C57BL / 6J mice were intraarticularly infected with MRSA ( 4 × 106 CFU ; n = 3 - 4 per group ) and left untreated for 24 hours . K . Paraffin - embedded tissues were sectioned and used to measure inflammation score and synovial hyperplasia . GLUT1 and MCT4 expression was detected , and the percentage of positively staining cells were determined ( Scale bar : 1 , 000 μm ) . Data information : Error bars show means ± SD with individual data points . Two - tailed unpaired t - test analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ) .

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

C57BL/6J mice were intraarticularly infected with MRSA (4 × 106 CFU; n = 3-4 per group) and left untreated for 24 hours. K. Paraffin-embedded tissues were sectioned and used to measure inflammation score and synovial hyperplasia. GLUT1 and MCT4 expression was detected, and the percentage of positively staining cells were determined (Scale bar: 1,000 μm). Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p<0.05 or **p <0.01; N.D. = not detected).


M . Human cartilage and synovial tissue were infected with MRSA ( 4 × 106 CFU ) for 24 hours . The co - expression and - localization levels of GLUT1 and MCT4 in human cartilage tissue was measured and confirmed by line profile analysis ( Scale bar : 100 μm ) . Expression of GLUT1 , MCT4 , IL - 1β , and MMP3 was measured in human synovial tissue ( Scale bar : 1 , 000 μm ) .

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

M. Human cartilage and synovial tissue were infected with MRSA (4 × 106 CFU) for 24 hours. The co-expression and -localization levels of GLUT1 and MCT4 in human cartilage tissue was measured and confirmed by line profile analysis (Scale bar: 100 μm). Expression of GLUT1, MCT4, IL-1β, and MMP3 was measured in human synovial tissue (Scale bar: 1,000 μm).


B . Physiological changes were identified and representative images were generated .

B. Physiological changes were identified and representative images were generated.


C . Blood was collected and complete blood counts ( CBC ) were measured . Data information : In vivo experiments were repeated in at least two independent experiments . Error bars show means ± SD with individual data points . One - way ANOVA with Tukey ' s post hoc analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ; N . S . = not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Blood was collected and complete blood counts (CBC) were measured. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected; N.S. = not significant).


D and E . Infiltrating immune cell counts within synovial fluid and MRSA synovial fluid bioburden were quantified . MRSA bioburden in synovial tissue was quantified . F . Paraffin - embedded tissues were sectioned and measured at 7 and 14 days with respect to inflammation score , synovial hyperplasia , synovial cellularity , OARSI score , proteoglycan depletion , osteophyte formation , bone erosion , and bone formation ( Scale bar : 1 , 000 μm ) . G and H . Lactate level in synovial fluid was measured at 14 days and expression of GLUT1 , MCT4 , cleaved - IL - 1β , and NLRP3 in synovial tissue was measured with GAPDH as a loading control . Data information : In vivo experiments were repeated in at least two independent experiments . Error bars show means ± SD with individual data points . One - way ANOVA with Tukey ' s post hoc analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ; N . S . = not significant ) .

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

D and E. Infiltrating immune cell counts within synovial fluid and MRSA synovial fluid bioburden were quantified. MRSA bioburden in synovial tissue was quantified. F. Paraffin-embedded tissues were sectioned and measured at 7 and 14 days with respect to inflammation score, synovial hyperplasia, synovial cellularity, OARSI score, proteoglycan depletion, osteophyte formation, bone erosion, and bone formation (Scale bar: 1,000 μm). G and H. Lactate level in synovial fluid was measured at 14 days and expression of GLUT1, MCT4, cleaved-IL-1β, and NLRP3 in synovial tissue was measured with GAPDH as a loading control. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected; N.S. = not significant).


B . Infiltrating immune cell counts within synovial fluid were quantified . Data information : In vivo experiments were repeated in at least two independent experiments . Error bars show means ± SD with individual data points . One - way ANOVA with Tukey ' s post hoc analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Infiltrating immune cell counts within synovial fluid were quantified. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected).


C . Serum and synovial fluid lactate levels were measured and the relative luminescence intensity is shown . Data information : In vivo experiments were repeated in at least two independent experiments . Error bars show means ± SD with individual data points . One - way ANOVA with Tukey ' s post hoc analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ) .

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

C. Serum and synovial fluid lactate levels were measured and the relative luminescence intensity is shown. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected).


D . Expression of GLUT1 and MMP3 in synovial tissue was measured using GAPDH as a loading control .

D. Expression of GLUT1 and MMP3 in synovial tissue was measured using GAPDH as a loading control.


E . Physiological changes were identified , and representative images were generated .

E. Physiological changes were identified, and representative images were generated.


F . Blood was collected , and complete blood counts ( CBC ) were measured . G . Paraffin - embedded tissues were sectioned and measured with respect to inflammation score , synovial hyperplasia , OARSI score , proteoglycan depletion , osteophyte formation , bone erosion , and bone formation ( Scale bar : 1 , 000 μm ) . The expression of GLUT1 was detected and the percentage of positively staining cells was determined . Data information : In vivo experiments were repeated in at least two independent experiments . Error bars show means ± SD with individual data points . One - way ANOVA with Tukey ' s post hoc analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ) .

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

F. Blood was collected, and complete blood counts (CBC) were measured. G. Paraffin-embedded tissues were sectioned and measured with respect to inflammation score, synovial hyperplasia, OARSI score, proteoglycan depletion, osteophyte formation, bone erosion, and bone formation (Scale bar: 1,000 μm). The expression of GLUT1 was detected and the percentage of positively staining cells was determined. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected).


C57BL / 6J mice were intraarticularly infected with MRSA ( 4 × 106 CFU ; n = 4 - 5 per group ) for 24 hours . Synovial fluid cells were collected and ( 5 × 105 ) seeded on antibiotic - free DMEM medium with or without DMF ( 300 µg / ml ) for 6 and 24 hours . B . MRSA expressive of green fluorescent protein ( GFP ) was detected and measured ( Scale bar : 100 μm ) . Data information : Error bars show means ± SD with individual data points . Two - tailed unpaired t - test analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 or * * * p < 0 . 001 or * * * * p < 0 . 0001 ; N . D . = not detected ; N . S . = not significant ) .

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

C57BL/6J mice were intraarticularly infected with MRSA (4 × 106 CFU; n = 4-5 per group) for 24 hours. Synovial fluid cells were collected and (5 × 105) seeded on antibiotic-free DMEM medium with or without DMF (300 µg/ml) for 6 and 24 hours. B. MRSA expressive of green fluorescent protein (GFP) was detected and measured (Scale bar: 100 μm). Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected; N.S. = not significant).


G . Inhibition zones in disk fusion assays using vancomycin ( 1 , 000 µg ) and DMF ( 75 to 300 µg ) were measured and presented in a histogram . Data information : Error bars show means ± SD with individual data points . Two - tailed unpaired t - test analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 or * * * p < 0 . 001 or * * * * p < 0 . 0001 ; N . D . = not detected ; N . S . = not significant ) .

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

G. Inhibition zones in disk fusion assays using vancomycin (1,000 µg) and DMF (75 to 300 µg) were measured and presented in a histogram. Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected; N.S. = not significant).


J . RAW264 . 7 cells were stained with CellTracker and infected with MRSA ( 4 × 106 CFU ) for 2 hours . The cells were then washed to remove extra - cellular MRSA . Medium containing DMF ( 700 µg / ml ) , vancomycin ( 100 µg / ml ) , and DMF ( 700 µg / ml ) with vancomycin ( 100 µg / ml ) was added and GFP intensity was measured at 1 - hour intervals for a total of 12 hours . Representative images of differential fluorescence with CellTracker and GFP at 4 - hour intervals for a total of 12 hours . Intracellular and extracellular MRSA were semi - quantitatively measured at 12 hours . Data information : Error bars show means ± SD with individual data points . Two - tailed unpaired t - test analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 or * * * p < 0 . 001 or * * * * p < 0 . 0001 ; N . D . = not detected ; N . S . = not significant ) .

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

J. RAW264.7 cells were stained with CellTracker and infected with MRSA (4 × 106 CFU) for 2 hours. The cells were then washed to remove extra-cellular MRSA. Medium containing DMF (700 µg/ml), vancomycin (100 µg/ml), and DMF (700 µg/ml) with vancomycin (100 µg/ml) was added and GFP intensity was measured at 1-hour intervals for a total of 12 hours. Representative images of differential fluorescence with CellTracker and GFP at 4-hour intervals for a total of 12 hours. Intracellular and extracellular MRSA were semi-quantitatively measured at 12 hours. Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected; N.S. = not significant).


C57BL / 6J mice were subcutaneously treated with vancomycin ( 30 mg / kg ) and rifampin ( 20 mg / kg ) for 3 days following MRSA ( 8 × 106 CFU ) infection , followed by systemic ( 14 mg / kg ) or local ( 70 μg / joint ) DMF treatment at 3 days interval for a total of 3 times ( n = 6 per group ) . D . Paraffin - embedded tissues were sectioned and measured with respect to inflammation score , synovial hyperplasia , synovial cellularity , proteoglycan depletion , osteophyte formation , bone erosion , and bone formation ( Scale bar : 2 , 000 or 500 μm ) . Bone resorption by osteoclasts was measured by tartrate - resistant acid phosphatase ( TRAP ) staining ( Scale bar : 2 , 000 μm ) . Lameness score was also measured . E . Illustrated summary of DMF ' s effect on MRSA - induced septic arthritis . Data information : In vivo experiments were repeated in at least two independent experiments . Error bars show means ± SD with individual data points . One - way ANOVA with Tukey ' s post hoc analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 or * * * p < 0 . 001 or * * * * p < 0 . 0001 ; N . D . = not detected ) .

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

C57BL/6J mice were subcutaneously treated with vancomycin (30 mg/kg) and rifampin (20 mg/kg) for 3 days following MRSA (8 × 106 CFU) infection, followed by systemic (14 mg/kg) or local (70 μg/joint) DMF treatment at 3 days interval for a total of 3 times (n = 6 per group). D. Paraffin-embedded tissues were sectioned and measured with respect to inflammation score, synovial hyperplasia, synovial cellularity, proteoglycan depletion, osteophyte formation, bone erosion, and bone formation (Scale bar: 2,000 or 500 μm). Bone resorption by osteoclasts was measured by tartrate-resistant acid phosphatase (TRAP) staining (Scale bar: 2,000 μm). Lameness score was also measured. E. Illustrated summary of DMF's effect on MRSA-induced septic arthritis. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected).


A Representative image of tumor - bearing mice and tumors after subcutaneous injection of Hepa1 - 6 hepatoma cells into WT and Tet2 - / - ( KO ) mice .

O O O O O O O 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 image of tumor-bearing mice and tumors after subcutaneous injection of Hepa1-6 hepatoma cells into WT and Tet2-/- (KO) mice.


B Mean tumor volumes in WT and Tet2 - / - mice ( n = 6 ) .

B Mean tumor volumes in WT and Tet2-/- mice (n=6).


C Tumor weights 18 days after tumor cell injection into WT and KO mice ( n = 6 ) .

C Tumor weights 18 days after tumor cell injection into WT and KO mice (n=6).


D Kaplan - Meier survival curves of tumor - bearing and tumor - free WT and KO mice ( n = 15 ) .

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 Kaplan-Meier survival curves of tumor-bearing and tumor-free WT and KO mice (n=15).


E Representative images of lungs with metastasis from WT and KO mice injected with Hepa1 - 6 cells via tail vein , as well as quantification of the number of lung metastasis ( n = 4 ) .

O O O O O O O O O O O O O O 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 lungs with metastasis from WT and KO mice injected with Hepa1-6 cells via tail vein, as well as quantification of the number of lung metastasis (n=4).


F Representative images of tumors from WT and KO mice subcutaneously injected with Py8119 breast cancer cells , as well as quantification of tumor weights ( n = 5 ) .

F Representative images of tumors from WT and KO mice subcutaneously injected with Py8119 breast cancer cells, as well as quantification of tumor weights (n=5).


A , B Immunohistochemistry analysis of CD8a , CD4 ( A ) , PCNA , and Foxp3 ( B ) in tumors from WT and Tet2 - / - ( KO ) mice subcutaneously injected with Hepa1 - 6 hepatoma cells . Corresponding bar charts display positive staining , quantified using Image Pro Plus 6 . 0 software ( n = 3 ) . Pictures were captured at 400X magnification . Scale bar = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B Immunohistochemistry analysis of CD8a, CD4 (A), PCNA, and Foxp3 (B) in tumors from WT and Tet2-/- (KO) mice subcutaneously injected with Hepa1-6 hepatoma cells. Corresponding bar charts display positive staining, quantified using Image Pro Plus 6.0 software (n=3). Pictures were captured at 400X magnification. Scale bar = 50 μm.


D Tumor growth curves in WT and KO mice treated with PBS or with anti - PD - 1 Ab ( n = 3 ) .

O O 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 Tumor growth curves in WT and KO mice treated with PBS or with anti-PD-1 Ab (n =3).


E Representatives images of day - 19 tumors from WT and KO from panel D .

E Representatives images of day-19 tumors from WT and KO from panel D.


F Tumor weights from WT and KO mice at day 19 .

F Tumor weights from WT and KO mice at day 19.


G Flow cytometry analysis of splenic CD8 + T cells in WT and KO mice with or without anti - PD - 1 treatment after tumor challenge .

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

G Flow cytometry analysis of splenic CD8+ T cells in WT and KO mice with or without anti-PD-1 treatment after tumor challenge.


H - K GSEA analysis showing the significant down - regulation of genes in GO biological processes : chemokine signaling pathway ( H ) , T cell receptor signaling pathway ( I ) , innate immune response ( J ) and cytokine receptor interaction ( K ) . The x - axis represents that the mouse genes are ranked from the most up - regulated ( left end ) to the most down - regulated ( right end ) in comparison of syngeneic tumor from Hepa1 - 6 tumor - bearing KO to WT mice ( n = 4 ) . The y - axis is an enrichment score ( ES ) , which goes up ( > 0 ) and down ( < 0 ) representing the enrichment of up - regulated genes and down - regulated genes , respectively . NES is the normalized enrichment score . p - value was calculated by permutation .

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

H-K GSEA analysis showing the significant down-regulation of genes in GO biological processes: chemokine signaling pathway (H), T cell receptor signaling pathway (I), innate immune response (J) and cytokine receptor interaction (K). The x-axis represents that the mouse genes are ranked from the most up-regulated (left end) to the most down-regulated (right end) in comparison of syngeneic tumor from Hepa1-6 tumor-bearing KO to WT mice (n=4). The y-axis is an enrichment score (ES), which goes up (>0) and down (<0) representing the enrichment of up-regulated genes and down-regulated genes, respectively. NES is the normalized enrichment score. p-value was calculated by permutation.


A Percentages of MDSCs ( CD11b + Gr1 + ) gated from the CD45 + cell population in peripheral blood and spleen [ n = 11 , tumor - free ( - ) WT and Tet2 - / - （ KO ） mice ; n = 12 , tumor - bearing ( + ) WT and KO mice , peripheral blood ; n = 10 , tumor - free ( - ) WT and KO mice ; n = 13 , tumor - bearing ( + ) WT and KO mice , spleen ] . B Percentages of G - MDSCs ( CD11b + Ly6G + ) gated from the CD45 + cell population in peripheral blood and spleen [ n = 14 , tumor - free ( - ) WT and KO mice ; n = 15 , tumor - bearing ( + ) WT and KO mice , peripheral blood ; n = 15 , tumor - free ( - ) WT and KO mice ; n = 15 , tumor - bearing ( + ) WT and KO mice , spleen ] . C Percentages of M - MDSCs ( CD11b + Ly6C + ) gated from the CD45 + cell population in peripheral blood and spleen [ n = 5 , tumor - free ( - ) WT and KO mice ; n = 6 , tumor - bearing ( + ) WT and KO mice , peripheral blood ; n = 6 , tumor - free ( - ) WT and KO mice ; n = 6 , tumor - bearing ( + ) WT and KO mice , spleen ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Percentages of MDSCs (CD11b+Gr1+) gated from the CD45+ cell population in peripheral blood and spleen [n=11, tumor-free (-) WT and Tet2-/-（KO） mice; n=12, tumor-bearing (+) WT and KO mice, peripheral blood; n=10, tumor-free (-) WT and KO mice; n=13, tumor-bearing (+) WT and KO mice, spleen]. B Percentages of G-MDSCs (CD11b+Ly6G+) gated from the CD45+ cell population in peripheral blood and spleen [n=14, tumor-free (-) WT and KO mice; n=15, tumor-bearing (+) WT and KO mice, peripheral blood; n=15, tumor-free (-) WT and KO mice; n=15, tumor-bearing (+) WT and KO mice, spleen]. C Percentages of M-MDSCs (CD11b+Ly6C+) gated from the CD45+ cell population in peripheral blood and spleen [n=5, tumor-free (-) WT and KO mice; n=6, tumor-bearing (+) WT and KO mice, peripheral blood; n=6, tumor-free (-) WT and KO mice; n=6, tumor-bearing (+) WT and KO mice, spleen].


D Representative images of Ly6g and Ly6c immunohistochemistry in Hepa1 - 6 cell tumors from WT and KO mice . Corresponding bar charts display quantification of positive staining using Image Pro Plus 6 . 0 software ( n = 3 ) . Pictures were captured at 400X magnification . Scale bar = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of Ly6g and Ly6c immunohistochemistry in Hepa1-6 cell tumors from WT and KO mice. Corresponding bar charts display quantification of positive staining using Image Pro Plus 6.0 software (n=3). Pictures were captured at 400X magnification. Scale bar = 50 μm.


E Percentages of CD8 + T cells gated from the CD45 + cell population in peripheral blood and spleen [ n = 11 , tumor - free ( - ) WT and KO mice ; n = 5 , tumor - bearing ( + ) WT and Tet2 - / - KO mice , peripheral blood ; n = 16 , tumor - free ( - ) WT and KO mice ; n = 12 , tumor - bearing ( + ) WT and KO mice , spleen ] . F Percentages of CD4 + T cells gated from the CD45 + cell population in peripheral blood and spleen [ n = 8 , tumor - free ( - ) WT and KO mice ; n = 5 , tumor - bearing ( + ) WT and KO mice , peripheral blood ; n = 16 , tumor - free ( - ) WT and KO mice ; n = 11 , tumor - bearing ( + ) WT and KO mice , spleen ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Percentages of CD8+ T cells gated from the CD45+ cell population in peripheral blood and spleen [n=11, tumor-free (-) WT and KO mice; n=5, tumor-bearing (+) WT and Tet2-/- KO mice, peripheral blood; n=16, tumor-free (-) WT and KO mice; n=12, tumor-bearing (+) WT and KO mice, spleen]. F Percentages of CD4+ T cells gated from the CD45+ cell population in peripheral blood and spleen [n=8, tumor-free (-) WT and KO mice; n=5, tumor-bearing (+) WT and KO mice, peripheral blood; n=16, tumor-free (-) WT and KO mice; n=11, tumor-bearing (+) WT and KO mice, spleen].


A Sorting efficiency of G - MDSCs ( CD11b + Ly6G + ) from spleens of tumor - bearing Tet2 - / - （ KO ） and WT mice .

O O O O O O 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 Sorting efficiency of G-MDSCs (CD11b+Ly6G+) from spleens of tumor-bearing Tet2-/- （KO） and WT mice.


B GSEA analysis showing the significant upregulation of genes in GO biological processes : DNA replication ( left ) , cell cycle G1 - S phase transition ( middle ) , and ribosome biogenesis ( right ) . The x - axis represents that the mouse genes are ranked from the most upregulated ( left end ) to the most downregulated ( right end ) in comparison of tumor - bearing KO to WT mice from G - MDSCs of spleen ( n = 2 ) . The y - axis is an enrichment score ( ES ) , which goes up ( > 0 ) and down ( < 0 ) representing the enrichment of upregulated genes and downregulated genes , respectively . NES is the normalized enrichment score . p - value was calculated by permutation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 GSEA analysis showing the significant upregulation of genes in GO biological processes: DNA replication (left), cell cycle G1-S phase transition (middle), and ribosome biogenesis (right). The x-axis represents that the mouse genes are ranked from the most upregulated (left end) to the most downregulated (right end) in comparison of tumor-bearing KO to WT mice from G-MDSCs of spleen (n=2). The y-axis is an enrichment score (ES), which goes up (>0) and down (<0) representing the enrichment of upregulated genes and downregulated genes, respectively. NES is the normalized enrichment score. p-value was calculated by permutation.


C G - MDSCs purified from spleen of tumor - bearing WT or Tet2 - / - mice were co - cultured with CD8 + T cells at the indicated cellular ratios for T cell activation assays , quantified by CFSE - labeling and dye dilution for 3 days in vitro . Representative histograms show CFSE signals in the in vitro T cell activation assays . D Quantification of CD8 + T cell proliferation in panel C . Each point represents a co - culture experiment ( n = 3 ) . ns , not significant . Graphs show mean ± SEM and statistical analysis by Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 G-MDSCs purified from spleen of tumor-bearing WT or Tet2-/- mice were co-cultured with CD8+ T cells at the indicated cellular ratios for T cell activation assays, quantified by CFSE-labeling and dye dilution for 3 days in vitro. Representative histograms show CFSE signals in the in vitro T cell activation assays. D Quantification of CD8+ T cell proliferation in panel C. Each point represents a co-culture experiment (n=3). ns, not significant. Graphs show mean ± SEM and statistical analysis by Student's t-test.


B Representative flow cytometry plots of splenic G - MDSCs measured in tumor - free untreated Tet2 - / - ( KO ) and WT mice and after anti - Ly6g or PBS injections into tumor - bearing mice . Flow cytometric analysis of T cells in Tet2 - / - mice and littermate control mice ( WT ) with or without anti - Ly6g treatment after tumor challenge .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative flow cytometry plots of splenic G-MDSCs measured in tumor-free untreated Tet2-/- (KO) and WT mice and after anti-Ly6g or PBS injections into tumor-bearing mice. Flow cytometric analysis of T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-Ly6g treatment after tumor challenge.


C Tumor growth curves in WT and KO mice treated with either PBS or anti - mouse Ly6g ( n = 5 ) .

C Tumor growth curves in WT and KO mice treated with either PBS or anti-mouse Ly6g (n=5).


D Day - 18 tumor weights at the endpoint of ( C ) [ n = 11 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 12 , anti - Ly6g antibody ( + ) injected tumor - bearing WT and KO mice ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Day-18 tumor weights at the endpoint of (C) [n=11, PBS (-) injected tumor-bearing WT and KO mice; n=12, anti-Ly6g antibody (+) injected tumor-bearing WT and KO mice].


E Percentages of CD8 + T cells from the spleens of tumor - bearing mice with or without anti - mouse Ly6g treatment [ n = 17 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 6 , anti - Ly6g ( + ) antibody injected tumor - bearing WT and KO mice ] . F Percentages of CD4 + T cells from the spleens of tumor - bearing mice with or without anti - mouse Ly6g treatment [ n = 16 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 6 , anti - Ly6g ( + ) antibody injected tumor - bearing WT and KO mice Flow cytometric analysis of T cells in Tet2 - / - mice and littermate control mice ( WT ) with or without anti - Ly6g treatment after tumor challenge .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Percentages of CD8+ T cells from the spleens of tumor-bearing mice with or without anti-mouse Ly6g treatment [n=17, PBS (-) injected tumor-bearing WT and KO mice; n=6, anti-Ly6g (+) antibody injected tumor-bearing WT and KO mice]. F Percentages of CD4+ T cells from the spleens of tumor-bearing mice with or without anti-mouse Ly6g treatment [n=16, PBS (-) injected tumor-bearing WT and KO mice; n=6, anti-Ly6g (+) antibody injected tumor-bearing WT and KO mice Flow cytometric analysis of T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-Ly6g treatment after tumor challenge.


A Gene set enrichment analysis of the IL - 6 / JAK / STAT signaling pathway in G - MDSCs from tumor - bearing Tet2 - / - ( KO ) versus WT mice ( n = 2 ) . p - value was calculated by permutation .

O O O O O O O O O O O O O O O O O O O O O O O O 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 Gene set enrichment analysis of the IL-6/JAK/STAT signaling pathway in G-MDSCs from tumor-bearing Tet2-/- (KO) versus WT mice (n=2). p-value was calculated by permutation.


B ELISA analysis of IL - 6 , IFN - γ , and IL - 2 levels in blood from WT and KO mice upon tumor challenge ( n = 4 , tumor - bearing WT mice ; n = 3 , tumor - bearing KO mice ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 ELISA analysis of IL-6, IFN-γ, and IL-2 levels in blood from WT and KO mice upon tumor challenge (n=4, tumor-bearing WT mice; n=3, tumor-bearing KO mice).


C Tumor growth curves in tumor - bearing WT and KO mice treated with PBS or anti - mouse IL - 6 antibody ( n = 5 ) .

O O O O O 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 Tumor growth curves in tumor-bearing WT and KO mice treated with PBS or anti-mouse IL-6 antibody (n=5).


D Day - 18 tumor weights at the endpoint of ( C ) [ n = 10 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 7 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Day-18 tumor weights at the endpoint of (C) [n=10, PBS (-) injected tumor-bearing WT and KO mice; n=7, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice].


E , F Percentages of CD11b + Ly6G + G - MDSCs gated from total cells in ( E ) spleen and ( F ) peripheral blood from tumor - bearing mice treated with PBS or anti - mouse IL - 6 antibody [ n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 4 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , spleen ; n = 4 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 4 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , peripheral blood ] . G , H Percentages of CD8 + T cells gated from total cells in ( G ) spleen and ( H ) peripheral blood from tumor - bearing mice treated with PBS or anti - mouse IL - 6 [ n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , spleen ; n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , peripheral blood ] . I , J Percentages of CD4 + T cells gated from total cells in ( I ) spleen and ( J ) peripheral blood from tumor - bearing mice treated with PBS or anti - mouse IL - 6 [ n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , spleen ; n = 4 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , peripheral blood ] . Flow cytometric analysis of G - MDSCs and T cells in Tet2 - / - mice and littermate control mice ( WT ) with or without anti - IL - 6 treatment after tumor challenge .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F Percentages of CD11b+ Ly6G+ G-MDSCs gated from total cells in (E) spleen and (F) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 antibody [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=4, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=4, PBS (-) injected tumor-bearing WT and KO mice; n=4, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood]. G, H Percentages of CD8+ T cells gated from total cells in (G) spleen and (H) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood]. I, J Percentages of CD4+ T cells gated from total cells in (I) spleen and (J) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=4, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood]. Flow cytometric analysis of G-MDSCs and T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-IL-6 treatment after tumor challenge.


( A , B ) Flag ‐ SENP3 or HA ‐ PELP1 was expressed in HeLa cells as indicated . Proteins were captured on Flag ‐ or HA ‐ beads and immunoblotted using antibodies directed against endogenous PELP1 or SENP3 as indicated .

O O O O O O O O O O O O O O O O O O O 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, B) Flag‐SENP3 or HA‐PELP1 was expressed in HeLa cells as indicated. Proteins were captured on Flag‐ or HA‐beads and immunoblotted using antibodies directed against endogenous PELP1 or SENP3 as indicated.


( C ) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of SENP3 by western blotting with an anti ‐ SENP3 antibody .

O O O O O O O O O O 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) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of SENP3 by western blotting with an anti‐SENP3 antibody.


( D - F ) Flag ‐ or HA ‐ tagged proteins were expressed in HeLa cells , captured on Flag ‐ or HA ‐ beads and subjected to immunoblotting as indicated . In all experiments , the inputs represent 2 . 5 % of the total cell lysate .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Flag‐ or HA‐tagged proteins were expressed in HeLa cells, captured on Flag‐ or HA‐beads and subjected to immunoblotting as indicated. In all experiments, the inputs represent 2.5% of the total cell lysate.


( A , B ) Flag ‐ PELP1 and HA ‐ WDR18 were expressed in HeLa cells as indicated . HA ‐ WDR18 was captured on HA ‐ beads and the bound material was probed by immunoblotting for the presence of ectopically expressed Flag ‐ tagged PELP1 or endogenous PELP1 as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B) Flag‐PELP1 and HA‐WDR18 were expressed in HeLa cells as indicated. HA‐WDR18 was captured on HA‐beads and the bound material was probed by immunoblotting for the presence of ectopically expressed Flag‐tagged PELP1 or endogenous PELP1 as indicated.


( C ) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of endogenous WDR18 by western blotting with an anti ‐ WDR18 antibody .

O O O O O O O O O O O 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) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of endogenous WDR18 by western blotting with an anti‐WDR18 antibody.


( D ) Flag ‐ TEX10 was expressed with HA ‐ PELP1 in HeLa cells , captured on Flag ‐ beads and proteins were immunoblotted as indicated .

O O O O 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) Flag‐TEX10 was expressed with HA‐PELP1 in HeLa cells, captured on Flag‐beads and proteins were immunoblotted as indicated.


( E ) HA ‐ TEX10 was expressed , captured on HA ‐ beads and associated proteins were probed for the presence of endogenous WDR18 using immunoblotting . The inputs represent 2 . 5 % of the total cell lysate .

O O O O O O O O O O O O O O O O O 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) HA‐TEX10 was expressed, captured on HA‐beads and associated proteins were probed for the presence of endogenous WDR18 using immunoblotting. The inputs represent 2.5% of the total cell lysate.


( A - C ) Immunofluorescence staining of endogenous PELP1 , SENP3 , UBF1 and NPM1 in HeLa cells . Nuclei were visualized using DAPI staining .

O O O 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-C) Immunofluorescence staining of endogenous PELP1, SENP3, UBF1 and NPM1 in HeLa cells. Nuclei were visualized using DAPI staining.


( B ) HeLa cells were transfected with siRNA duplexes targeting PELP1 , TEX10 , WDR18 , MDN1 or a control siRNA as indicated . Downregulation of the respective proteins was analysed by western blotting as indicated ( WB , three lower panels ) . At 72 h after transfection , cells were pulse labelled with 32P ‐ orthophosphate for 1 h and chased for 2 . 5 h . RNA was separated on a denaturing agarose gel and detected by ethidium bromide ( EtBr ) staining and autoradiography after drying of the gel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected with siRNA duplexes targeting PELP1, TEX10, WDR18, MDN1 or a control siRNA as indicated. Downregulation of the respective proteins was analysed by western blotting as indicated (WB, three lower panels). At 72 h after transfection, cells were pulse labelled with 32P‐orthophosphate for 1 h and chased for 2.5 h. RNA was separated on a denaturing agarose gel and detected by ethidium bromide (EtBr) staining and autoradiography after drying of the gel.


( C ) The signal intensities of the 28S and 32S rRNA forms were quantified by phosphoimager analysis and the ratio of 28S : 32S rRNA was calculated . Values represent the average of four ( two for siMDN1 ) independent experiments . Error bars indicate s . d .

(C) The signal intensities of the 28S and 32S rRNA forms were quantified by phosphoimager analysis and the ratio of 28S:32S rRNA was calculated. Values represent the average of four (two for siMDN1) independent experiments. Error bars indicate s.d.


( D ) PELP1 is associated with ITS2 ‐ containing RNA species . Endogenous PELP1 was immunoprecipitated from HeLa cells . In total , 10 % of the immunoprecipitated material was analysed by western blotting ( lower panel ) . The remaining material was used for RNA isolation and cDNA synthesis . The cDNA was used as template for PCR amplification using primers within the ITS2 / 28S region or GAPDH as a control . Input corresponds to 10 % of the cell lysate . Vertical line in the upper panel indicates removal of irrelevant adjacent lanes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PELP1 is associated with ITS2‐containing RNA species. Endogenous PELP1 was immunoprecipitated from HeLa cells. In total, 10% of the immunoprecipitated material was analysed by western blotting (lower panel). The remaining material was used for RNA isolation and cDNA synthesis. The cDNA was used as template for PCR amplification using primers within the ITS2/28S region or GAPDH as a control. Input corresponds to 10% of the cell lysate. Vertical line in the upper panel indicates removal of irrelevant adjacent lanes.


( E ) Pre ‐ ribosomal particles were separated by sucrose gradient centrifugation of nuclear extracts . Proteins were recovered from the fractions by TCA precipitation and further analysed by SDS - PAGE and western blotting using antibodies as indicated . Vertical lines solely indicate loading onto two separate gels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pre‐ribosomal particles were separated by sucrose gradient centrifugation of nuclear extracts. Proteins were recovered from the fractions by TCA precipitation and further analysed by SDS-PAGE and western blotting using antibodies as indicated. Vertical lines solely indicate loading onto two separate gels.


( F ) Nucleolar export of pre ‐ 60S particles is strongly compromised in cells depleted of PELP1 , TEX10 , WDR18 , MDN1 or SENP3 . HeLa cells expressing YFP ‐ RpL27 from a tetracycline ‐ inducible promoter were transfected with the indicated siRNAs . After 24 h , expression of YFP ‐ RpL27 was induced and 72 h later the localization of RpL27 was monitored . Pictures were taken using identical exposure times .

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

(F) Nucleolar export of pre‐60S particles is strongly compromised in cells depleted of PELP1, TEX10, WDR18, MDN1 or SENP3. HeLa cells expressing YFP‐RpL27 from a tetracycline‐inducible promoter were transfected with the indicated siRNAs. After 24 h, expression of YFP‐RpL27 was induced and 72 h later the localization of RpL27 was monitored. Pictures were taken using identical exposure times.


( A ) HA ‐ PELP1 was expressed alone or in combination with SUMO1 or SUMO2 in the presence or absence of SV5 ‐ tagged SENP3 in HeLa cells . Expression of the respective proteins was verified by western blotting . Detection of β ‐ tubulin served as loading control .

(A) HA‐PELP1 was expressed alone or in combination with SUMO1 or SUMO2 in the presence or absence of SV5‐tagged SENP3 in HeLa cells. Expression of the respective proteins was verified by western blotting. Detection of β‐tubulin served as loading control.


( B ) SUMO modification of endogenous PELP1 was monitored by anti ‐ PELP1 immunoblotting in HeLa cells transfected with siRNAs as indicated . Depletion of the respective proteins was verified by western blotting .

(B) SUMO modification of endogenous PELP1 was monitored by anti‐PELP1 immunoblotting in HeLa cells transfected with siRNAs as indicated. Depletion of the respective proteins was verified by western blotting.


( C ) HeLa cells were transfected with siRNAs and plasmids as indicated . His ‐ SUMO conjugates were recovered on Ni ‐ NTA beads and subjected to western blotting using anti ‐ PELP1 antibodies .

O O O O O O O O O O O O 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) HeLa cells were transfected with siRNAs and plasmids as indicated. His‐SUMO conjugates were recovered on Ni‐NTA beads and subjected to western blotting using anti‐PELP1 antibodies.


( D ) HA ‐ PELP1 or HA ‐ PELP1K826R was expressed in the presence of SUMO1 or SUMO2 in HeLa cells . Expression of the respective proteins was analysed by western blotting . Detection of β ‐ tubulin served as loading control .

O O O O O O O O O O O O O O O O O O O O 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) HA‐PELP1 or HA‐PELP1K826R was expressed in the presence of SUMO1 or SUMO2 in HeLa cells. Expression of the respective proteins was analysed by western blotting. Detection of β‐tubulin served as loading control.


( E , F ) Flag ‐ p14ARF and SUMO were expressed in HeLa cells and SUMO modification of ectopically expressed HA ‐ PELP1 or endogenous PELP1 was monitored by immunoblotting with anti ‐ HA or anti ‐ PELP1 antibodies as indicated . Arrowheads indicate SUMO - PELP1 conjugates .

(E, F) Flag‐p14ARF and SUMO were expressed in HeLa cells and SUMO modification of ectopically expressed HA‐PELP1 or endogenous PELP1 was monitored by immunoblotting with anti‐HA or anti‐PELP1 antibodies as indicated. Arrowheads indicate SUMO-PELP1 conjugates.


( A ) Flag ‐ SUMO2 was expressed in HeLa cells by transient transfection . Flag ‐ SUMO2 ‐ expressing cells were visualized by indirect immunofluorescence . The localization of endogenous PELP1 was monitored in Flag ‐ SUMO2 ‐ positive cells ( green arrowheads ) or untransfected cells ( white arrowheads ) using anti ‐ PELP1 antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flag‐SUMO2 was expressed in HeLa cells by transient transfection. Flag‐SUMO2‐expressing cells were visualized by indirect immunofluorescence. The localization of endogenous PELP1 was monitored in Flag‐SUMO2‐positive cells (green arrowheads) or untransfected cells (white arrowheads) using anti‐PELP1 antibody.


( B - D ) HeLa cells were transfected with siRNAs as indicated . After fixation and permeabilization , the localization of PELP1 , LAS1L , TEX10 or PES1 was monitored by indirect immunofluorescence using the corresponding antibodies .

O O O O O O O O O O O O O O O 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-D) HeLa cells were transfected with siRNAs as indicated. After fixation and permeabilization, the localization of PELP1, LAS1L, TEX10 or PES1 was monitored by indirect immunofluorescence using the corresponding antibodies.


( E ) HeLa cells were depleted of endogenous PELP1 and reconstituted with siRNA resistant constructs expressing either wild ‐ type HA ‐ tagged PELP1 or a Flag ‐ tagged PELP1 - SUMO2 fusion protein . The localization of the respective proteins was monitored by indirect immunofluorescence using either anti ‐ HA ‐ or anti ‐ Flag ‐ antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were depleted of endogenous PELP1 and reconstituted with siRNA resistant constructs expressing either wild‐type HA‐tagged PELP1 or a Flag‐tagged PELP1-SUMO2 fusion protein. The localization of the respective proteins was monitored by indirect immunofluorescence using either anti‐HA‐ or anti‐Flag‐antibodies.


( F ) The localization of LAS1L or SENP3 was analysed in cells expressing Flag ‐ PELP1 - SUMO2 . Indirect immunofluorescence was done using anti ‐ LAS1L or anti ‐ SENP3 antibodies . Nuclei were counterstained with DAPI .

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

(F) The localization of LAS1L or SENP3 was analysed in cells expressing Flag‐PELP1-SUMO2. Indirect immunofluorescence was done using anti‐LAS1L or anti‐SENP3 antibodies. Nuclei were counterstained with DAPI.


( A ) LAS1L was generated by in vitro transcription / translation and incubated with recombinant E1 and E2 enzymes and SUMO1 or SUMO2 , respectively , in the presence of ATP . In the control reaction ( lane 1 ) SUMO was not added .

O O O O O O O O O O O O O O O O O O O O O O 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) LAS1L was generated by in vitro transcription/translation and incubated with recombinant E1 and E2 enzymes and SUMO1 or SUMO2, respectively, in the presence of ATP. In the control reaction (lane 1) SUMO was not added.


( B ) HeLa cells were transfected with siRNAs and plasmids as indicated . Expression of the respective proteins was verified by western blotting . Detection of β ‐ tubulin served as loading control .

(B) HeLa cells were transfected with siRNAs and plasmids as indicated. Expression of the respective proteins was verified by western blotting. Detection of β‐tubulin served as loading control.


( C ) HeLa cells were transfected with siRNAs and plasmids as indicated . His ‐ SUMO2 conjugates were captured on magnetic Ni ‐ NTA beads and subjected to western blotting using anti ‐ LAS1L antibodies . Vertical lines indicate removal of irrelevant neighbouring lanes from the initial gel ( see Supplementary Figure 14 ) .

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(C) HeLa cells were transfected with siRNAs and plasmids as indicated. His‐SUMO2 conjugates were captured on magnetic Ni‐NTA beads and subjected to western blotting using anti‐LAS1L antibodies. Vertical lines indicate removal of irrelevant neighbouring lanes from the initial gel (see Supplementary Figure 14).


C ) A representative selection of 2D classes of UBR5 . UBR - UBR box ,

C) A representative selection of 2D classes of UBR5. UBR - UBR box,


B ) Sucrose gradient sedimentation profiles of GFPAKIRIN2 in the presence or absence of UBR5 . Gels were imaged using fluorescent imaging ( 488nm ) to visualise the recombinant GFP tag , and stain - free imaging to visualise all proteins . Gels of assays are a representative of at least three experimental replicates . Reactions in B were performed at 37 ° C . The final concentration of E3 enzyme in B : 0 .

B) Sucrose gradient sedimentation profiles of GFPAKIRIN2 in the presence or absence of UBR5. Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels of assays are a representative of at least three experimental replicates. Reactions in B were performed at 37°C. The final concentration of E3 enzyme in B : 0.3µM


C ) Ubiquitination of GFPAKIRIN2 . D ) Ubiquitination of GFPUbAKIRIN2 . E ) Ubiquitination activity of wild - type and mutant UBR5ΔUBA on GFPUbAKIRIN2 substrate . Gels were imaged using fluorescent imaging ( 488nm ) to visualise the recombinant GFP tag , and stain - free imaging to visualise all proteins . Gels were subsequently stained using Coomassie staining to visualise ubiquitin levels . Gels of assays are a representative of at least three experimental replicates . Reactions C were performed at 37 ° C . Reactions in D performed at 25 ° C . The final concentration of E3 enzyme in C , D : 0 .

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C) Ubiquitination of GFPAKIRIN2. D) Ubiquitination of GFPUbAKIRIN2. E) Ubiquitination activity of wild-type and mutant UBR5ΔUBA on GFPUbAKIRIN2 substrate. Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels were subsequently stained using Coomassie staining to visualise ubiquitin levels. Gels of assays are a representative of at least three experimental replicates. Reactions C were performed at 37°C. Reactions in D performed at 25°C. The final concentration of E3 enzyme in C, D: 0.3µM


F ) Sucrose gradient sedimentation profiles of GFPUbAKIRIN2 in the absence ( upper panel ) or in the presence of UBR5wt ( middle panel ) , and UBR5ΔUBA ( lower panel ) . Gels were imaged using fluorescent imaging ( 488nm ) to visualise the recombinant GFP tag , and stain - free imaging to visualise all proteins . Gels of assays are a representative of at least three experimental replicates .

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F) Sucrose gradient sedimentation profiles of GFPUbAKIRIN2 in the absence (upper panel) or in the presence of UBR5wt (middle panel), and UBR5ΔUBA (lower panel). Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels of assays are a representative of at least three experimental replicates.


G ) Ubiquitination activity of wild - type and oligomerisation - defective mutants on GFPUbAKIRIN2 . Gels were imaged using fluorescent imaging ( 488nm ) to visualise the recombinant GFP tag , and stain - free imaging to visualise all proteins . Gels were subsequently stained using Coomassie staining to visualise ubiquitin levels . Gels of assays are a representative of at least three experimental replicates . Reactions in G were performed at 25 ° C . The final concentration of E3 enzyme , in G : 0 . 2µM .

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G) Ubiquitination activity of wild-type and oligomerisation-defective mutants on GFPUbAKIRIN2. Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels were subsequently stained using Coomassie staining to visualise ubiquitin levels. Gels of assays are a representative of at least three experimental replicates. Reactions in G were performed at 25°C. The final concentration of E3 enzyme , in G: 0.2µM.


C ) A selection of 2D classes from Fig 1C . Arrows point to the position of the HECT domain . Scale bar : 100Å . Reactions in C were performed at 25 °

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C) A selection of 2D classes from Fig 1C. Arrows point to the position of the HECT domain. Scale bar: 100Å. Reactions in C were performed at 25°C


F ) Ubiquitination of GFPUbAKIRIN2 by UBR5 and UBR5ΔPL and G ) isolated UBR5HECT . Gels were imaged as described in Figure 3 . Gels of assays are a representative of at least two experimental replicates . All reactions were performed using a final concentration of 0 . 3µM E3 .

F) Ubiquitination of GFPUbAKIRIN2 by UBR5 and UBR5ΔPL and G) isolated UBR5HECT. Gels were imaged as described in Figure 3. Gels of assays are a representative of at least two experimental replicates. All reactions were performed using a final concentration of 0.3µM E3.


( A ) Multiple sequence alignment of Atg8 homologues . Amino ‐ acid sequences of Atg8 and its mammalian homologues are aligned using the Clustal _ W program . Underlined residues represent mutated amino acids in Atg8 .

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(A) Multiple sequence alignment of Atg8 homologues. Amino‐acid sequences of Atg8 and its mammalian homologues are aligned using the Clustal_W program. Underlined residues represent mutated amino acids in Atg8.


( B ) Alignment of Atg8 predicted three ‐ dimensional structure with its mammalian homologues . SWISS MODEL was used to predict Atg8 structure and PyMol was used for presentation . Orange represents the predicted Atg8 structure , and red , blue and green represent GATE ‐ 16 , GABARAP and LC3 , respectively .

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(B) Alignment of Atg8 predicted three‐dimensional structure with its mammalian homologues. SWISS MODEL was used to predict Atg8 structure and PyMol was used for presentation. Orange represents the predicted Atg8 structure, and red, blue and green represent GATE‐16, GABARAP and LC3, respectively.


( C ) Three ‐ dimensional model of Atg8 . The predicted structure contains a ubiquitin fold decorated by two further amino ‐ terminal helices ( amino acids Tyr 49 , Leu 50 , Phe 77 and Phe 79 are marked in green ) .

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(C) Three‐dimensional model of Atg8. The predicted structure contains a ubiquitin fold decorated by two further amino‐terminal helices (amino acids Tyr 49, Leu 50, Phe 77 and Phe 79 are marked in green).


( A ) Cells were grown to mid ‐ exponential phase and then incubated in the presence of 3 . 5 % galactose to induce expression of GFP - Atg8 . Aminopeptidase I ( API ) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti ‐ Atg8 ( right bottom panel ) , anti ‐ GFP ( left bottom panel ) or anti ‐ API antibodies ( upper panel ) .

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(A) Cells were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies (upper panel).


( B ) Cells were pulse - labelled with [ 35S ] methionine and chased on non - radioactive starvation medium . Aliquots were taken at the indicated times and acid - soluble small peptides generated by proteolysis were determined .

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(B) Cells were pulse-labelled with [35S]methionine and chased on non-radioactive starvation medium. Aliquots were taken at the indicated times and acid-soluble small peptides generated by proteolysis were determined.


( C ) Cells were grown in control or in starvation medium and visualized by light microscopy . Arrows point to the pre ‐ autophagosomal structure ( PAS ) , and arrowheads point to GFP within the vacuoles .

(C) Cells were grown in control or in starvation medium and visualized by light microscopy. Arrows point to the pre‐autophagosomal structure (PAS), and arrowheads point to GFP within the vacuoles.


( D ) Cell extracts were subjected to SDS - polyacrylamide gel electrophoresis followed by immunoblot analysis using anti ‐ GFP antibodies . GFP , green fluorescent protein ; mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

(D) Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis using anti‐GFP antibodies. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


( A ) Atg8WT - HA , Atg8F77A - HA , Atg8F79A - HA or Atg8F77 / 79A - HA was transformed into Δatg8 cells . Cell extracts were prepared and subjected to 13 . 5 % SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) containing 6 M urea , followed by immunoblot analysis with anti - Atg8 antibodies .

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(A) Atg8WT-HA, Atg8F77A-HA, Atg8F79A-HA or Atg8F77/79A-HA was transformed into Δatg8 cells. Cell extracts were prepared and subjected to 13.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) containing 6 M urea, followed by immunoblot analysis with anti-Atg8 antibodies.


( B ) Prediction of protein - protein interaction sites in Atg8 ( Neuvirth et al , 2004 ) . A three ‐ dimensional model of Atg8 is coloured according to ProMate prediction ( see supplementary information online ) with red representing the predicted protein - protein binding sites , and Phe 77 and Phe 79 are marked in green within the prediction site .

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(B) Prediction of protein-protein interaction sites in Atg8 ( Neuvirth et al, 2004). A three‐dimensional model of Atg8 is coloured according to ProMate prediction ( see supplementary information online) with red representing the predicted protein-protein binding sites, and Phe 77 and Phe 79 are marked in green within the prediction site.


( C ) Pull ‐ down assay of Atg8 mutants with Atg4 . His - Atg4 was attached to cobalt beads ( Talon metal ‐ affinity resin ) , washed and incubated with total cell extracts of Atg8 mutants . Elution was subjected to SDS - PAGE followed by western blot analysis using anti ‐ Atg8 and anti ‐ Atg4 antibodies .

(C) Pull‐down assay of Atg8 mutants with Atg4. His-Atg4 was attached to cobalt beads (Talon metal‐affinity resin), washed and incubated with total cell extracts of Atg8 mutants. Elution was subjected to SDS-PAGE followed by western blot analysis using anti‐Atg8 and anti‐Atg4 antibodies.


( D ) Localization of green fluorescent protein ( GFP ) - Atg8wt and GFP - Atg877 / 79 cells carrying Atg4 plasmid under normal ( control ) and starvation conditions . Arrows point to the pre ‐ autophagosomal structure , and arrowheads point to GFP within the vacuoles .

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(D) Localization of green fluorescent protein (GFP)-Atg8wt and GFP-Atg877/79 cells carrying Atg4 plasmid under normal (control) and starvation conditions. Arrows point to the pre‐autophagosomal structure, and arrowheads point to GFP within the vacuoles.


( E ) Aminopeptidase I ( API ) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti ‐ API antibodies . mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

(E) Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐API antibodies. mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


( A ) Cell extracts were subjected to 13 . 5 % SDS - polyacrylamide gel electrophoresis containing 6 M urea , followed by immunoblot analysis using anti ‐ Atg8 ‐ 15N peptide antibodies .

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(A) Cell extracts were subjected to 13.5% SDS-polyacrylamide gel electrophoresis containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐15N peptide antibodies.


( B ) Cells were grown in control or in starvation medium and visualized using fluorescence microscopy . Arrows point to the pre ‐ autophagosomal structure , whereas arrowheads point to GFP within the vacuoles .

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(B) Cells were grown in control or in starvation medium and visualized using fluorescence microscopy. Arrows point to the pre‐autophagosomal structure, whereas arrowheads point to GFP within the vacuoles.


( C ) Aminopeptidase I ( API ) maturation and GFP cleavage were tested as described above . Asterisk represents nonspecific band . GFP , green fluorescent protein ; mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

(C) Aminopeptidase I (API) maturation and GFP cleavage were tested as described above. Asterisk represents nonspecific band. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


( A ) Δatg8 cells transformed with GFP - Atg8WT , GFP - Atg8L50A or GFP - Atg8Y49A constructs were grown to mid ‐ exponential phase and then incubated in the presence of 3 . 5 % galactose to induce expression of GFP - Atg8 . API maturation was tested using anti ‐ Atg8 ( right bottom panel ) , anti ‐ GFP ( left bottom panel ) or anti ‐ API antibodies .

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(A) Δatg8 cells transformed with GFP-Atg8WT, GFP-Atg8L50A or GFP-Atg8Y49A constructs were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. API maturation was tested using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies.


( B ) Starvation ‐ induced protein degradation of Δatg8 harbouring the different mutants was tested as described above . API , aminopeptidase I ; GFP , green fluorescent protein ; mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

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(B) Starvation‐induced protein degradation of Δatg8 harbouring the different mutants was tested as described above. API, aminopeptidase I; GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


( A ) GFP - Atg8 localization was determined using fluorescence microscopy ( arrows depict pre ‐ autophagosomal structure , and arrowheads point to GFP within vacuoles ) . Cells were also tested for the appearance of autophagic bodies by using light microscopy .

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(A) GFP-Atg8 localization was determined using fluorescence microscopy (arrows depict pre‐autophagosomal structure, and arrowheads point to GFP within vacuoles). Cells were also tested for the appearance of autophagic bodies by using light microscopy.


( B ) Immunoblot analysis of different cell extracts was carried out using anti ‐ GFP antibodies ( asterisk represents nonspecific band ) .

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(B) Immunoblot analysis of different cell extracts was carried out using anti‐GFP antibodies (asterisk represents nonspecific band).


( C ) Atg8WT - HA , Atg8L50A - HA or Atg8Y49A - HA was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and cell extracts were subjected to SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) followed by immunoblot analysis using anti ‐ HA antibodies ( lower panel ; asterisk represents nonspecific band ) .

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(C) Atg8WT-HA, Atg8L50A-HA or Atg8Y49A-HA was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and cell extracts were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblot analysis using anti‐HA antibodies (lower panel; asterisk represents nonspecific band).


( D ) Atg8WT , Atg8L50A or Atg8Y49A was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and grown under control or starvation conditions for 4 h . Cell extracts were subjected to 13 . 5 % SDS - PAGE containing 6 M urea , followed by immunoblot analysis using anti ‐ Atg8 ‐ N15 antibodies . GFP , green fluorescent protein ; WT , wild type .

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(D) Atg8WT, Atg8L50A or Atg8Y49A was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and grown under control or starvation conditions for 4 h. Cell extracts were subjected to 13.5% SDS-PAGE containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐N15 antibodies. GFP, green fluorescent protein; WT, wild type.


A Western blot analysis of LC3 in SH - SY5Y cells after IU1 treatment ( 100 μM , 24h ) . Graphs represent mean ± SEM . N = 8

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A Western blot analysis of LC3 in SH-SY5Y cells after IU1 treatment (100 μM, 24h). Graphs represent mean± SEM. N= 8


B - D Electron microscope images were taken to quantify the number of autophagosomes and autolysosome present in control ( DMSO ) or IU1 treated cells . At least 55 cells were analysed from 3 different experiments . Graphs represent mean ± SEM

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B-D Electron microscope images were taken to quantify the number of autophagosomes and autolysosome present in control (DMSO) or IU1 treated cells. At least 55 cells were analysed from 3 different experiments. Graphs represent mean± SEM


E Western blot analysis of mitochondrial marker proteins ATP5a , HSP60 and normalised by actin after 24h of DMSO ( C ) or IU1 ( 100 μM ) treatment . N = 3 . Graphs represent mean ± SEM

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E Western blot analysis of mitochondrial marker proteins ATP5a, HSP60 and normalised by actin after 24h of DMSO (C) or IU1 (100 μM) treatment. N=3. Graphs represent mean± SEM


F - I Confocal images of cells transfected with mito - YFP and treated with DMSO ( F , control ) or IU1 ( G ) for 24h . Fragmentation index ( H ) and total mitochondrial volume / cell were measured and represented in ( I ) , mean ± SEM . At least 35 cells were analysed from 3 different experimental conditions . Scale bar 10 μm

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F-I Confocal images of cells transfected with mito-YFP and treated with DMSO (F, control) or IU1 (G) for 24h. Fragmentation index (H) and total mitochondrial volume / cell were measured and represented in (I), mean ±SEM. At least 35 cells were analysed from 3 different experimental conditions. Scale bar 10 μm


J - L Mito - YFP transfected cells were treated with IU1 ( 100 μM , 24h ) , fixed and immuno labelled for LC3 . Number of LC3 dots / cell ( K ) and LC3 dot overlapping mitochondria ( L ) were counted . At least 35 cells from 3 independent experiments were analysed . Scale bar : 10 μm

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J-L Mito-YFP transfected cells were treated with IU1 (100 μM, 24h), fixed and immuno labelled for LC3. Number of LC3 dots / cell (K) and LC3 dot overlapping mitochondria (L) were counted. At least 35 cells from 3 independent experiments were analysed. Scale bar: 10 μm


M - N Cells were transfected with mito - keima and treated with DMSO or IU1 for 24h and subjected to FACS analysis , counting 10 , 000 cells per experiment . ( M ) represents the shift of the signal intensity in IU1 treated cells when excited at 560 nm ( false colour ) . ( N ) represents the average intensity of the mito - keima protein when excited at 560 nm and the ratio between 560 : 405 nm . Graphs represent mean ± SEM from 3 different sets of

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M-N Cells were transfected with mito-keima and treated with DMSO or IU1 for 24h and subjected to FACS analysis, counting 10,000 cells per experiment. (M) represents the shift of the signal intensity in IU1 treated cells when excited at 560 nm (false colour). (N) represents the average intensity of the mito-keima protein when excited at 560 nm and the ratio between 560: 405 nm. Graphs represent mean ± SEM from 3 different sets of experiments


A - B Western blot analysis of the indicated proteins in cell lysates from SH - SY5Y cells , treated with different concentrations of IU1 ( 1 - 100 μM ) for 24h . Bar graphs represents mean ± SEM . Blots are representative of three independent experiments . ANOVA followed by Dunnets test

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A-B Western blot analysis of the indicated proteins in cell lysates from SH-SY5Y cells, treated with different concentrations of IU1 (1-100 μM) for 24h. Bar graphs represents mean ± SEM. Blots are representative of three independent experiments. ANOVA followed by Dunnets test


C - D Representative images of MEF cells knocked out for the indicated mitochondrial dynamics protein and transfected with mito - YFP ( C ) with / without IU1 treatment ( 100 μM , 48h ) . Scale bar 10 μm . ( D ) Mitochondrial volume / cell was measured as described and represented as mean ± SEM . At least 35 cells were

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C-D Representative images of MEF cells knocked out for the indicated mitochondrial dynamics protein and transfected with mito-YFP (C) with / without IU1 treatment (100 μM, 48h). Scale bar 10 μm. (D) Mitochondrial volume / cell was measured as described and represented as mean ±SEM. At least 35 cells were evaluated


E - F Western blot analysis of HSP60 and ATP5a , as mitochondrial marker in the indicated knock out MEF cells ( with / without the indicated DRP1 variant ) after 48h of IU1 treatment . Bar graphs represent mean ± SEM , n = at least 3 . Student ' s t test ;

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E-F Western blot analysis of HSP60 and ATP5a, as mitochondrial marker in the indicated knock out MEF cells (with / without the indicated DRP1 variant) after 48h of IU1 treatment. Bar graphs represent mean ± SEM, n= at least 3. Student's t test;


A - D Wild type MEF ( A ) , Hela ( B ) and PINK1 KO MEF ( C ) cells were treated with IU1 ( 48h , 100 μM ) and electron microscope images were evaluated for autophagic vesicle formation . ( D ) bar graphs represents mean ± SEM number of autophagosomes and autolysosome / cell from 3 different set of experiments ; at least 40 cells were analysed from each

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A-D Wild type MEF (A), Hela (B) and PINK1 KO MEF (C) cells were treated with IU1 (48h, 100 μM) and electron microscope images were evaluated for autophagic vesicle formation. (D) bar graphs represents mean± SEM number of autophagosomes and autolysosome / cell from 3 different set of experiments; at least 40 cells were analysed from each group


E Western blot analysis of HSP60 protein with or without IU1 treatment in the indicated cell type . Bar graphs represent mean ± SEM . N = at least 3 independent experiments . Student ' s t test . * P ≤ 0 . 05 , * * P ≤ 0 . 01

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E Western blot analysis of HSP60 protein with or without IU1 treatment in the indicated cell type. Bar graphs represent mean ± SEM. N= at least 3 independent experiments. Student's t test. *P ≤ 0.05, **P ≤ 0.01


A - B SH - SY5Y cells were immunostained for ATP5a ( green ) and 20S proteasome core ( red ) after IU1 treatment ( 6h - 24h ) . Scale bar 10 μm . Colour mapping of the co - localisation of HSP60 and 20S proteasome from the selected regions were generated by image j and represented in the bottom panel . Intensity of the co - localisation is represented in the colour map scale at the bottom of the panel . ( B ) 20S proteasome subunit and mitochondria co - localisation was quantified by measuring Mander ' s coefficient . Bar graphs represent mean fold change ( mean ± SEM ) from 3 different experiments . At least 30 cells were evaluated for the calculations . ANOVA followed by Dunnet ' s test

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A-B SH-SY5Y cells were immunostained for ATP5a (green) and 20S proteasome core (red) after IU1 treatment (6h-24h). Scale bar 10 μm. Colour mapping of the co-localisation of HSP60 and 20S proteasome from the selected regions were generated by image j and represented in the bottom panel. Intensity of the co-localisation is represented in the colour map scale at the bottom of the panel. (B) 20S proteasome subunit and mitochondria co-localisation was quantified by measuring Mander's coefficient. Bar graphs represent mean fold change (mean ± SEM) from 3 different experiments. At least 30 cells were evaluated for the calculations. ANOVA followed by Dunnet's test


C Electron microscopy images showing subcellular localization of immuno - gold labelled 20S subunit of proteasome complex after 12h or 24h of IU1 treatment . Experiments were repeated twice in two biological replicates with similar results . The contrast in the magnified region is enhanced from the original image to highlight the immunogold labelled signals

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C Electron microscopy images showing subcellular localization of immuno- gold labelled 20S subunit of proteasome complex after 12h or 24h of IU1 treatment. Experiments were repeated twice in two biological replicates with similar results. The contrast in the magnified region is enhanced from the original image to highlight the immunogold labelled signals


D - E Protein proximity ligation assay for Prohibitin2 ( PHB2 ) and LC3 after 24h IU1 treatment . Images showing signal are merged with bright field images to count the signal dots ( cyan false colour ) as depicted in the figure . In the inset only fluorescence signal is shown , without merging with the bright field from the selected regions . Scale bar 10 μm . ( E ) Bar graphs represent mean ± SEM of the number of signal dots per cell from 3 different experiments . Student ' s t test ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001

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D-E Protein proximity ligation assay for Prohibitin2 (PHB2) and LC3 after 24h IU1 treatment. Images showing signal are merged with bright field images to count the signal dots (cyan false colour) as depicted in the figure. In the inset only fluorescence signal is shown, without merging with the bright field from the selected regions. Scale bar 10 μm. (E) Bar graphs represent mean ±SEM of the number of signal dots per cell from 3 different experiments. Student's t test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001


Electron microscope images of mitochondria from SH - SY5Y e , as indicated in the figure , with / without USP14 inhibition / knock down . Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point . Bar graphs represent mean ( ± SEM ) of percentage ruptured mitochondria from at least 3 different experiments . For cells , total 150 - 300 mitochondria for the control group and 150 - 190 mitochondria from the IU1 treated groups were

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Electron microscope images of mitochondria from SH-SY5Y e, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted


Representative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60 , ATP5a and TOM20 . Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins ( with : without trypsin ) from at least 3 independent

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Representative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experiments


Electron microscope images of mitochondria fro MEF WT , MEF PINK1 K as indicated in the figure , with / without USP14 inhibition / knock down . Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point . Bar graphs represent mean ( ± SEM ) of percentage ruptured mitochondria from at least 3 different experiments . For cells , total 150 - 300 mitochondria for the control group and 150 - 190 mitochondria from the IU1 treated groups were

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Electron microscope images of mitochondria fro MEF WT, MEF PINK1 K as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted


Electron microscope images of mitochondria fro Hela an e , as indicated in the figure , with / without USP14 inhibition / knock down . Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point . Bar graphs represent mean ( ± SEM ) of percentage ruptured mitochondria from at least 3 different experiments . For cells , total 150 - 300 mitochondria for the control group and 150 - 190 mitochondria from the IU1 treated groups were

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Electron microscope images of mitochondria fro Hela an e, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted


Electron microscope images of mitochondria fro Drosophila thoracic muscle , as indicated in the figure , with / without USP14 inhibition / knock down . Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point . Bar graphs represent mean ( ± SEM ) of percentage ruptured mitochondria from at least 3 different experiments For flies , more than 200 mitochondria from each group were counted from 5 different

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Electron microscope images of mitochondria fro Drosophila thoracic muscle, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments For flies, more than 200 mitochondria from each group were counted from 5 different experiments


A Life span analysis of flies of the indicated genetic background . At least 50 flies were monitored from each group . Log - rank test ( Mantel - Cox test , Mantel - Haenszel test , p < 0 . 0001 )

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A Life span analysis of flies of the indicated genetic background. At least 50 flies were monitored from each group. Log-rank test (Mantel-Cox test, Mantel-Haenszel test, p < 0.0001)


B Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotypes . n = 4 - 7 . ANOVA followed by Newman Keuls test

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B Graph bar shows mean± SEM of the climbing performance of flies of the indicated genotypes. n=4-7. ANOVA followed by Newman Keuls test


C Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes . Graph shows respiratory control rate ( RCR ) calculated as described in materials and methods . Graphs represent mean ± SEM . N = 4 - 5 , ANOVA followed by Newman Keuls test

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C Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes. Graph shows respiratory control rate (RCR) calculated as described in materials and methods. Graphs represent mean± SEM. N=4-5, ANOVA followed by Newman Keuls test


D Dopamine content in the fly heads ( 15 day old ) was measured by HPLC and the individual fold change values are represented in the scatter graph . Graphs represent mean ± SEM . N = 14 , 11 and 11 fly heads for control , PINK1 and PINK1 + USP14 KD fly respectively . ANOVA followed by Newman Keuls test

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D Dopamine content in the fly heads (15 day old) was measured by HPLC and the individual fold change values are represented in the scatter graph. Graphs represent mean± SEM. N= 14, 11 and 11 fly heads for control, PINK1 and PINK1+USP14 KD fly respectively. ANOVA followed by Newman Keuls test


E Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes . The experiment was repeated 3 times in replicates . Bar graphs represent quantification of electron dense mitochondria ( represented as grey bar ) and depolarised mitochondrial with ruptured cristae ( represented as white bars ) from the indicated genotypes

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E Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes. The experiment was repeated 3 times in replicates. Bar graphs represent quantification of electron dense mitochondria (represented as grey bar) and depolarised mitochondrial with ruptured cristae (represented as white bars) from the indicated genotypes


( A ) Life span analysis of flies of the indicated genetic background . At least 50 flies were analysed for each group . Log - rank test ( Mantel - Cox test , Mantel - Haenszel test , p < 0 . 0001 )

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(A) Life span analysis of flies of the indicated genetic background. At least 50 flies were analysed for each group. Log-rank test (Mantel-Cox test, Mantel-Haenszel test, p < 0.0001)


( B ) Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotypes . One way ANOVA , Newman Keuls test , n = 3

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(B) Graph bar shows mean± SEM of the climbing performance of flies of the indicated genotypes. One way ANOVA, Newman Keuls test, n=3


( C ) Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes . Graph shows ( mean ± SEM , n = 3 independent experiments ) respiratory control rate ( RCR ) calculated as described . One way ANOVA , Newman Keuls test

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(C) Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes. Graph shows (mean± SEM, n=3 independent experiments) respiratory control rate (RCR) calculated as described. One way ANOVA, Newman Keuls test


( D ) Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes . The experiment was repeated three times in replicates . Bar graphs represent quantification of electron dense mitochondria ( represented as grey bar ) and mitochondrial with ruptured cristae ( represented as white bars ) from the indicated genotypes

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(D) Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes. The experiment was repeated three times in replicates. Bar graphs represent quantification of electron dense mitochondria (represented as grey bar) and mitochondrial with ruptured cristae (represented as white bars) from the indicated genotypes


, B . survival of Ssrp1fl / fl ; CreERT2 + / + mice of different ages ( weeks ) after the start of tamoxifen ( TMX ) treatment . B . Kaplan - Meier survival curves . Log - rank test p = 0 . 032 for the oldest male age group versus all other male groups ; p = 0 . 013 for all females versus all males ; for all other comparisons p > 0 . 05 . n = 6 - 10 mice .

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, B. survival of Ssrp1fl/fl; CreERT2+/+ mice of different ages (weeks) after the start of tamoxifen (TMX) treatment. B. Kaplan-Meier survival curves. Log-rank test p = 0.032 for the oldest male age group versus all other male groups; p = 0.013 for all females versus all males; for all other comparisons p > 0.05. n = 6-10 mice.


C . Normalized blood cell counts in Ssrp1fl / fl ; CreERT2 + / + mice treated with vehicle or tamoxifen on day 7 after start of treatment . Mean ± SD , * indicates p < 0 . 05 ( unpaired t - test ) . n = 3 mice .

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C. Normalized blood cell counts in Ssrp1fl/fl; CreERT2+/+ mice treated with vehicle or tamoxifen on day 7 after start of treatment. Mean ± SD, * indicates p < 0.05 (unpaired t-test). n = 3 mice.


A . H & E staining of mouse organ sections . Scale bars : spleen , colon 100 μM , bone marrow - 50 μM .

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A. H&E staining of mouse organ sections. Scale bars: spleen, colon 100 μM, bone marrow - 50 μM.


A , B . Effect of tamoxifen on replication in the spleen ( A ) and intestine ( B ) of Ssrp1fl / fl ; CreERT2 + / + mice . EdU was given to mice three days after the end of tamoxifen treatment ( 1 hour before tissue collection ) . Organ sections were stained for total DNA ( Hoechst ) , EdU , and SSRP1 . Scale bars - 100 μM . C . Quantitation of EdU staining . Mean number of EdU positive cells per field of view + / - SD . n > 5 fields of view per mouse . 3 mice were analyzed . D . Quantitation of cleaved caspase - 3 staining . Mean number of positive cells per fields of view + / - SD , n > 5 fields of view per mouse Two male and female mice were analyzed . Data information : P - values for C and D were calculated using the unpaired t - test .

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A, B. Effect of tamoxifen on replication in the spleen (A) and intestine (B) of Ssrp1fl/fl; CreERT2+/+ mice. EdU was given to mice three days after the end of tamoxifen treatment (1 hour before tissue collection). Organ sections were stained for total DNA (Hoechst), EdU, and SSRP1. Scale bars - 100 μM. C. Quantitation of EdU staining. Mean number of EdU positive cells per field of view +/- SD. n > 5 fields of view per mouse. 3 mice were analyzed. D. Quantitation of cleaved caspase-3 staining. Mean number of positive cells per fields of view +/- SD, n > 5 fields of view per mouse Two male and female mice were analyzed. Data information: P-values for C and D were calculated using the unpaired t-test.


D . Fold - change in the proportion of cells in each cluster shown in panel A in samples from tamoxifen - treated versus vehicle - treated mice . Cell types are identified by color as in panel B .

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D. Fold-change in the proportion of cells in each cluster shown in panel A in samples from tamoxifen-treated versus vehicle-treated mice. Cell types are identified by color as in panel B.


D . UMAP plots with color - coded intestinal stem cells identified by transcriptional signatures of intestinal stem cells or by expression of Lgr5 or Gkn3 genes .

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D. UMAP plots with color-coded intestinal stem cells identified by transcriptional signatures of intestinal stem cells or by expression of Lgr5 or Gkn3 genes.