using WST1 in PC3-R-caba cells after SCR or OGX-011 transfection and treatment with 10 nM cabazitaxel for 48h. Error bar represent mean ± SEM, n=3, *** p<0.001 by one way ANOVA followed by Bonferroni's post hoc correction.


( D ) survival measured using WST1 in PC3 resistant after transfection with siSCR or siCLU followed by treatment with cabazitaxel ( 10 nM ; C10 ) , MK - 1775 ( 10 nM ; MK10 ) or both for 72 hours . . Error bar represent mean ± , n = 3 , * * * p < 0 . 001 by one way ANOVA followed by Bonferroni ' s post hoc correction .

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(D) Cell survival measured using WST1 in PC3 resistant cells after transfection with siSCR or siCLU followed by treatment with cabazitaxel (10 nM; C10), MK-1775 (10 nM; MK10) or both for 72 hours. .Error bar represent the mean ± SEM, n=3, *** p<0.001 by one way ANOVA followed by Bonferroni's post hoc correction.


( E ) PARP cleavage was assessed by western blot in treated as in D its relative expression was quantified by densitometry using ImageJ . Vinculin was used as loading

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(E) PARP cleavage was assessed by western blot in cells treated as in D and its relative expression was quantified by densitometry using ImageJ. Vinculin was used as loading control


( F ) Left panel : of tumour volume over 52 days in nude mice xenotransplanted with PC3 - docetaxel resistant treated with either MK - 1775 ( M , black ) , MK - 1775 micellar taxane ( MT , blue ) , micellar taxane OGX - 011 ( TO , red ) or MK - 1775 , micellar taxane OGX - 011 ( MTO , green ) . error bars indicate mean ± , n = 8 . Right panel : Slopes of growth between groups , were compared by ANOVA followed by Dunnett ' s post hoc . . * p < 0 . 05 , * * * p < 0 . 001 . p = 0 . 0488 ( M to TO ) ; p = 0 . 0007 ( M to MTO ) .

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(F) Left panel: Quantification of tumour volume over 52 days in nude male mice xenotransplanted with PC3-docetaxel resistant cells and treated with either MK-1775 (M, black line), MK-1775 and micellar taxane (MT, blue line), micellar taxane and OGX-011 (TO, red line) or MK-1775, micellar taxane and OGX-011 (MTO, green line). The error bars indicate mean ± SEM, n=8. Right panel: Slopes of tumor growth between groups, were compared by ANOVA followed by Dunnett's post hoc analysis..*p<0.05, ***p<0.001 .p=0.0488 (M to TO); p=0.0007 (M to MTO).


A . Identification of FASTKD2 interacting by AP - MS BioID . 26 high confidence interactors detected by both techniques are listed on right .

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A. Identification of FASTKD2 interacting proteins by AP-MS and BioID. 26 high confidence interactors detected by both techniques are listed on the right.


B . Prey specificity graph for BioID interactome of TRUB2 . Prey specificity was calculated as relative enrichment of interaction of preys TRUB2 , compared to their interaction with 138 other baits ( 42 mitochondrial baits , 96 baits from other compartments ) .

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B. Prey specificity graph for BioID interactome of TRUB2 protein. Prey specificity was calculated as the relative enrichment of interaction of individual preys and TRUB2, compared to their interaction with 138 other baits (42 mitochondrial baits, 96 baits from other cellular compartments).


A . Immunoblot of indicated in control siRNA treated .

A. Immunoblot analysis of indicated proteins in control and siRNA treated cells.


B . BN - PAGE of siRNA - mediated depletion shows an OXPHOS defect as revealed by subunit - specific antibodies against OXPHOS complexes . C . of of OXPHOS complexes normalized to II . graph represents relative abundance of complexes in treated with specified siRNA versus controls . bars represent mean ± of 3 - 7 independent experiments . P - values were calculated using paired two - t - ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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B. BN-PAGE analysis of siRNA-mediated depletion shows an OXPHOS defect as revealed by subunit-specific antibodies against individual OXPHOS complexes.C. Quantification of the levels of individual OXPHOS complexes normalized to Complex II levels. The graph represents the relative abundance of individual complexes in cells treated with the specified siRNA versus controls. The bars represent the mean ± SEM of 3-7 independent experiments. P-values were calculated using paired two-tailed t-test (* P<0.05; ** P<0.01; ***P<0.001).


D , E . - labeling mitochondrial translation experiment of 13 mitochondria - encoded polypeptides ( seven subunits of I [ ND ] , three subunits of IV [ COX ] , two subunits of V [ ATP ] one subunit of III [ cyt b ] ) in control siRNA treated . ( E ) Detail of a mitochondrial - labeling experiment showing a severe decrease in translation of ATP6 ATP8 in TRUB2 depleted .

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D, E. Pulse-labeling mitochondrial translation experiment of the 13 mitochondria-encoded polypeptides (seven subunits of complex I [ND], three subunits of complex IV [COX], two subunits of complex V [ATP] and one subunit of complex III [cyt b]) in control and siRNA treated cells. (E) Detail of a mitochondrial pulse-labeling experiment showing a severe decrease in the translation of ATP6 and ATP8 in TRUB2 depleted cells.


A , B . Identification of mitochondrial ribosomal pseudouridine synthase interacting by sucrose gradient centrifugation in control ( A ) fractions were separated by SDS - PAGE immunoblotted with indicated antibodies . migration of mt - SSU ( 28S ) , mt - LSU ( 39S ) mitochondrial monosome ( 55S ) are shown .

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A, B. Identification of mitochondrial ribosomal proteins and pseudouridine synthase interacting proteins by sucrose gradient centrifugation in control cells (A) Individual fractions were separated by SDS-PAGE and immunoblotted with the indicated antibodies. The migration of the mt-SSU (28S), the mt-LSU (39S) and the mitochondrial monosome (55S) are shown.


A , B . Identification of mitochondrial ribosomal pseudouridine synthase interacting by sucrose gradient centrifugation in treated with siRNA ( B ) . fractions were separated by SDS - PAGE immunoblotted with indicated antibodies . migration of mt - SSU ( 28S ) , mt - LSU ( 39S ) mitochondrial monosome ( 55S ) are shown .

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A, B. Identification of mitochondrial ribosomal proteins and pseudouridine synthase interacting proteins by sucrose gradient centrifugation in cells treated with siRNA (B). Individual fractions were separated by SDS-PAGE and immunoblotted with the indicated antibodies. The migration of the mt-SSU (28S), the mt-LSU (39S) and the mitochondrial monosome (55S) are shown.


C . of of mt - SSU ( 28S ) , mt - LSU ( 39S ) mitochondrial monosome ( 55S ) normalized to SDHA . graph represents relative abundance of subunits in treated with specified siRNA versus controls . bars represent mean ± .

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C. Quantification of the levels of the mt-SSU (28S), the mt-LSU (39S) and the mitochondrial monosome (55S) normalized to the SDHA levels. The graph represents the relative abundance of individual subunits in cells treated with specified siRNA versus controls. The bars represent the mean ± SEM.


D . 2D - immunoblot ( BN - PAGE / SDS - PAGE ) of pseudouridine synthase interacting . migration ( sizes in kDa ) of known complexes in first dimension is indicated on top of blot .

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D. 2D-immunoblot analysis (BN-PAGE/SDS-PAGE) of pseudouridine synthase interacting proteins. The migration (sizes in kDa) of known protein complexes in the first dimension is indicated on the top of the blot.


B . of Δ ( difference between rate of deletions in CMC treated vs un - treated ) for ψ sites in control siRNA treated . bars represent mean ± of two independent experiments . P - values were calculated using a one - t - ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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B. Quantification of Δ (the difference between the rate of deletions in CMC treated vs un-treated cells) for individual ψ sites in control and siRNA treated cells. The bars represent the mean ± SEM of two independent experiments. P-values were calculated using a one-tailed t-test (* P<0.05; ** P<0.01; ***P<0.001).


SILAC - proteomics data identify known ( red ) & novel ( green ) hypoxia - induced in SW620 . One sample T - was performed .

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SILAC-based proteomics data identify known (red) & novel (green) hypoxia-induced proteins in SW620 cells. One sample T-test was performed.


Western blotting confirmed GPRC5A as a hypoxia - induced in SILAC lysates .

Western blotting confirmed GPRC5A as a hypoxia-induced protein in SILAC lysates.


Validation of GPRC5A western blot data using siRNA . * Non - specific band of ~ 60kDa not depleted by GPRC5A siRNA .

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Validation of GPRC5A western blot data using siRNA. *Non-specific band of ~60kDa not depleted by GPRC5A siRNA.


Confocal microscopy showing plasma GPRC5A expression in hypoxic SW620 ( scale bars : 75um ) .

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Confocal microscopy showing plasma membrane GPRC5A expression in hypoxic SW620 cells (scale bars: 75um).


Western blotting showing GPRC5A upregulation by hypoxia in a panel of colorectal tumour lines .

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Western blotting showing GPRC5A upregulation by hypoxia in a panel of colorectal tumour cell lines.


Basal & hypoxia - induced GPRC5A expression was decreased by HIF - 1 / 2α depletion .

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Basal & hypoxia-induced GPRC5A protein expression was decreased by HIF-1/2α depletion.


Depletion of HIF - 1β decreased GPRC5A upregulation in hypoxia .

Depletion of HIF-1β decreased GPRC5A protein upregulation in hypoxia.


Hypoxia mimetic DMOG induced HIF - 1 / 2α , CA9 & GPRC5A expression . Dual HIF - 1 / 2α depletion reduced GPRC5A induction by DMOG .

Hypoxia mimetic DMOG induced HIF-1/2α, CA9 & GPRC5A protein expression. Dual HIF-1/2α depletion reduced GPRC5A induction by DMOG.


qRT - PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia . GPRC5A was normalised to HPRT ( error bars ± SD ) .

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qRT-PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia. GPRC5A was normalised to HPRT (error bars ± SD).


qRT - PCR demonstrating that HIF - 1 / 2α depletion decreased GPRC5A induction during hypoxia . GPRC5A was normalised to HPRT ( error bars ± SD ) .

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qRT-PCR demonstrating that HIF-1/2α depletion decreased GPRC5A induction during hypoxia. GPRC5A was normalised to HPRT (error bars ± SD).


ChIP / PCR analyses identify HIF - 1α a binding to GPRC5A promoter region containing a putative optimal HRE ( error bars ± SD ) .

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ChIP/PCR analyses identify HIF-1α a binding to the GPRC5A promoter region containing a putative optimal HRE (error bars ± SD).


Expression of CA9 in formalin fixed paraffin embedded hypoxic SW620 by . Reduced CA9 expression with siRNA confirms antibody specificity ( scale bars : 200um ) .

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Expression of CA9 in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced CA9 expression with siRNA confirms antibody specificity (scale bars: 200um).


Expression of ; GPRC5A in formalin fixed paraffin embedded hypoxic SW620 by . Reduced ; GPRC5A expression with siRNA confirms antibody specificity ( scale bars : 200um ) .

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Expression of ; GPRC5A in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced ; GPRC5A expression with siRNA confirms antibody specificity (scale bars: 200um).


of serial sections from human colorectal tissue from with mesenteric ischaemia ( strangulated colon ) . GPRC5A is co - expressed with CA9 in colonic epithelial ( scale bars : 50um ) .

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IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia (strangulated colon). GPRC5A is co-expressed with CA9 in the colonic epithelial cells (scale bars: 50um).


Quantitative RT - PCR of mouse intestinal tissue . Gene expression was normalised to housekeeping gene Tbp . Raw data from three independent experiments ( n = 3 mice ) are shown ( error bars ± ) .

Quantitative RT-PCR analysis of mouse intestinal tissue. Gene expression was normalised to housekeeping gene Tbp. Raw data from three independent experiments (n=3 mice) are shown (error bars ± SEM).


Tg [ fli1 : eGFP ; vhl - / - ] & Tg [ fli1 : eGFP ] zebrafish ( 5 days post - fertilisation ) demonstrate excessive angiogenesis & increased expression of HIF target genes ( scale bars : 100um ) .

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Tg[fli1:eGFP; vhl-/-] & Tg[fli1:eGFP] zebrafish embryos (5 days post-fertilisation) demonstrate excessive angiogenesis & increased expression of HIF target genes (scale bars: 100um).


gprc5ba was induced in vhl mutant zebrafish & fli1 : eGFP zebrafish exposed to 5 % O2 ( vs normoxia ) for 24 hours ( RT - PCR ) .

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gprc5ba was induced in vhl mutant zebrafish embryos & fli1:eGFP zebrafish embryos exposed to 5% O2 (vs normoxia) for 24 hours (RT-PCR).


Kaplan - Meier curve following of transcriptomics dataset GSE24551 . Event - free survival is significantly reduced in with tumours expressing high of GPRC5A mRNA .

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Kaplan-Meier curve following analysis of transcriptomics dataset GSE24551. Event-free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA.


GPRC5A depletion markedly increases caspase - 3 activation / PARP cleavage during hypoxia .

GPRC5A depletion markedly increases caspase-3 activation/PARP cleavage during hypoxia.


Three independent siRNA sequences targeting GPRC5A induce caspase - 3 activation / PARP cleavage during hypoxia .

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Three independent siRNA sequences targeting GPRC5A induce caspase-3 activation/PARP cleavage during hypoxia.


GPRC5A depletion reduces hypoxic growth / survival . Crystal violet assays show reduced growth / survival in GPRC5A depleted during hypoxia [ n = 3 ] .

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GPRC5A depletion reduces hypoxic cell growth/survival. Crystal violet cell assays show reduced cell growth/survival in GPRC5A depleted cells during hypoxia [n=3].


Expression of an siRNA - resistant GPRC5A cDNA rescues hypoxic GPRC5A - depleted from apoptosis . Upper : doxycycline - induced expression GPRC5Asi1R rescues increases caspase - 3 / PARP cleavage induced by GPRC5A depletion in hypoxia . Lower : generation of an siRNA1 resistant GPRC5A cDNA by synonymous mutations .

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Expression of an siRNA-resistant GPRC5A cDNA rescues hypoxic GPRC5A-depleted cells from apoptosis. Upper: doxycycline-induced expression GPRC5Asi1R rescues increases caspase-3/PARP cleavage induced by GPRC5A depletion in hypoxia. Lower: generation of an siRNA1 resistant GPRC5A cDNA by synonymous mutations.


Caspase inhibitor QVD prevents caspase - 3 activation / PARP cleavage by GPRC5A depletion in hypoxia .

Caspase inhibitor QVD prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia.


GPRC5A depletion in hypoxia induces apoptosis as determined by violet ratiometric asymmetry probe / dead apoptosis flow

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GPRC5A depletion in hypoxia induces apoptosis as determined by the violet ratiometric membrane asymmetry probe/dead cell apoptosis assay and flow cytometry


Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted .

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Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells.


Hypoxia - induced localisation of YAP was attenuated in GPRC5A depleted .

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Hypoxia-induced nuclear localisation of YAP was attenuated in GPRC5A depleted cells.


Hypoxia activated TEAD activity ( 8x GTIIC - luc reporter ) but this was reduced by GPRC5A depletion . A representative triplicate experiment is shown ( n = 3 ) .

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Hypoxia activated TEAD activity (8x GTIIC-luc reporter) but this was reduced by GPRC5A depletion. A representative triplicate experiment is shown (n=3).


Hypoxia reduced LATS activity expression but this was prevented by GPRC5A depletion .

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Hypoxia reduced LATS activity and expression but this was prevented by GPRC5A depletion.


Constitutively active RhoA ( G14V ) expression restored YAP stabilisation ( Ser397 dephosphorylation ) by hypoxia in GPRC5A depleted .

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Constitutively active RhoA (G14V) expression restored YAP stabilisation (Ser397 dephosphorylation) by hypoxia in GPRC5A depleted cells.


YAP / TEAD inhibitor verteporfin selectively inhibited cancer survival in hypoxia by crystal violet ( n = 3 independent experiments ) .

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The YAP/TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay (n=3 independent experiments).


YAP knockdown was sufficient to induce caspase - 3 activation / PARP cleavage in hypoxia was not further enhanced by GPRC5A depletion .

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YAP knockdown was sufficient to induce caspase-3 activation/PARP cleavage in hypoxia and was not further enhanced by GPRC5A depletion.


Crystal violet assays show that YAP was required downstream of GPRC5A to promote survival . GPRC5A depleted were rescued by expression of an siRNA - resistant GPRC5A cDNA ( GPRC5Asi1R ) but this was prevented by co - depletion of YAP ( n = 3 independent experiments ) .

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Crystal violet assays show that YAP was required downstream of GPRC5A to promote cell survival. GPRC5A depleted cells were rescued by expression of an siRNA-resistant GPRC5A cDNA (GPRC5Asi1R) but this was prevented by co-depletion of YAP (n=3 independent experiments).


Expression of an siRNA - resistant GPRC5A rescued critical of GPRC5A depletion . GPRC5Asi1R expression prevented PARP cleavage in hypoxia as well as restoring hypoxia - induced YAP stabilisation ( Ser397 dephosphorylation ) ; these were reversed by YAP depletion .

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Expression of an siRNA-resistant GPRC5A rescued the critical phenotypes of GPRC5A depletion. GPRC5Asi1R expression prevented PARP cleavage in hypoxia as well as restoring hypoxia-induced YAP stabilisation (Ser397 dephosphorylation); these phenotypes were reversed by YAP depletion.


GPRC5A depletion attenuated hypoxia - induced BCL - XL expression .

GPRC5A depletion attenuated hypoxia-induced BCL-XL expression.


Constitutively active YAP ( S127A ) expression induced BCL - XL expression prevents caspase - 3 activation / PARP cleavage by GPRC5A depletion in hypoxia .

Constitutively active YAP (S127A) expression induced BCL-XL expression and prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia.


GPRC5Asi1R expression restored BCL - XL expression prevents appearance of caspase - 3 induced by GPRC5A depletion in hypoxia .

GPRC5Asi1R expression restored BCL-XL expression and prevents the appearance of cleaved caspase-3 induced by GPRC5A depletion in hypoxia.


A CUEDC2protein in mouse tissues . Adult ( 12 - week - old ) mouse was sacrifices total was extracted from liver , kidney , lung , spleen , heart , stomach , intestine , prostate , testis , brain , thymus .

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A CUEDC2protein levels in mouse tissues. Adult (12-week-old) mouse was sacrifices and total protein was extracted from the liver, kidney, lung, spleen, heart, stomach, intestine, prostate, testis, brain, and thymus.


B ( top ) In vivo cardiac ischemia - reperfusion model protocol ( 30 - minute ischemia followed by indicated times of reperfusion ) . ( bottom ) Mice were subjected to reversible ischemia in vivo for 30 minutes reperfused for different time period . was extracted from at risk of heart at indicated time points subjected to immunoblot of CUEDC2 CUEDC1 . Representative results were shown from 2 mice at each time point , experiments were repeated for three times .

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B (top) In vivo cardiac ischemia-reperfusion model protocol (30-minute ischemia followed by indicated times of reperfusion). (bottom) Mice were subjected to reversible ischemia in vivo for 30 minutes and reperfused for different time period. Protein was extracted from the area at risk of heart at the indicated time points and subjected to immunoblot analysis of CUEDC2 and CUEDC1. Representative results were shown from 2 mice at each time point, and experiments were repeated for three times.


C Isolated ratneonatal cardiomyocytes were treated to hypoxia with serum free - medium for 6h reoxygenated accompanying adding serum back for different time period in vitro level of CUEDC2 was tested at different time points during reoxygenation . Each experiment was repeated for three times .

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C Isolated primary ratneonatal cardiomyocytes were treated to hypoxia with serum free-medium for 6h and reoxygenated accompanying adding serum back for different time period in vitro and the protein level of CUEDC2 was tested at different time points during reoxygenation. Each experiment was repeated for three times.


D CUEDC2 level was plotted using immunohistochemical scores as described in methods . ( left ) Plot of CUEDC2 scores in each BZ DZ ( n = 9 ) . ( right ) Representative from immunohistochemistry for CUEDC2 in tissues from who suffered acute myocardial infarctions . boxed areas in left are magnified in middle right . DZ , distant zone ; BZ , border zone . Scale bars , 250 μm ( left ) , 50 μm ( middle right ) . * p = 0 . 001 .

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D CUEDC2 protein level was plotted using the immunohistochemical scores as described in methods. (left) Plot of CUEDC2 scores in each BZ and DZ (n = 9 patients). (right) Representative images from immunohistochemistry staining for CUEDC2 in tissues from patients who suffered acute myocardial infarctions. The boxed areas in the left images are magnified in the middle and right images. DZ, distant zone; BZ, border zone. Scale bars, 250 μm (left), 50 μm (middle and right). * p = 0.001.


A mousecardiomyocytes were treated with H2O2 ( 1 mM ) for 3 hours cardiomyocyteviability was determined by flow cytometry . Representative results of flow cytometry .

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A Primary mousecardiomyocytes were treated with H2O2 (1 mM) for 3 hours and cardiomyocyteviability was determined by flow cytometry. Representative results of flow cytometry analysis.


B Quantitative of PI - positive treated with H2O2 was shown ( 42 . 9 ± 3 . 1 % in wild - type group vs . 10 . 1 ± 4 . 4 % in Cuedc2 - / - group , * p = 0 . 0004 , n = 5 wells per group repeated for three times ) .

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B Quantitative analysis of PI-positive cells treated with H2O2 was shown (42.9 ± 3.1% in wild-type group vs. 10.1 ± 4.4% in Cuedc2-/- group, * p = 0.0004, n = 5 wells per group and repeated for three times).


C Mouse neonatal cardiomyocytes were subjected to hypoxia for 6 hours then reoxygenation for 30 minutes . Cardiomyocytes were then stained with 5 μM CellROX ® Deep Red Reagent analyzed by flow cytometry . Representative results of flow cytometry are shown , experiments were repeated for three times . D ROS were quantitated summarized . * p = 0 . 0108 , n = 3 wells per group repeated for three times .

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C Mouse neonatal cardiomyocytes were subjected to hypoxia for 6 hours and then reoxygenation for 30 minutes. Cardiomyocytes were then stained with 5 μM CellROX® Deep Red Reagent and analyzed by flow cytometry. Representative results of flow cytometry analysis are shown, and all experiments were repeated for three times.D ROS levels were quantitated and summarized. * p = 0.0108, n = 3 wells per group and repeated for three times.


E HPLC detection of a superoxide probe oxidized dihydroethidium ( DHE ) product in sham I / R injury heart tissue ( 30 - min ischemia followed by 30 - min reperfusion ) , 2 - hydroxyethidium ( EOH ) , a specific product for superoxide anion radical . * p < 0 . 0001 , n = 5 in each group . F In same samples of ( E ) , HPLC detection of ethidium ( E ) , oxidized by other reactive oxygen species such as H2O2 ( mainly ) ONOO . * p = 0 . 0001 , n = 5 in each group .

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E HPLC detection of a superoxide probe oxidized dihydroethidium (DHE) product in sham and I/R injury heart tissue (30-min ischemia followed by 30-min reperfusion), 2-hydroxyethidium (EOH), a specific product for superoxide anion radical. *p < 0.0001, n = 5 in each group.F In the same samples of (E), HPLC detection of ethidium (E), oxidized by other reactive oxygen species such as H2O2 (mainly) and ONOO. *p = 0.0001, n = 5 in each group.


A was extracted from at risk of left ventricle from wild - type or Cuedc2 - / - mice with 30 - minute ischemia followed by 30 - minute reperfusion subjected to immunoblot . Representative results from 2 mice are shown , each experiment was repeated for three times .

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A Protein was extracted from the area at risk of left ventricle from wild-type or Cuedc2-/- mice with 30-minute ischemia followed by 30-minute reperfusion and subjected to immunoblot analysis. Representative results from 2 mice are shown, and each experiment was repeated for three times.


B Neonatal mousecardiomyocytes were subjected to hypoxia for 6 hours , followed by indicated times of reoxygenation , before was extracted subjected to immunoblot .

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B Neonatal mousecardiomyocytes were subjected to hypoxia for 6 hours, followed by the indicated times of reoxygenation, before protein was extracted and subjected to immunoblot analysis.


C Luciferase of WT or Cuedc2 - / - MEFs transiently transfected with an NF - κB - responsive luciferase reporter then , 1 d later , treated for 6 h with TNF ( 10 ng / ml ; TNF ) or for H / R . * p = 0 . 0032 , n = 3 per group , * * p = 0 . 013 , n = 3 per group .

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

C Luciferase assay of WT or Cuedc2-/- MEFs transiently transfected with an NF-κB-responsive luciferase reporter and then, 1 d later, treated for 6 h with TNF (10 ng/ml; TNF) or for H/R. * p = 0.0032, n = 3 per group, ** p = 0.013, n = 3 per group.


D Wild - type Cuedc2 - / - mice were treated with a sham or I / R operation in vivo . Three hours after onset of reperfusion , mRNA of necrosis factor α ( TNFα ) , interleukin 6 ( IL - 6 ) interleukin 23 ( IL - 23 ) were tested . * p = 0 . 0005 , * * p = 0 . 0001 , n = 3 per group .

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

D Wild-type and Cuedc2-/- mice were treated with a sham or I/R operation in vivo. Three hours after the onset of reperfusion, the mRNA levels of tumor necrosis factor α (TNFα), interleukin 6 (IL-6) and interleukin 23 (IL-23) were tested. * p = 0.0005, ** p = 0.0001, n = 3 per group.


A was extracted from left ventricle of wild - type or Cuedc2 - / - adult mice ( 12 week - old ) subjected to immunoblot ( 3 mice per group ) .

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

A Protein was extracted from the left ventricle of wild-type or Cuedc2-/- adult mice (12 week-old) and subjected to immunoblot analysis (3 mice per group).


B Wild - type or Cuedc2 - / - mice were subjected to a heart ischemia for 30 - minute in vivo , followed by reperfusion for indicated times . was extracted from at risk of left ventricle subjected to immunoblot .

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

B Wild-type or Cuedc2-/- mice were subjected to a heart ischemia for 30-minute in vivo, followed by reperfusion for the indicated times. Protein was extracted from the area at risk of left ventricle and subjected to immunoblot analysis.