Figure 2A . 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 ) ) .

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

Figure 2A. 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)).


Figure 3A , 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 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 .

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

Figure 3A, 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 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.


Figure 6A . 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 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 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 ) .

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

Figure 6A. 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 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 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).


Figure 7A . 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 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 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 ) .

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

Figure 7A. 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 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 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).


Figure 1Overexpression 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 .

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Figure 1Overexpression 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.


Figure 2H1299 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 .

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Figure 2H1299 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.


Figure 3USP36 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 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 .

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Figure 3USP36 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 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.


Figure 4USP36 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 .

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Figure 4USP36 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.


Figure 5USP36 does not change the levels of endogenous snoRNP proteins . HeLa cells infected with scrambled or the indicated USP36 shRNA lentiviruses were assayed by IB .

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Figure 5USP36 does not change the levels of endogenous snoRNP proteins. HeLa cells infected with scrambled or the indicated USP36 shRNA lentiviruses were assayed by IB.


Figure 6USP36 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 ) .

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Figure 6USP36 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).


Figure 7 ( 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 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 .

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

Figure 7(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 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.


Figure 1 ( 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 : 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 : 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 .

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

Figure 1(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: 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: 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.


Figure 2 ( 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 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 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

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

Figure 2(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 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 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


Figure 3 ( 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 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 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

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

Figure 3(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 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 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


Figure 4 ( 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 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 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 .

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Figure 4(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 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 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.


Figure 6 ( 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 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 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 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 .

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

Figure 6(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 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 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 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.


Figure 7 ( 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 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 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 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 .

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

Figure 7(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 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 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 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.


Figure 8 ( 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 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 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 )

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Figure 8(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 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 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)


Figure 9 ( 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 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 ) 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 ) 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 cells treated as in D and its relative expression was quantified by densitometry using ImageJ . Vinculin was used as loading control ( 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 ) .

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Figure 9(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 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) 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) 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 cells treated as in D and its relative expression was quantified by densitometry using ImageJ. Vinculin was used as loading control(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).


Figure 1A . 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 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 ) .

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Figure 1A. 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 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).


Figure 2A . Immunoblot analysis of indicated proteins in control and siRNA treated cells . 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 . 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 .

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Figure 2A. Immunoblot analysis of indicated proteins in control and siRNA treated cells.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. 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.


Figure 3A , 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 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 . 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 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 .

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Figure 3A, 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 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. 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 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.


Figure 4B . 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 ) .

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Figure 4B. 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).


Figure 1SILAC - 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 protein in SILAC lysates . Validation of GPRC5A western blot data using siRNA . * Non - specific band of ~ 60kDa not depleted by GPRC5A siRNA . 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 cell lines . Basal & hypoxia - induced GPRC5A protein expression was decreased by HIF - 1 / 2α depletion . Depletion of HIF - 1β decreased GPRC5A protein upregulation in hypoxia . 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 ) . 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 the GPRC5A promoter region containing a putative optimal HRE ( error bars ± SD ) .

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Figure 1SILAC-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 protein in SILAC lysates.Validation of GPRC5A western blot data using siRNA. *Non-specific band of ~60kDa not depleted by GPRC5A siRNA.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 cell lines.Basal & hypoxia-induced GPRC5A protein expression was decreased by HIF-1/2α depletion.Depletion of HIF-1β decreased GPRC5A protein upregulation in hypoxia.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).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 the GPRC5A promoter region containing a putative optimal HRE (error bars ± SD).


Figure 2Expression 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 cells by IHC . Reduced ; GPRC5A expression with siRNA confirms antibody specificity ( scale bars : 200um ) . 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 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 embryos ( 5 days post - fertilisation ) demonstrate excessive angiogenesis & increased expression of HIF target genes ( scale bars : 100um ) . 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 analysis of transcriptomics dataset GSE24551 . Event - free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA .

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Figure 2Expression 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 cells by IHC. Reduced ; GPRC5A expression with siRNA confirms antibody specificity (scale bars: 200um).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 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 embryos (5 days post-fertilisation) demonstrate excessive angiogenesis & increased expression of HIF target genes (scale bars: 100um).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 analysis of transcriptomics dataset GSE24551. Event-free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA.


Figure 3GPRC5A depletion markedly increases caspase - 3 activation / PARP cleavage during hypoxia . Three independent siRNA sequences targeting GPRC5A induce caspase - 3 activation / PARP cleavage during hypoxia . 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 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 . GPRC5A depletion in hypoxia induces apoptosis as determined by the violet ratiometric membrane asymmetry probe / dead cell apoptosis assay and flow

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Figure 3GPRC5A depletion markedly increases caspase-3 activation/PARP cleavage during hypoxia.Three independent siRNA sequences targeting GPRC5A induce caspase-3 activation/PARP cleavage during hypoxia.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 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.GPRC5A depletion in hypoxia induces apoptosis as determined by the violet ratiometric membrane asymmetry probe/dead cell apoptosis assay and flow cytometry


Figure 4Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells . 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 ) . 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 cells . 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 and was not further enhanced by GPRC5A depletion . 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 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 . 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 and prevents the appearance of cleaved caspase - 3 induced by GPRC5A depletion in hypoxia .

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Figure 4Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells.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).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 cells.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 and was not further enhanced by GPRC5A depletion.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 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.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 and prevents the appearance of cleaved caspase-3 induced by GPRC5A depletion in hypoxia.


Figure 1A 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 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 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 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 .

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Figure 1A 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 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 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 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.


Figure 2A 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 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 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 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 .

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Figure 2A 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 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 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 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.


Figure 3A 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 the indicated times of reoxygenation , before protein was extracted and subjected to immunoblot analysis . 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 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 .

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Figure 3A 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 the indicated times of reoxygenation, before protein was extracted and subjected to immunoblot analysis.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 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.


Figure 4A 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 the indicated times . Protein was extracted from the area at risk of left ventricle and subjected to immunoblot analysis . C GPX1 protein level was plotted using the immunohistochemical scores as described in methods . ( left ) representative images from immunohistochemistry staining for GPX1 in tissues from patients who suffered acute myocardial infarctions . ( right ) plot of GPX1 scores in each BZ and DZ ( n = 9 patients ) . DZ , distant area ; BZ , border zone . Scale bars , 250 μm ( left ) . * p = 0 . 0039 . D Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were treated with H2O2 ( 1 mM ) for 3 hours . Cell viability was detected by FACS . * p = 0 . 0001 ; * * p = 0 . 0275 , n = 3 wells per group . NS , not significant . E Primary neonatal mousecardiomyocytes were transfected with lentivirus carrying control or GPX1 shRNA , and the efficiency of GPX1 knockdown was tested by immunoblotting . F Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were subjected to H2O2 ( 1 mM ) for 30 minutes and then stained with 5 μM CellROX ® Deep Red Reagent and analyzed by flow cytometry . * p = 0 . 0056 ; * * p = 0 . 0218 , n = 3 wells per group , n = 3 wells per group . NS , not significant .

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Figure 4A 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 the indicated times. Protein was extracted from the area at risk of left ventricle and subjected to immunoblot analysis.C GPX1 protein level was plotted using the immunohistochemical scores as described in methods. (left) representative images from immunohistochemistry staining for GPX1 in tissues from patients who suffered acute myocardial infarctions. (right) plot of GPX1 scores in each BZ and DZ (n = 9 patients). DZ, distant area; BZ, border zone. Scale bars, 250 μm (left). * p = 0.0039.D Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were treated with H2O2 (1 mM) for 3 hours. Cell viability was detected by FACS. * p = 0.0001; ** p = 0.0275, n = 3 wells per group. NS, not significant.E Primary neonatal mousecardiomyocytes were transfected with lentivirus carrying control or GPX1 shRNA, and the efficiency of GPX1 knockdown was tested by immunoblotting.F Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were subjected to H2O2 (1 mM) for 30 minutes and then stained with 5 μM CellROX® Deep Red Reagent and analyzed by flow cytometry. * p = 0.0056; ** p = 0.0218, n = 3 wells per group, n = 3 wells per group. NS, not significant.


Figure 5A Lysates from MEF - WT or MEF - Cuedc2 - / - cells treated with 20μM cycloheximide ( CHX ) for the indicated times were subjected to immunoblotting ( left ) . Relative GPX1 levels were quantified by densitometry ( right ) . B MEF - Cuedc2 - / - cells were transfected with increasing amounts of FLAG - CUEDC2 ( mouse ) plasmids ( 1 μg and 2 μg ) . At 24 hours after transfection , the cells were treated with the proteasome inhibitor MG - 132 ( 10 μM ) . Cells were cultured for additional 6 hours and subjected to immunoblotting . C HEK293T cells were transfected with the FLAG - GPX1 , HA - CUEDC2 , and Myc - ubiquitin plasmids as indicated and treated with MG - 132 ( 10 µM ) for 6 hours before harvest . Cell lysates were immunoprecipitated ( IP ) with anti - FLAG ( M2 ) . The immunoprecipitates were analyzed by western blot using an anti - GPX1 antibody . Whole - cell lysates ( WCL ) were analyzed by Western blots with anti - FLAG or anti - HA antibody to determine the protein of Flag - GPX1 and HA - CUEDC2 . D The heart tissue lysates from wide - type and Cuedc2 - / - mice were incubated with IgG or anti - GPX1 antibody , and then captured on protein G - Sepharose beads . The immunoprecipitates were analyzed by immunoblotting with an anti - ubiquitin antibody . E HEK293T cells were transfected with the Flag - CUEDC2 or Flag - CUEDC2 △ CUE ( deletion of the CUE domain of CUEDC2 ) , and treated with MG - 132 ( 10 µM ) for 6 hours before harvest . Cell lysates were immunoprecipitated ( IP ) with anti - Flag ( M2 ) . The immunoprecipitates were analyzed by western blot using an anti - GPX1 antibody . Whole - cell lysates ( WCL ) were analyzed by Western blots with anti - Flag or anti - GPX1 antibody to determine the protein of Flag - CUEDC2 and GPX1 . v : vector .

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Figure 5A Lysates from MEF-WT or MEF-Cuedc2-/- cells treated with 20μM cycloheximide (CHX) for the indicated times were subjected to immunoblotting (left). Relative GPX1 levels were quantified by densitometry (right).B MEF-Cuedc2-/- cells were transfected with increasing amounts of FLAG-CUEDC2 (mouse) plasmids (1 μg and 2 μg). At 24 hours after transfection, the cells were treated with the proteasome inhibitor MG-132 (10 μM). Cells were cultured for additional 6 hours and subjected to immunoblotting.C HEK293T cells were transfected with the FLAG-GPX1, HA-CUEDC2, and Myc-ubiquitin plasmids as indicated and treated with MG-132 (10 µM) for 6 hours before harvest. Cell lysates were immunoprecipitated (IP) with anti-FLAG (M2). The immunoprecipitates were analyzed by western blot using an anti-GPX1 antibody. Whole-cell lysates (WCL) were analyzed by Western blots with anti-FLAG or anti-HA antibody to determine the protein of Flag-GPX1 and HA-CUEDC2.D The heart tissue lysates from wide-type and Cuedc2-/- mice were incubated with IgG or anti-GPX1 antibody, and then captured on protein G-Sepharose beads. The immunoprecipitates were analyzed by immunoblotting with an anti-ubiquitin antibody.E HEK293T cells were transfected with the Flag-CUEDC2 or Flag-CUEDC2△CUE (deletion of the CUE domain of CUEDC2), and treated with MG-132 (10 µM) for 6 hours before harvest. Cell lysates were immunoprecipitated (IP) with anti-Flag (M2). The immunoprecipitates were analyzed by western blot using an anti-GPX1 antibody. Whole-cell lysates (WCL) were analyzed by Western blots with anti-Flag or anti-GPX1 antibody to determine the protein of Flag-CUEDC2 and GPX1. v: vector.


Figure 6A HEK293 cells were transfected with increasing amounts of Flag - TRIM33 expression vectors and 48 hours after transfection , cell lysates were subjected to immunoblotting ( IB ) using antibodies as indicated . B HEK293 cells were transfected with increasing amounts of Flag - FBXW7 expression vectors and 48 hours after transfection , cell lysates were subjected to immunoblotting ( IB ) . C HEK293T cells were transfected as indicated , at 18 h after transfection , the cells were treated with the proteasome inhibitor MG132 ( 20μM ) for 6 hours and then harvested . Cell lysates were immunoprecipitated by Flag - antibody ( M2 beads ) and Ubiquitin - conjugated GPX1 was detected by Western blotting with anti - Myc antibody . D HEK293 cells were transfected with either control or CUEDC2 siRNA and increasing amounts of Flag - TRIM33 expression vectors , cell lysates were subjected to immunoblotting ( IB ) using antibodies as indicated .

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Figure 6A HEK293 cells were transfected with increasing amounts of Flag-TRIM33 expression vectors and 48 hours after transfection, cell lysates were subjected to immunoblotting (IB) using antibodies as indicated.B HEK293 cells were transfected with increasing amounts of Flag-FBXW7 expression vectors and 48 hours after transfection, cell lysates were subjected to immunoblotting (IB).C HEK293T cells were transfected as indicated, at 18 h after transfection, the cells were treated with the proteasome inhibitor MG132 (20μM) for 6 hours and then harvested. Cell lysates were immunoprecipitated by Flag-antibody (M2 beads) and Ubiquitin-conjugated GPX1 was detected by Western blotting with anti-Myc antibody.D HEK293 cells were transfected with either control or CUEDC2 siRNA and increasing amounts of Flag-TRIM33 expression vectors, cell lysates were subjected to immunoblotting (IB) using antibodies as indicated.


Figure 7A CUEDC2 ablation reduced I / R injury , which was induced by 30 minutes of ischemia followed by 24 hours reperfusion . AAR , area at risk ; LV , left ventricular area , IRI , area of I / R injury . The ratios of AAR to LV , IRI to AAR and IRI to LV are shown . * p < 0 . 0001 compared with WT , n = 15 mice per group . ( top panel ) Representative cross - sectional slices derived from the hearts stained by TTC . Area of I / R injury is indicated in light pink . B Representative images of ventricular myocardium sections from WT and Cuedc2 - / - mice exposed to sham operation or I / R injury ( 2 hours after the onset of reperfusion following 30 - min ischemia ) . Green , TUNEL - positive myocytenuclei ; red , Troponin I - stained cardiomyocytes ; blue , DAPI - stained nuclei . Scale bar , 50 μm . C Quantitative analysis of apoptosis . * p < 0 . 0001 , n = 8 mice per group . D SerumLDH levels in sham - operated mice or those subjected to ischemia and a 4 - hour reperfusion . Data from 3 separate experiments was analyzed by one - way analysis of variance . * p < 0 . 0001 , n = 13 mice per group . E Echocardiography analysis of left ventricular dimensions and cardiac function at 1 week post - I / R in mice . * * p < 0 . 0001 , n = 15 mice in the sham - operated group and n = 10 mice in the I / R group .

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Figure 7A CUEDC2 ablation reduced I/R injury, which was induced by 30 minutes of ischemia followed by 24 hours reperfusion. AAR, area at risk; LV, left ventricular area, IRI, area of I/R injury. The ratios of AAR to LV, IRI to AAR and IRI to LV are shown. * p < 0.0001 compared with WT, n = 15 mice per group. (top panel) Representative cross-sectional slices derived from the hearts stained by TTC. Area of I/R injury is indicated in light pink.B Representative images of ventricular myocardium sections from WT and Cuedc2-/- mice exposed to sham operation or I/R injury (2 hours after the onset of reperfusion following 30-min ischemia). Green, TUNEL-positive myocytenuclei; red, Troponin I-stained cardiomyocytes; blue, DAPI-stained nuclei. Scale bar, 50 μm.C Quantitative analysis of apoptosis. * p < 0.0001, n = 8 mice per group.D SerumLDH levels in sham-operated mice or those subjected to ischemia and a 4-hour reperfusion. Data from 3 separate experiments was analyzed by one-way analysis of variance. * p < 0.0001, n = 13 mice per group.E Echocardiography analysis of left ventricular dimensions and cardiac function at 1 week post-I/R in mice. ** p < 0.0001, n = 15 mice in the sham-operated group and n = 10 mice in the I/R group.


Figure 8A Immunoblotting for CUEDC2 protein level . 4 weeks after tail - vein injection of rAAV9 - CUEDC2 , indicated mice tissues were harvested and subjected to immunoblotting to detect CUEDC2 protein level . Ctl : heart of rAAV9 - GFP injected mice . B Animals were injected by rAAV9 - CUEDC2 in a different amount of viral genomes ( VG ) . Heart tissues were then analyzed by immunoblotting to detect CUEDC2 transduction . Tubulin protein level was examined for normalization purposes . C The ratios of AAR to LV , IRI to AAR and IRI to LV are shown . n = 10 mice in WT + rAAV - GFP group and Cuedc2 - / - + rAAV - GFP group ; n = 12 mice in Cuedc2 - / - + rAAV - CUEDC2 ( 1 × 1010 VG ) group and Cuedc2 - / - + rAAV - CUEDC2 ( 1 × 1011 VG ) group . * p = 0 . 0055 , * * p = 0 . 0146 , * * * p = 0 . 0200 , * * * * p = 0 . 0377 . D Effect of rAAV9 - CUEDC2 treatment on echocardiography indices of left ventricle function recovery post - I / R measured by fractional shortening ( FS ) and ejection fraction ( EF ) . WT + rAAV - GFP ( n = 10 ) ; Cuedc2 - / - + rAAV - GFP ( n = 10 ) ; Cuedc2 - / - + rAAV - CUEDC2 ( Low dose ) ( n = 12 ) ; Cuedc2 - / - + rAAV - CUEDC2 ( High dose ) ( n = 12 ) . * * p < 0 . 0001 .

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Figure 8A Immunoblotting for CUEDC2 protein level. 4 weeks after tail-vein injection of rAAV9-CUEDC2, indicated mice tissues were harvested and subjected to immunoblotting to detect CUEDC2 protein level. Ctl: heart of rAAV9-GFP injected mice.B Animals were injected by rAAV9-CUEDC2 in a different amount of viral genomes (VG). Heart tissues were then analyzed by immunoblotting to detect CUEDC2 transduction. Tubulin protein level was examined for normalization purposes.C The ratios of AAR to LV, IRI to AAR and IRI to LV are shown. n = 10 mice in WT + rAAV-GFP group and Cuedc2-/- + rAAV-GFP group; n = 12 mice in Cuedc2-/- + rAAV-CUEDC2 (1×1010 VG) group and Cuedc2-/- + rAAV-CUEDC2 (1×1011 VG) group. * p = 0.0055, ** p = 0.0146, *** p = 0.0200, **** p = 0.0377.D Effect of rAAV9-CUEDC2 treatment on echocardiography indices of left ventricle function recovery post-I/R measured by fractional shortening (FS) and ejection fraction (EF). WT+ rAAV-GFP (n = 10); Cuedc2-/- + rAAV-GFP (n = 10); Cuedc2-/- + rAAV-CUEDC2 (Low dose) (n = 12); Cuedc2-/- + rAAV-CUEDC2 (High dose) (n = 12). ** p < 0.0001.