Control or KO ATAD3B HeLa cells were treated with 4 mM 3 - NPA for 2 h . Cells were washed with fresh medium and incubated for another 1 h . Cells were then fixed and immunostained with DAPI and anti - 8 - oxo - dG antibodies and were analyzed by confocal microscopy . Scale bar , 10 µm . Quantification of the relative 8 - oxo - dG fluorescence intensity in cells described in ( D ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 .

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Control or KO ATAD3B HeLa cells were treated with 4 mM 3-NPA for 2 h. Cells were washed with fresh medium and incubated for another 1 h. Cells were then fixed and immunostained with DAPI and anti-8-oxo-dG antibodies and were analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in cells described in (D). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test , N.S., not significant, **p < 0.01.


PINK1 KO HeLa cells stably expressing mito - Keima were infected with control or shATAD3B . Five days later , cells were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h . Cells were then analyzed and imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima by confocal microscopy . Scale bar , 10 µm . Quantification of the relative ratio of red to green fluorescence intensity ( 561nm / 458nm ) of the cells described in ( A ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 .

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

PINK1 KO HeLa cells stably expressing mito-Keima were infected with control or shATAD3B. Five days later, cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were then analyzed and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561nm/458nm) of the cells described in (A). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01.


HeLa cells were treated with DMSO , 4 mM 3 - NPA , 8 mM 3 - NPA , or 10 µM CCCP for 2 h , and cell lysates were then analyzed by Western blotting using the indicated antibodies .

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HeLa cells were treated with DMSO, 4 mM 3-NPA, 8 mM 3-NPA, or 10 µM CCCP for 2 h, and cell lysates were then analyzed by Western blotting using the indicated antibodies.


PINK1 KO HeLa cells stably expressing ATAD3A - Flag ( 3A - Flag ) or ATAD3B - Flag ( 3B - Flag ) were mixed with control PINK1 KO cells respectively , and incubated for 24 h . Cells were treated with 4 mM 3 - NPA ( D ) or 200 µM H2O2 ( F ) for 2 h , and then washed with fresh medium and incubated for another 1 h . Cells were fixed and immunostained with anti - 8 - oxo - dG and anti - Flag antibodies and analyzed by confocal microscopy . 8 - oxo - dG fluorescence intensity of cells treated with 3 - NPA ( E ) or H2O2 ( G ) was quantified by ImageJ software . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by individual two - tailed student ' s t - test , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 . Scale bar , 10 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 O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PINK1 KO HeLa cells stably expressing ATAD3A-Flag (3A-Flag) or ATAD3B-Flag (3B-Flag) were mixed with control PINK1 KO cells respectively, and incubated for 24 h. Cells were treated with 4 mM 3-NPA (D) or 200 µM H2O2 (F) for 2 h, and then washed with fresh medium and incubated for another 1 h. Cells were fixed and immunostained with anti-8-oxo-dG and anti-Flag antibodies and analyzed by confocal microscopy. 8-oxo-dG fluorescence intensity of cells treated with 3-NPA (E) or H2O2 (G) was quantified by ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by individual two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. Scale bar, 10 µm.


Control ( GFP + ) and shATAD3B ( GFP - ) PINK1 KO HeLa cells were mixed and incubated for 24 h . Cells were then treated with 4 mM 3 - NPA ( H ) or 200 µM H2O2 ( J ) for 2 h , and washed with fresh medium , and incubated for another 1 h . Cells were fixed and immunostained with anti - 8 - oxo - dG and DAPI , then analyzed by confocal microscopy . Control or shATAD3B cells are circled by white dashed lines . 8 - oxo - dG fluorescence intensity of cells treated with 3 - NPA ( I ) or H2O2 ( K ) was quantified using ImageJ software . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , * * p < 0 . 01 . Scale bar , 10 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Control (GFP+) and shATAD3B (GFP-) PINK1 KO HeLa cells were mixed and incubated for 24 h. Cells were then treated with 4 mM 3-NPA (H) or 200 µM H2O2 (J) for 2 h, and washed with fresh medium, and incubated for another 1 h. Cells were fixed and immunostained with anti-8-oxo-dG and DAPI, then analyzed by confocal microscopy. Control or shATAD3B cells are circled by white dashed lines. 8-oxo-dG fluorescence intensity of cells treated with 3-NPA (I) or H2O2 (K) was quantified using ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, **p < 0.01. Scale bar, 10 µm.


293T cells were treated with H2O2 for 2 h , and cell lysates were then immunoprecipitated with Dynabeads Protein G pre - coupled with rabbit IgG ( control ) or anti - ATAD3 antibody , followed by Western blotting with anti - LC3B or anti - ATAD3 antibodies .

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293T cells were treated with H2O2 for 2 h, and cell lysates were then immunoprecipitated with Dynabeads Protein G pre-coupled with rabbit IgG (control) or anti-ATAD3 antibody, followed by Western blotting with anti-LC3B or anti-ATAD3 antibodies.


ATAD3 DKO HeLa cells expressing GFP - LC3B were transiently transfected with control , ATAD3A - Flag or ATAD3B - Flag . 48 h after transfection , cell lysates were immunoprecipitated ( IP ) with anti - Flag M2 affinity gel , and analyzed by Western blotting using anti - Flag or anti - GFP antibodies .

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ATAD3 DKO HeLa cells expressing GFP-LC3B were transiently transfected with control, ATAD3A-Flag or ATAD3B-Flag. 48 h after transfection, cell lysates were immunoprecipitated (IP) with anti-Flag M2 affinity gel, and analyzed by Western blotting using anti-Flag or anti-GFP antibodies.


HeLa cells were treated with 200 µM H2O2 for 2 h , and cell lysates were incubated with the indicated GST - ATAD3A or GST - ATAD3B protein fragments ( expressed in E . coli ) coupled to glutathione agarose beads ( Pierce ) for GST pull - down assay . Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B .

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HeLa cells were treated with 200 µM H2O2 for 2 h, and cell lysates were incubated with the indicated GST-ATAD3A or GST-ATAD3B protein fragments (expressed in E.coli) coupled to glutathione agarose beads (Pierce) for GST pull-down assay. Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B.


The indicated GST - ATAD3A or GST - ATAD3B protein fragments and His - LC3B were expressed in E . coli and purified . GST pull - down assay was performed using glutathione agarose beads . Eluted protein samples were analyzed by Western blotting using anti - GST or anti - His antibodies .

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The indicated GST-ATAD3A or GST-ATAD3B protein fragments and His-LC3B were expressed in E.coli and purified. GST pull-down assay was performed using glutathione agarose beads. Eluted protein samples were analyzed by Western blotting using anti-GST or anti-His antibodies.


The indicated GST - ATAD3B ( WT ) or GST - ATAD3B ( LIR mutations ) and His - LC3 were expressed in E . coli and purified . GST pull - down assay was performed using the glutathione agarose beads . Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B .

The indicated GST-ATAD3B(WT) or GST-ATAD3B (LIR mutations) and His-LC3 were expressed in E.coli and purified. GST pull-down assay was performed using the glutathione agarose beads. Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B.


HeLa expressing GFP - LC3 monoclonal cell line was infected with lentivirus particles containing control or shATAD3B . Five days later , cells were treated with DMSO , or 200 µM H2O2 for 2 h . Cells were fixed and immunostained with anti - Tom20 and imaged by confocal microscopy . The white arrows indicate the LC3 puncta colocalizing or contacting with Tom20 ( mitochondria ) . Scale bar , 10 µm .

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HeLa expressing GFP-LC3 monoclonal cell line was infected with lentivirus particles containing control or shATAD3B. Five days later, cells were treated with DMSO, or 200 µM H2O2 for 2 h. Cells were fixed and immunostained with anti-Tom20 and imaged by confocal microscopy. The white arrows indicate the LC3 puncta colocalizing or contacting with Tom20 (mitochondria). Scale bar, 10 µm.


Quantification of the GFP - LC3 puncta colocalized or contacted with Tom20 ( mitochondria ) in cells described in ( G ) . Data are presented as mean ± SD ( n = 30 cells from 3 coverslips ) , statistical significance was assessed by a two - way ANOVA , N . S . , not significant , * * * p < 0 . 001 .

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Quantification of the GFP-LC3 puncta colocalized or contacted with Tom20 (mitochondria) in cells described in (G). Data are presented as mean ± SD (n =30 cells from 3 coverslips), statistical significance was assessed by a two-way ANOVA, N.S., not significant, ***p < 0.001.


293T cells transiently transfected with control or ATAD3B - Flag were treated with or without 200 µM H2O2 for 2 h . Cell lysates were then immunoprecipitated with Dynabeads Protein G pre - coupled with anti - Flag antibody , followed by Western blotting with anti - Flag or anti - LC3B antibodies . Relative protein levels of LC3B and ATAD3B - Flag were further evaluated by

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

293T cells transiently transfected with control or ATAD3B-Flag were treated with or without 200 µM H2O2 for 2 h. Cell lysates were then immunoprecipitated with Dynabeads Protein G pre-coupled with anti-Flag antibody, followed by Western blotting with anti-Flag or anti-LC3B antibodies. Relative protein levels of LC3B and ATAD3B-Flag were further evaluated by densitometry


Mic10 KO COS7 cells stably expressing ATAD3A - Flag or ATAD3B - Flag were treated with DMSO or 200 µM H2O2 for 2 h , and then fixed and immunostained with anti - Tom20 and anti - Flag antibodies , and analyzed by confocal microscopy . Scale bar , 10 µm .

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Mic10 KO COS7 cells stably expressing ATAD3A-Flag or ATAD3B-Flag were treated with DMSO or 200 µM H2O2 for 2 h, and then fixed and immunostained with anti-Tom20 and anti-Flag antibodies, and analyzed by confocal microscopy. Scale bar, 10 µm.


293T cells treated with DMSO ( B ) or H2O2 ( 200 µM , 2 h ) ( D ) , were harvested for mitochondrial isolation . Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice , and then were analyzed by Western blotting with anti - ATAD3 , anti - Tom20 ( OMM ) , anti - Tim23 ( IMS ) , and anti - HSP60 ( matrix ) . Relative protein levels of proteins in ( B ) or ( D ) were further evaluated by densitometry analysis using ImageJ software . Relative trends of proteolysis of indicated mitochondrial proteins from DMSO - or H2O2 - treated cells were shown ( C and E ) . Data are presented as mean ± SD ( n = 3 independent experiments ) .

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293T cells treated with DMSO (B) or H2O2 (200 µM, 2 h) (D), were harvested for mitochondrial isolation. Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice, and then were analyzed by Western blotting with anti-ATAD3, anti-Tom20 (OMM), anti-Tim23 (IMS), and anti-HSP60 (matrix). Relative protein levels of proteins in (B) or (D) were further evaluated by densitometry analysis using ImageJ software. Relative trends of proteolysis of indicated mitochondrial proteins from DMSO- or H2O2-treated cells were shown (C and E). Data are presented as mean ± SD (n = 3 independent experiments).


Control , ATAD3A KO , ATAD3B KO , or ATAD3 DKO HeLa cells were harvested for mitochondrial isolation . Purified mitochondria were treated with DMSO or EDC ( 20 mM ) for 30 min , and then were analyzed by Western blotting with the indicated 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

Control, ATAD3A KO, ATAD3B KO, or ATAD3 DKO HeLa cells were harvested for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min, and then were analyzed by Western blotting with the indicated antibodies.


293T cells were treated with DMSO , 200 µM H2O2 , 10 mM NAC , or 200 µM H2O2 plus 10 mM NAC for 2 h . Cells were then harvested and used for mitochondrial isolation . Purified mitochondria were treated with DMSO or EDC ( 20 mM ) for 30 min at 37 ℃ , and then were analyzed by Western blotting with the indicated antibodies . Quantification of the relative protein level of ATAD3 oligomers to HSP60 described in ( F ) . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * p < 0 . 05 .

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

293T cells were treated with DMSO, 200 µM H2O2, 10 mM NAC, or 200 µM H2O2 plus 10 mM NAC for 2 h. Cells were then harvested and used for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37 ℃, and then were analyzed by Western blotting with the indicated antibodies. Quantification of the relative protein level of ATAD3 oligomers to HSP60 described in (F). Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.


143B , 143B ρ0 cells were harvested for mitochondrial isolation . Purified mitochondria were treated with DMSO or EDC ( 20 mM ) for 30 min at 37 ℃ , and then were analyzed by Western blotting with the indicated antibodies .

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143B, 143B ρ0 cells were harvested for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37 ℃, and then were analyzed by Western blotting with the indicated antibodies.


293T cells were treated with DMSO or ddC ( 50 µM ) plus EB ( 0 . 5 µg / ml ) for 10 days . Cells were then harvested and used for mitochondrial isolation . Purified mitochondria were treated with DMSO or EDC ( 20 mM ) for 30 min at 37 ℃ , and then were analyzed by Western blotting with the indicated antibodies .

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

293T cells were treated with DMSO or ddC (50 µM) plus EB (0.5 µg/ml) for 10 days. Cells were then harvested and used for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37℃, and then were analyzed by Western blotting with the indicated antibodies.


ATAD3A KO HeLa cells were harvested for mitochondrial isolation . Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice , and then were analyzed by Western blotting with anti - ATAD3 , anti - Tom20 ( OMM ) , anti - Tim23 ( IMS ) , and anti - HSP60 ( matrix ) ( K ) . Relative protein levels of proteins in ( K ) were further evaluated by densitometry analysis using ImageJ software . Relative trends of proteolysis of indicated mitochondrial proteins from ATAD3A KO HeLa cells were shown ( L ) . Data are presented as mean ± SD ( n = 3 independent experiments ) .

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

ATAD3A KO HeLa cells were harvested for mitochondrial isolation. Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice, and then were analyzed by Western blotting with anti-ATAD3, anti-Tom20 (OMM), anti-Tim23 (IMS), and anti-HSP60 (matrix) (K). Relative protein levels of proteins in (K) were further evaluated by densitometry analysis using ImageJ software. Relative trends of proteolysis of indicated mitochondrial proteins from ATAD3A KO HeLa cells were shown (L). Data are presented as mean ± SD (n = 3 independent experiments).


ρ0206 _ A and ρ0206 _ B cells were stained with mitoSOX and analyzed by confocal microscopy . DIC , Differential Interference Contrast . Scale bar , 25 µm . Quantification of the MitoSOX fluorescence intensity described in ( A ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by student ' s t - test , * * p < 0 . 01 .

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ρ0206_A and ρ0206_B cells were stained with mitoSOX and analyzed by confocal microscopy. DIC, Differential Interference Contrast. Scale bar, 25 µm. Quantification of the MitoSOX fluorescence intensity described in (A). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by student's t-test, **p < 0.01.


Mitochondria carrying the 3243A > G mutation in the tRNA Leu ( UUR ) gene from the same MELAS myoblasts were transferred into ρ0 206 cell line to generate ρ0206 _ A ( 100 % wild - type mtDNA ) and ρ0206 _ B ( ~ 90 % m . 3243A > G mtDNA ) cell lines . ρ0206 _ A and ρ0206 _ B cell lysates were analyzed by Western blotting with anti - ATAD3 or anti - Tubulin antibodies . Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software . Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by student ' s t - test , * * * p < 0 . 001 .

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Mitochondria carrying the 3243A>G mutation in the tRNA Leu(UUR) gene from the same MELAS myoblasts were transferred into ρ0 206 cell line to generate ρ0206_A (100% wild-type mtDNA) and ρ0206_B (~90% m.3243A>G mtDNA) cell lines. ρ0206_A and ρ0206_B cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-Tubulin antibodies. Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software. Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by student's t-test, ***p < 0.001.


Normal or MELAS patient fibroblasts cell lysates were analyzed by Western blotting with anti - ATAD3 or anti - Tubulin antibodies . Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software . Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by student ' s t - test , * * * p < 0 . 001 .

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

Normal or MELAS patient fibroblasts cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-Tubulin antibodies. Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software. Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by student's t-test, ***p < 0.001.


ρ0206 _ A , ρ0206 _ B , and ρ0206 - B cells pretreated with ddC ( 50µM ) plus EB ( 0 . 5µg / ml ) for 10 days , and then harvested and lysed . Cell lysates were analyzed by Western blotting with anti - ATAD3 or anti - GAPDH antibodies .

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ρ0206_A, ρ0206_B, and ρ0206-B cells pretreated with ddC (50µM) plus EB (0.5µg/ml) for 10 days, and then harvested and lysed. Cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-GAPDH antibodies.


ρ0206 _ B cells were infected with control , ATAD3B - Flag or ATAD3B ( mLIR ) - Flag . After two weeks , total DNA from cells was isolated and used to calculate the mutation rate of 3243A > G by quantitative real - time PCR using the TaqMan Probe . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * p < 0 . 05 .

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ρ0206_B cells were infected with control, ATAD3B-Flag or ATAD3B (mLIR)-Flag. After two weeks, total DNA from cells was isolated and used to calculate the mutation rate of 3243A>G by quantitative real-time PCR using the TaqMan Probe. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.


ρ0206 _ B cells were infected with control , ATAD3A - Flag , ATAD3B - Flag , or ATAD3B ( mLIR ) - Flag . After two weeks , the cellular ATP levels were measured . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * p < 0 . 05 .

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ρ0206_B cells were infected with control, ATAD3A-Flag, ATAD3B-Flag, or ATAD3B (mLIR)-Flag. After two weeks, the cellular ATP levels were measured. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.


MELAS patient fibroblasts were infected with control , ATAD3A - Flag , ATAD3B - Flag or ATAD3B ( mLIR ) - Flag . After two weeks , total DNA was isolated from fibroblasts and used to calculate the mutation rate of 3243A > G by quantitative real - time PCR using the TaqMan Probe . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * * p < 0 . 01 .

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

MELAS patient fibroblasts were infected with control, ATAD3A-Flag, ATAD3B-Flag or ATAD3B(mLIR)-Flag. After two weeks, total DNA was isolated from fibroblasts and used to calculate the mutation rate of 3243A>G by quantitative real-time PCR using the TaqMan Probe. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, **p < 0.01.


Confocal images of the GFP fluorescent patterns in seedlings expressing ATG5 - GFP or GFP - ATG8 . ( a , b ) Control conditions : cytosolic signal associated with rare fluorescent puncta . ( c , d ) High increase of fluorescent puncta after 2 h of nutrient starvation . Note ring - like ATG5 structures in c . ( e , f ) Wortmannin treatment blocks the occurrence of fluorescent ATG5 or ATG8 structures . ( g , h ) Maximum intensity projections of vacuolar focal planes , allowing visualization of autophagic bodies after 4 h of nutrient starvation in the presence of concanamycin A . Vacuolar ATG5 - GFP fluorescence is not detected ( g ) , while GFP - ATG8 signals are abundant ( h ) . An inset in g , corresponding to a cortical focal plane , shows that formation of cytosolic ATG5 structures in the cytosol is not inhibited by concanamycin A treatment . Maximum intensity projections of 10 - μm - deep deconvolved stacks , of cortical ( a - f , and inset in g ) or vacuolar focal planes ( g , h ) . Scale bar , 10 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal images of the GFP fluorescent patterns in seedlings expressing ATG5-GFP or GFP-ATG8. (a,b) Control conditions: cytosolic signal associated with rare fluorescent puncta. (c,d) High increase of fluorescent puncta after 2 h of nutrient starvation. Note ring-like ATG5 structures in c. (e,f) Wortmannin treatment blocks the occurrence of fluorescent ATG5 or ATG8 structures. (g,h) Maximum intensity projections of vacuolar focal planes, allowing visualization of autophagic bodies after 4 h of nutrient starvation in the presence of concanamycin A. Vacuolar ATG5-GFP fluorescence is not detected (g), while GFP-ATG8 signals are abundant (h). An inset in g, corresponding to a cortical focal plane, shows that formation of cytosolic ATG5 structures in the cytosol is not inhibited by concanamycin A treatment. Maximum intensity projections of 10-μm-deep deconvolved stacks, of cortical (a-f, and inset in g) or vacuolar focal planes (g,h). Scale bar, 10 μm.


( a ) Differential interference contrast micrographs of root epidermal cells incubated for 8 h with concanamycin A , in nutrient starvation conditions . Autophagic bodies accumulation in the vacuole is clearly visible in wild - type seedlings , absent in the atg5 - 1 mutant and restored in atg5 - 1 seedlings expressing ATG5 - GFP . ( b ) Confocal microscopy . The ATG5 - GFP fluorescence pattern in the atg5 - 1 background is identical to that observed in wild - type seedling , including ring - like structures . Scale bar , 15 μm .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Differential interference contrast micrographs of root epidermal cells incubated for 8 h with concanamycin A, in nutrient starvation conditions. Autophagic bodies accumulation in the vacuole is clearly visible in wild-type seedlings, absent in the atg5-1 mutant and restored in atg5-1 seedlings expressing ATG5-GFP. (b) Confocal microscopy. The ATG5-GFP fluorescence pattern in the atg5-1 background is identical to that observed in wild-type seedling, including ring-like structures. Scale bar, 15 μm.


( a ) Dual observation of ATG5 - GFP and mCh - ATG8 under nutrient starvation reveals a partial colocalization of the two reporters . Merge of the GFP ( green ) and ATG8 ( magenta ) channels of maximum intensity projections of 20 - μm - deep deconvolved stacks . Scale bar , 3 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

(a) Dual observation of ATG5-GFP and mCh-ATG8 under nutrient starvation reveals a partial colocalization of the two reporters. Merge of the GFP (green) and ATG8 (magenta) channels of maximum intensity projections of 20-μm-deep deconvolved stacks. Scale bar, 3 μm.


( b ) Real - time imaging of an individual autophagosomal structure , showing one deconvolved image per 20 s . The ATG5 ring formation phase is defined as the steps up to establishment of the maximum diameter of the ring structure . The following steps , defined as the decay phase , being more dynamic , are depicted also at higher time resolution . Single - plane merged images of ATG5 - GFP ( green ) and mCh - ATG8 ( magenta ) channels , deconvolved . Scale bar , 1 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 O O O O O O O O O O O O O O O

(b) Real-time imaging of an individual autophagosomal structure, showing one deconvolved image per 20 s. The ATG5 ring formation phase is defined as the steps up to establishment of the maximum diameter of the ring structure. The following steps, defined as the decay phase, being more dynamic, are depicted also at higher time resolution. Single-plane merged images of ATG5-GFP (green) and mCh-ATG8 (magenta) channels, deconvolved. Scale bar, 1 μm.


( c ) Representative FRAP experiment on mCh - ATG8 during the ring formation phase . Deconvolved images . Scale bar , 1 μm . The corresponding fluorescence recovery kinetic , measured on non - deconvolved images , is shown on the right .

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

(c) Representative FRAP experiment on mCh-ATG8 during the ring formation phase. Deconvolved images. Scale bar, 1 μm. The corresponding fluorescence recovery kinetic, measured on non-deconvolved images, is shown on the right.


( d ) Representative FRAP experiment of mCh - ATG8 during the ring decay phase , presented as in c .

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(d) Representative FRAP experiment of mCh-ATG8 during the ring decay phase, presented as in c.


( e ) Box and whiskers representations of fluorescence recoveries during ATG5 ring formation ( n = 11 ) and decay ( n = 17 ) . Whiskers comprise the 10 - 90 percentile , the box the two central quartiles . The mean recovery values are indicated by an horizontal line . Recoveries during ATG5 ring formation and ring decay were significantly different according to the Mann - Whitney statistical test ( * * * P0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Box and whiskers representations of fluorescence recoveries during ATG5 ring formation (n=11) and decay (n=17). Whiskers comprise the 10-90 percentile, the box the two central quartiles. The mean recovery values are indicated by an horizontal line. Recoveries during ATG5 ring formation and ring decay were significantly different according to the Mann-Whitney statistical test (***P0.001).


( a ) Correlative light - electron microscopy of GFP - ATG8 - labelled autophagosomal structures . Correlation of a TEM micrograph of a root transverse section ( 1 ) with fluorescent imaging of GFP - ATG8 ( arrowheads , 2 ) confirms the association of ATG8 signal with autophagosome ( 3 ) . A closer view shows close contacts with ER - like membranes ( empty arrowheads , 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 O O O O O O O O O O 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) Correlative light-electron microscopy of GFP-ATG8-labelled autophagosomal structures. Correlation of a TEM micrograph of a root transverse section (1) with fluorescent imaging of GFP-ATG8 (arrowheads, 2) confirms the association of ATG8 signal with autophagosome (3). A closer view shows close contacts with ER-like membranes (empty arrowheads, 4).


( b ) Immunogold labelling of GFP - ATG8 on cryosections , employing an anti - GFP antibody ( arrowheads ) . Similar interactions between ER membranes and autophagosomal structures can be detected either on cryofixed ( samples c ) or after chemical fixation ( d ) . Empty arrowheads : connections with ER - like membranes . Scale bars , 5 μm in a ( 1 - 3 ) ; 0 . 5 μm in a ( 4 ) and b ( 1 - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunogold labelling of GFP-ATG8 on cryosections, employing an anti-GFP antibody (arrowheads). Similar interactions between ER membranes and autophagosomal structures can be detected either on cryofixed (samples c) or after chemical fixation (d). Empty arrowheads: connections with ER-like membranes. Scale bars, 5 μm in a(1-3); 0.5 μm in a(4) and b(1-3).


( A ) p53 - dependent dephosphorylation of Ulk1 at Ser637 . p53 + / + and p53 − / − MEFs were treated with etoposide ( 10 µM ) , and the expression of each protein was examined by western blotting . Dead cells ( % ) indicates the population of apoptotic cells assessed by propidium iodide ( PI ) staining . α - Tubulin was used as a loading control . A semiquantitative analysis of protein expression is shown in Fig . EV1 .

O 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

(A) p53-dependent dephosphorylation of Ulk1 at Ser637. p53+/+ and p53−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells assessed by propidium iodide (PI) staining. α-Tubulin was used as a loading control. A semiquantitative analysis of protein expression is shown in Fig. EV1.


( B , C ) PPM1D - dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy . PPM1D + / + MEFs and PPM1D − / − MEFs were treated with etoposide ( 10 µM ) , and the expression of each protein was examined by western blotting . Dead cells ( % ) indicates the population of apoptotic cells .

O O O O O O O O O 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

(B, C) PPM1D-dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy. PPM1D+/+MEFs and PPM1D−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells.


( B , C ) PPM1D - dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy . PPM1D + / + MEFs and PPM1D − / − MEFs were treated with etoposide ( 10 µM ) , and the expression of each protein was examined by western blotting . Dead cells ( % ) indicates the population of apoptotic cells . ( C ) Semiquantitative analysis of protein expression is shown ( n = 3 ; mean ± SD ) . * p < 0 . 05 , * * p < 0 . 01 , NS : not significant . ( D - I ) Suppression of etoposide - induced autophagy in PPM1D − / − MEFs .

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

(B, C) PPM1D-dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy. PPM1D+/+MEFs and PPM1D−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells. (C) Semiquantitative analysis of protein expression is shown (n = 3; mean ± SD). *p < 0.05, **p < 0.01, NS: not significant. (D-I) Suppression of etoposide-induced autophagy in PPM1D−/−MEFs.


( D , E ) The indicated MEFs were treated with etoposide ( 10 µM ) for 6 hr and then immunostained with an anti - LC3 antibody ( green ) . Representative images are shown in ( D ) . LC3 puncta are observed in etoposide - treated PPM1D + / + MEFs . ( E ) The proportion of cells with LC3 puncta was calculated ( n > 100 cells in each experiment ) . Data are shown as the mean ± SD ( n = 3 experiments ) . * p < 0 . 05 , * * p < 0 . 01 .

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 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

(D, E) The indicated MEFs were treated with etoposide (10 µM) for 6 hr and then immunostained with an anti-LC3 antibody (green). Representative images are shown in (D). LC3 puncta are observed in etoposide-treated PPM1D+/+MEFs. (E) The proportion of cells with LC3 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ±  SD (n = 3 experiments). *p < 0.05, **p < 0.01.


( F , G ) The indicated MEFs were treated with etoposide ( 10 µM ) for 6 hr and then analyzed using electron microscopy . Representative images are shown in ( F ) . Many autophagic vacuoles ( arrows ) can be seen in etoposide - treated PPM1D + / + cells ( upper panel ) . " N " indicates the nucleus . Bar = 2 µm . A representative autophagosome ( AP ) and autolysosome ( AL ) are shown in the insets . ( G ) The number of autophagosomes and autolysosomes in each cell were counted ( n > 8 cells ) . Red lines indicate the mean value . * p < 0 . 05 .

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 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

(F, G) The indicated MEFs were treated with etoposide (10 µM) for 6 hr and then analyzed using electron microscopy. Representative images are shown in (F). Many autophagic vacuoles (arrows) can be seen in etoposide-treated PPM1D+/+cells (upper panel). "N" indicates the nucleus. Bar = 2 µm. A representative autophagosome (AP) and autolysosome (AL) are shown in the insets. (G) The number of autophagosomes and autolysosomes in each cell were counted (n > 8 cells). Red lines indicate the mean value. *p < 0.05.


( H , I ) Analysis of autophagic flux . The indicated MEFs were treated with etoposide ( 10 µM ) for 6 hr in the presence or absence of bafilomycin A1 ( 10 nM ) , and the expression of each protein was examined by western blotting . α - Tubulin was included as a loading control . ( I ) Semiquantitative analyses of protein expression ( n = 3 ; mean ± SD ) . * p < 0 . 05 , * * p < 0 . 01 , NS : 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H, I) Analysis of autophagic flux. The indicated MEFs were treated with etoposide (10 µM) for 6 hr in the presence or absence of bafilomycin A1 (10 nM), and the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. (I) Semiquantitative analyses of protein expression (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant.


( A , B ) Effect of stable expression of PPM1D in PPM1D − / − MEFs . PPM1D − / − MEFs and PPM1D - transfected PPM1D − / − MEFs were treated with etoposide ( 10 µM ) , and the expression of each protein was examined by western blotting . ( B ) Semiquantitative analyses of protein expression in ( A ) ( n = 3 ; mean ± SD ) . * p < 0 . 05 , * * p < 0 . 01 , NS : 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 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

(A, B) Effect of stable expression of PPM1D in PPM1D−/−MEFs. PPM1D−/−MEFs and PPM1D-transfected PPM1D−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. (B) Semiquantitative analyses of protein expression in (A) (n = 3; mean ±  SD). *p < 0.05, **p < 0.01, NS: not significant.


( C , D ) Effect of a PPM1D inhibitor on etoposide - treated PPM1D + / + MEFs . PPM1D + / + MEFs were treated with etoposide ( 10 µM ) in the presence or absence of GSK2830371 ( 30 µM ) , and the expression of each protein was examined by western blotting . ( D ) Semiquantitative analysis of protein expression in ( C ) ( n = 3 ; mean ± SD ) . * p < 0 . 05 , * * p < 0 . 01 , NS : not significant .

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 B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) Effect of a PPM1D inhibitor on etoposide-treated PPM1D+/+MEFs. PPM1D+/+MEFs were treated with etoposide (10 µM) in the presence or absence of GSK2830371 (30 µM), and the expression of each protein was examined by western blotting. (D) Semiquantitative analysis of protein expression in (C) (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant.


( E , F ) Effect of the transient overexpression of PPM1D . PPM1D + / + MEFs were transfected with a PPM1D - Flag plasmid in the presence or absence of bafilomycin A1 ( 10 nM ) , and the expression of each protein was examined by western blotting . ( F ) Semiquantitative analyses of protein expression in ( E ) ( n = 3 ; mean ± SD ) . * p < 0 . 05 , * * p < 0 . 01 , NS : not significant .

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(E, F) Effect of the transient overexpression of PPM1D. PPM1D+/+MEFs were transfected with a PPM1D-Flag plasmid in the presence or absence of bafilomycin A1 (10 nM), and the expression of each protein was examined by western blotting. (F) Semiquantitative analyses of protein expression in (E) (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant.


( A ) Physical interaction between endogenous Ulk1 and endogenous PPM1D . PPM1D + / + MEFs were treated with etoposide ( 10 µM ) or left untreated for 6 hr . Cells were then lysed and immunoprecipitated with an anti - Ulk1 antibody or a control IgG . Immune complexes and total lysates ( 1 . 8 % input ) were analyzed by western blotting using anti - PPM1D and anti - Ulk1 antibodies .

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

(A) Physical interaction between endogenous Ulk1 and endogenous PPM1D. PPM1D+/+MEFs were treated with etoposide (10 µM) or left untreated for 6 hr. Cells were then lysed and immunoprecipitated with an anti-Ulk1 antibody or a control IgG. Immune complexes and total lysates (1.8% input) were analyzed by western blotting using anti-PPM1D and anti-Ulk1 antibodies.


( B ) Interaction between endogenous Ulk1 and recombinant PPM1D . Lysates from healthy PPM1D + / + MEFs were incubated with GST - PPM1D or GST . Binding molecules were then analyzed by western blotting using anti - Ulk1 and anti - GST antibodies . " Total lysate " indicates 7 % of the lysates that were incubated with GST fusion protein .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Interaction between endogenous Ulk1 and recombinant PPM1D. Lysates from healthy PPM1D+/+ MEFs were incubated with GST-PPM1D or GST. Binding molecules were then analyzed by western blotting using anti-Ulk1 and anti-GST antibodies. "Total lysate" indicates 7% of the lysates that were incubated with GST fusion protein.


( C ) In vitro Ulk1 dephosphorylation assay . Immunoprecipitated Ulk1 from PPM1D + / + MEF lysates was incubated with GST - PPM1D ( 1 µg ) with or without a phosphatase inhibitor cocktail for 1 hr . Then , the extent of Ulk1 dephosphorylation was analyzed by western blotting using anti - phospho - Ulk1637 and anti - phospho - Ulk1757 antibodies . Semiquantitative analyses are shown in Fig . EV2B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro Ulk1 dephosphorylation assay. Immunoprecipitated Ulk1 from PPM1D+/+ MEF lysates was incubated with GST-PPM1D (1 µg) with or without a phosphatase inhibitor cocktail for 1 hr. Then, the extent of Ulk1 dephosphorylation was analyzed by western blotting using anti-phospho-Ulk1637 and anti-phospho-Ulk1757 antibodies. Semiquantitative analyses are shown in Fig. EV2B.


( D ) The presence of cytosolic PPM1D puncta in etoposide - treated PPM1D + / + MEFs . PPM1D + / + MEFs were treated with etoposide ( 10 µM ) for 6 hr , immunostained with an anti - PPM1D antibody , and their nuclei stained with Hoechst 33342 ( 50 ng / ml ) . Representative images of anti - PPM1D ( green ; upper panels ) and Hoechst 33342 ( blue ; lower panels ) are shown . Arrowheads indicate cytoplasmic PPM1D puncta .

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 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

(D) The presence of cytosolic PPM1D puncta in etoposide-treated PPM1D+/+MEFs. PPM1D+/+MEFs were treated with etoposide (10 µM) for 6 hr, immunostained with an anti-PPM1D antibody, and their nuclei stained with Hoechst 33342 (50 ng/ml). Representative images of anti-PPM1D (green; upper panels) and Hoechst 33342 (blue; lower panels) are shown. Arrowheads indicate cytoplasmic PPM1D puncta.


( E ) Colocalization of endogenous PPM1D and endogenous Ulk1 in etoposide - treated PPM1D + / + MEFs . PPM1D + / + MEFs were treated with etoposide ( 10 µM ) for 6 hr , immunostained with anti - PPM1D and anti - Ulk1 antibodies , and their nuclei stained with Hoechst 33342 ( 50 ng / ml ) . Representative images of anti - PPM1D ( red ; left ) , anti - Ulk1 ( green ; middle ) , and a merged image ( right ) are shown . Magnified images of the areas within the dashed squares are shown at the bottom . Arrowheads indicate cytoplasmic PPM1D colocalized with Ulk1 .

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(E) Colocalization of endogenous PPM1D and endogenous Ulk1 in etoposide-treated PPM1D+/+MEFs. PPM1D+/+MEFs were treated with etoposide (10 µM) for 6 hr, immunostained with anti-PPM1D and anti-Ulk1 antibodies, and their nuclei stained with Hoechst 33342 (50 ng/ml). Representative images of anti-PPM1D (red; left), anti-Ulk1 (green; middle), and a merged image (right) are shown. Magnified images of the areas within the dashed squares are shown at the bottom. Arrowheads indicate cytoplasmic PPM1D colocalized with Ulk1.


( F - J ) The crucial role of Ulk1 dephosphorylation at Ser637 in etoposide - induced autophagy . ( F - H ) Ulk1 / Ulk2 DKO MEFs that were stably transfected with HA - Ulk1 or its mutants , S637D and S637A , were treated with etoposide ( 10 µM ) . Then , the cell lysates were collected time - dependently , and the expression of each protein was examined by western blotting . Asterisks in the Ulk1 blots are nonspecific bands . Semiquantitative analyses are shown in Fig . EV2E .

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 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

(F-J) The crucial role of Ulk1 dephosphorylation at Ser637 in etoposide-induced autophagy. (F-H) Ulk1/Ulk2 DKO MEFs that were stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM). Then, the cell lysates were collected time-dependently, and the expression of each protein was examined by western blotting. Asterisks in the Ulk1 blots are nonspecific bands. Semiquantitative analyses are shown in Fig. EV2E.


( F - J ) The crucial role of Ulk1 dephosphorylation at Ser637 in etoposide - induced autophagy . ( I , J ) The indicated MEFs were treated with or without etoposide ( 10 µM ) for 6 hr , followed by immunostaining with an anti - LC3 antibody . Representative images are shown in ( I ) . LC3 puncta are markedly observed in DKO MEFs transfected with wild - type HA - Ulk1 . Puncta were absent and weakly observed in MEFs expressing the mutants S637D and S637A , respectively . ( J ) The population of cells with LC3 puncta was calculated ( n > 100 cells in each experiment ) . Data are shown as the mean ± SD ( n = 3 experiments ) . * p < 0 . 05 , * * p < 0 . 01 .

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 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

(F-J) The crucial role of Ulk1 dephosphorylation at Ser637 in etoposide-induced autophagy. (I, J) The indicated MEFs were treated with or without etoposide (10 µM) for 6 hr, followed by immunostaining with an anti-LC3 antibody. Representative images are shown in (I). LC3 puncta are markedly observed in DKO MEFs transfected with wild-type HA-Ulk1. Puncta were absent and weakly observed in MEFs expressing the mutants S637D and S637A, respectively. (J) The population of cells with LC3 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ±  SD (n = 3 experiments). *p < 0.05, **p < 0.01.


( A - D ) Ulk1 puncta formation was induced by etoposide in a PPM1D - dependent manner . The indicated MEFs were treated with etoposide ( 10 µM ) with or without GSK2830371 ( 20 µM ) for the indicated times , followed by immunostaining with an anti - Ulk1 antibody . Representative images are shown in ( A ) . Ulk1 puncta are observed in etoposide - treated PPM1D + / + MEFs . ( B - D ) The proportion of cells with Ulk1 puncta was calculated ( n > 100 cells in each experiment ) . Data are shown as the mean ± SD ( n = 3 experiments ) . * p < 0 . 05 , * * p < 0 . 01 , NS : not significant .

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 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 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

(A-D) Ulk1 puncta formation was induced by etoposide in a PPM1D-dependent manner. The indicated MEFs were treated with etoposide (10 µM) with or without GSK2830371 (20 µM) for the indicated times, followed by immunostaining with an anti-Ulk1 antibody. Representative images are shown in (A). Ulk1 puncta are observed in etoposide-treated PPM1D+/+MEFs. (B-D) The proportion of cells with Ulk1 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, **p < 0.01, NS: not significant.


( E - I ) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide - induced Ulk1 puncta formation and Atg13 phosphorylation . Ulk1 / Ulk2 DKO MEFs stably transfected with HA - Ulk1 or its mutants , S637D and S637A , were treated with etoposide ( 10 µM ) for the indicated times . ( E , F ) Cells were immunostained with an anti - Ulk1 antibody ( E ) , and the population of cells with Ulk1 puncta was calculated ( n > 100 cells in each experiment ) ( F ) . Data are shown as the mean ± SD ( n = 3 experiments ) . * p < 0 . 05 , * * p < 0 . 01 .

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 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

(E-I) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide-induced Ulk1 puncta formation and Atg13 phosphorylation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM) for the indicated times. (E, F) Cells were immunostained with an anti-Ulk1 antibody (E), and the population of cells with Ulk1 puncta was calculated (n > 100 cells in each experiment) (F). Data are shown as the mean  ±  SD (n = 3 experiments). *p < 0.05, **p < 0.01.


( E - I ) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide - induced Ulk1 puncta formation and Atg13 phosphorylation . Ulk1 / Ulk2 DKO MEFs stably transfected with HA - Ulk1 or its mutants , S637D and S637A , were treated with etoposide ( 10 µM ) for the indicated times . ( G - I ) Cell lysates were collected in a time - dependent manner , and Atg13 protein levels and its phosphorylation at Ser317 were examined by western blotting . Semiquantitative analyses are shown in Fig . EV3D .

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 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

(E-I) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide-induced Ulk1 puncta formation and Atg13 phosphorylation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM) for the indicated times. (G-I) Cell lysates were collected in a time-dependent manner, and Atg13 protein levels and its phosphorylation at Ser317 were examined by western blotting. Semiquantitative analyses are shown in Fig. EV3D.


( J ) EGFP - DFCP1 - expressing PPM1D + / + MEFs were treated with etoposide ( 10 µM ) for the indicated times , followed by immunostaining with an anti - Ulk1 antibody . Representative images of EGFP - DFCP1 ( green ; left ) , anti - Ulk1 ( red ; center ) , and a merged image ( right ) are shown . The nuclei were stained with Hoechst 33342 in the merged image . A magnified image of the area within the dashed square is also shown . Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta .

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

(J) EGFP-DFCP1-expressing PPM1D+/+MEFs were treated with etoposide (10 µM) for the indicated times, followed by immunostaining with an anti-Ulk1 antibody. Representative images of EGFP-DFCP1 (green; left), anti-Ulk1 (red; center), and a merged image (right) are shown. The nuclei were stained with Hoechst 33342 in the merged image. A magnified image of the area within the dashed square is also shown. Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta.


( K , L ) The role of Ulk1 dephosphorylation at Ser637 on etoposide - induced DFCP1 puncta formation . Ulk1 / Ulk2 DKO MEFs stably transfected with HA - Ulk1 or its mutants , S637D and S637A , were transfected with EGFP - DFCP1 and treated with etoposide ( 10 µM ) for the indicated hours , followed by immunostaining with an anti - HA antibody . Representative merged images of EGFP - DFCP1 ( green ) , anti - HA ( red ) , and Hoechst 33342 ( blue ) are shown in ( K ) . Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta . The number of DFCP1 puncta in each cell was calculated and is shown in ( L ) ( n > 8 cells in each experiment ) . Red lines indicate the mean values . * * p < 0 . 01 , NS : not significant .

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 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

(K, L) The role of Ulk1 dephosphorylation at Ser637 on etoposide-induced DFCP1 puncta formation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were transfected with EGFP-DFCP1 and treated with etoposide (10 µM) for the indicated hours, followed by immunostaining with an anti-HA antibody. Representative merged images of EGFP-DFCP1 (green), anti-HA (red), and Hoechst 33342 (blue) are shown in (K). Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta. The number of DFCP1 puncta in each cell was calculated and is shown in (L) (n > 8 cells in each experiment). Red lines indicate the mean values. **p < 0.01, NS: not significant.


( A ) PPM1D - dependent dephosphorylation of Ulk1 at Ser637 , induction of autophagy , and degradation of Noxa . PPM1D + / + and PPM1D − / − primary thymocytes were X - rayirradiated ( 5 Gy ) , and 3 hr later , thymocytes were lysed and the expression of each protein was examined by western blotting . α - Tubulin was used as a loading control . Semiquantitative analyses are shown in Fig . EV5B .

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(A) PPM1D-dependent dephosphorylation of Ulk1 at Ser637, induction of autophagy, and degradation of Noxa. PPM1D+/+ and PPM1D−/− primary thymocytes were X-rayirradiated (5 Gy), and 3 hr later, thymocytes were lysed and the expression of each protein was examined by western blotting. α-Tubulin was used as a loading control. Semiquantitative analyses are shown in Fig. EV5B.


( B - F ) Suppression of X - ray - induced autophagy in PPM1D − / − thymocytes . ( B , C ) The indicated thymocytes were X - rayirradiated ( 5 Gy ) , and 3 hr later , thymocytes were immunostained with an anti - LC3 antibody . Nuclei were stained with Hoechst 33342 . Representative images are shown in ( B ) . LC3 puncta can be seen in irradiatedPPM1D + / + thymocytes ( arrowheads ) . In ( C ) , the proportion of cells with LC3 puncta was calculated ( n > 100 cells in each experiment ) . Data are shown as the mean ± SD ( n = 3 experiments ) . * p < 0 . 05 .

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(B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (B, C) The indicated thymocytes were X-rayirradiated (5 Gy), and 3 hr later, thymocytes were immunostained with an anti-LC3 antibody. Nuclei were stained with Hoechst 33342. Representative images are shown in (B). LC3 puncta can be seen in irradiatedPPM1D+/+thymocytes (arrowheads). In (C), the proportion of cells with LC3 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05.


( B - F ) Suppression of X - ray - induced autophagy in PPM1D − / − thymocytes . ( D ) The indicated thymocytes were X - ray irradiated ( 5 Gy ) , and 3 hr later , thymocytes were subjected to EM analysis . An autophagic vacuole ( arrowhead ) can be seen in an irradiated PPM1D + / + thymocyte .

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(B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (D) The indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, thymocytes were subjected to EM analysis. An autophagic vacuole (arrowhead) can be seen in an irradiated PPM1D+/+thymocyte.


( B - F ) Suppression of X - ray - induced autophagy in PPM1D − / − thymocytes . ( E ) The indicated thymocytes were X - ray irradiated ( 5 Gy ) , and 3 hr later , thymocytes were stained with Cyto - IDTM . Representative flow cytometry data are shown . Quantitative data are shown in Fig . EV5C . Cyto - ID fluorescence in irradiated PPM1D + / + thymocytes was higher than that in the other thymocytes .

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(B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (E) The indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, thymocytes were stained with Cyto-IDTM. Representative flow cytometry data are shown. Quantitative data are shown in Fig. EV5C. Cyto-ID fluorescence in irradiated PPM1D+/+thymocytes was higher than that in the other thymocytes.


( B - F ) Suppression of X - ray - induced autophagy in PPM1D − / − thymocytes . ( F ) Analysis of autophagic flux . The indicated thymocytes were X - rayirradiated ( 5 Gy ) in the presence or absence of chloroquine ( 120 µM ) , and the expression of each protein was examined by western blotting . α - Tubulin was included as a loading control . Semiquantitative analyses are shown in Figure EV4D .

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(B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (F) Analysis of autophagic flux. The indicated thymocytes were X-rayirradiated (5 Gy) in the presence or absence of chloroquine (120 µM), and the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. Semiquantitative analyses are shown in Figure EV4D.


( G - J ) Contribution of PPM1D / Ulk1 - dependent autophagy to irradiation - induced apoptosis . ( G ) Thymocytes were X - ray irradiated ( 5 Gy ) , and 6 hr later , cell death was determined using the PI assay . Data represent the mean ± SD ( n = 3 independent thymi ) . * p < 0 . 05 , * * p < 0 . 01 , NS : 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

(G-J) Contribution of PPM1D/Ulk1-dependent autophagy to irradiation-induced apoptosis. (G) Thymocytes were X-ray irradiated (5 Gy), and 6 hr later, cell death was determined using the PI assay. Data represent the mean  ± SD (n = 3 independent thymi). *p < 0.05, **p < 0.01, NS: not significant.


( G - J ) Contribution of PPM1D / Ulk1 - dependent autophagy to irradiation - induced apoptosis . ( H , I ) Thymocytes were X - rayirradiated ( 5 Gy ) , and 3 hr later , the expression of each protein was examined by western blotting . α - Tubulin was included as a loading control . ( I ) Semiquantitative analyses are shown ( n = 3 ; mean ± SD ) . * p < 0 . 05 , * * p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Contribution of PPM1D/Ulk1-dependent autophagy to irradiation-induced apoptosis. (H, I) Thymocytes were X-rayirradiated (5 Gy), and 3 hr later, the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. (I) Semiquantitative analyses are shown (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01.


( G - J ) Contribution of PPM1D / Ulk1 - dependent autophagy to irradiation - induced apoptosis . ( J ) Indicated thymocytes were X - ray irradiated ( 5 Gy ) , and 3 hr later , caspase3 / 7 activity was examined using the Caspase - Glo 3 / 7 assay ( Promega ) according to the manufacturer ' s protocol . Data are shown as the mean ± SD ( n = 3 experiments ) . * p < 0 . 05 , * * p < 0 . 01 .

(G-J) Contribution of PPM1D/Ulk1-dependent autophagy to irradiation-induced apoptosis. (J) Indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, caspase3/7 activity was examined using the Caspase-Glo 3/7 assay (Promega) according to the manufacturer's protocol. Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, **p < 0.01.


( K ) The effect of Noxa siRNA on thymocytes . The indicated thymocytes were transfected with the pmaxGFP vector and siRNAs . After 12 hr , cells were X - ray irradiated ( 5 Gy ) , and 6 hr or 10 hr later , the population of dead cells among the GFP - positive cells was determined using the PI assay . Data are shown as the mean ± SD ( n = 3 experiments ) . * p < 0 . 05 , NS : 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 O

(K) The effect of Noxa siRNA on thymocytes. The indicated thymocytes were transfected with the pmaxGFP vector and siRNAs. After 12 hr, cells were X-ray irradiated (5 Gy), and 6 hr or 10 hr later, the population of dead cells among the GFP-positive cells was determined using the PI assay. Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, NS: not significant.


( A , B ) Net charge distribution ( black ) of αSyn ( A ) and Aß ( B ) , plotted together with the binding distribution of wild - type SERF2 ( blue ) and the SERF2 charge mutant ( red ) . Blue boxes represent the SERF2 interacting regions .

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(A,B) Net charge distribution (black) of αSyn (A) and Aß (B), plotted together with the binding distribution of wild-type SERF2 (blue) and the SERF2 charge mutant (red). Blue boxes represent the SERF2 interacting regions.


( C ) ThT - monitored amyloid kinetics of 50uM alpha - synuclein in the presence of either wild - type SERF2 or mutant SERF2 in a 1 : 1 ratio . The average of three biological replicates is represented and error bars indicate mean ± SD .

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(C) ThT-monitored amyloid kinetics of 50uM alpha-synuclein in the presence of either wild-type SERF2 or mutant SERF2 in a 1:1 ratio. The average of three biological replicates is represented and error bars indicate mean ± SD.


( D ) ThT - monitored amyloid kinetics of 1uM amyloid - beta in the presence of either wild - type SERF2 or mutant SERF2 in a 1 : 4 ratio . The average of four technical replicates is represented and error bars indicate mean ± SD .

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(D) ThT-monitored amyloid kinetics of 1uM amyloid-beta in the presence of either wild-type SERF2 or mutant SERF2 in a 1:4 ratio. The average of four technical replicates is represented and error bars indicate mean ± SD.


( E ) Net charge distribution of hIAPP ( black ) , plotted together with the binding distribution of wild - type SERF2 ( blue ) and the SERF2 charge mutant ( red ) .

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(E) Net charge distribution of hIAPP (black), plotted together with the binding distribution of wild-type SERF2 (blue) and the SERF2 charge mutant (red).


( F ) ThT - monitored amyloid kinetics of 2uM human islet amyloid polypeptide in the presence of either wild - type SERF2 or mutant SERF2 in a 1 : 4 ratio . The average of five technical replicates is represented and error bars indicate mean ± SD .

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(F) ThT-monitored amyloid kinetics of 2uM human islet amyloid polypeptide in the presence of either wild-type SERF2 or mutant SERF2 in a 1:4 ratio. The average of five technical replicates is represented and error bars indicate mean ± SD.


( G ) Model and charge distribution graph of HA - Q74 and muHA - Q74 constructs .

(G) Model and charge distribution graph of HA-Q74 and muHA-Q74 constructs.


( H ) Mutations and charge distribution in the SERF2 control mutant and SERF2 charge mutant proteins .

(H) Mutations and charge distribution in the SERF2 control mutant and SERF2 charge mutant proteins.


Filter Trap with 5 - fold serial dilution and quantification of crude protein extract from SERF2 CRISPR deletion mutant cells transiently transfected with HA - polyQ74 and either wild - type SERF2 , empty vector or SERF2 charge mutant ( I ) , Western blots for HA - Q74 , SERF2 and Tubulin expression were included as controls For all Filter Trap assays , the depicted blots are from one representative experiment of four biological replicates . The average of four biological replicates with each three or four technical replicates is represented in the graphs . Data are represented as mean ± SD , significance was calculated using a one - way ANOVA , followed by a post - hoc Bonferroni multiple comparisons test . ( I ) p < 0 . 0001 , * * p < 0 . 01 ; * * * p < 0 . 001 .

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Filter Trap with 5-fold serial dilution and quantification of crude protein extract from SERF2 CRISPR deletion mutant cells transiently transfected with HA-polyQ74 and either wild-type SERF2, empty vector or SERF2 charge mutant (I), Western blots for HA-Q74, SERF2 and Tubulin expression were included as controls For all Filter Trap assays, the depicted blots are from one representative experiment of four biological replicates. The average of four biological replicates with each three or four technical replicates is represented in the graphs. Data are represented as mean ± SD, significance was calculated using a one-way ANOVA, followed by a post-hoc Bonferroni multiple comparisons test. (I) p<0.0001, **p < 0.01; ***p < 0.001.


Filter Trap with 5 - fold serial dilution and quantification of crude protein extract from SERF2 CRISPR deletion mutant cells transiently transfected with HA - polyQ74 and either wild - type SERF2 , empty vector or SERF2 control mutant ( J ) or SERF2 charge mutant . Western blots for HA - Q74 , muHA - Q74 , SERF2 and Tubulin expression were included as controls For all Filter Trap assays , the depicted blots are from one representative experiment of four biological replicates . The average of four biological replicates with each three or four technical replicates is represented in the graphs . Data are represented as mean ± SD , significance was calculated using a one - way ANOVA , followed by a post - hoc Bonferroni multiple comparisons test . ( J ) = 0 . 0003 , ( K ) p = 0 . 9130 * * p < 0 . 01 ; * * * p < 0 . 001 .

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Filter Trap with 5-fold serial dilution and quantification of crude protein extract from SERF2 CRISPR deletion mutant cells transiently transfected with HA-polyQ74 and either wild-type SERF2, empty vector or SERF2 control mutant (J) or SERF2 charge mutant. Western blots for HA-Q74, muHA-Q74, SERF2 and Tubulin expression were included as controls For all Filter Trap assays, the depicted blots are from one representative experiment of four biological replicates. The average of four biological replicates with each three or four technical replicates is represented in the graphs. Data are represented as mean ± SD, significance was calculated using a one-way ANOVA, followed by a post-hoc Bonferroni multiple comparisons test. (J)=0.0003, (K) p=0.9130 **p < 0.01; ***p < 0.001.


( B ) Representative images of Q40 and Q40 ; moag - 4 charge mutant animals . Scale bar , 75 mm . ( C ) Representative quantification of the number of aggregates in Q40 worms with moag - 4 deletion or expression of either wild - type moag - 4 , moag - 4 charge mutant or moag - 4 ctrl mutant . The results shown are representative experiments of three biological replicates of n = 20 worms in L4 stage . Data are represented as mean ± SD and significance was calculated using a one - way ANOVA , followed by a post - hoc Bonferroni multiple comparisons test . * * * * p < 0 . 0001 .

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(B) Representative images of Q40 and Q40;moag-4 charge mutant animals. Scale bar, 75 mm. (C) Representative quantification of the number of aggregates in Q40 worms with moag-4 deletion or expression of either wild-type moag-4, moag-4 charge mutant or moag-4 ctrl mutant. The results shown are representative experiments of three biological replicates of n=20 worms in L4 stage. Data are represented as mean ± SD and significance was calculated using a one-way ANOVA, followed by a post-hoc Bonferroni multiple comparisons test. ****p < 0.0001.


( D ) Representative TCSPC - FLIM images from the indicated strains . The dashed lines mark the retro vesicular ganglion and arrowheads indicate aggregates . Scale bar is 50 µm .

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(D) Representative TCSPC-FLIM images from the indicated strains. The dashed lines mark the retro vesicular ganglion and arrowheads indicate aggregates. Scale bar is 50 µm.


( E ) Number of aggregates per retro vesicular ganglion from three independent cohorts of the indicated Aβ1 - 42 strains with moag - 4 deletion mutant ( del ( tm4909 ) ) n = 11 , moag - 4 control mutant ( ctrl mutant ) n = 12 , moag - 4 charge mutant ( charge mutant ) n = 17 , and wild - type n = 15 . Data are represented as mean ± SD . One - way ANOVA + Bonferroni post - hoc test for significance ( * p < 0 . 05 , * * * * p < 0 . 0001 ) .

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(E) Number of aggregates per retro vesicular ganglion from three independent cohorts of the indicated Aβ1-42 strains with moag-4 deletion mutant (del (tm4909)) n=11, moag-4 control mutant (ctrl mutant) n=12, moag-4 charge mutant (charge mutant) n=17, and wild-type n=15. Data are represented as mean ± SD. One-way ANOVA + Bonferroni post-hoc test for significance (* p < 0.05, **** p < 0.0001).


( B and C ) IC50 values for MPA - response curves using cell viability assay were shown . Human AML cell lines ( with MLL - fusions : MV4 ; 11 , MOLM13 , NOMO1 and THP1 , without MLL - fusions : HL60 , Kasumi - 1 , OCI - AML3 and U937 ) , human cord blood ( CB ) CD34 + cells , and those expressing MLL - AF9 ( CB - MLL - AF9 # 1 , 2 , 3 ) or MLL - ENL ( CB # MLL - ENL - 1 , 2 ) , and patient cells with or without MLL - fusions ( AML # 1 , 2 , 3 or AML # 4 , 5 , 6 ) were treated with titrating doses of MPA ( 1 - 100 μM ) for 72 hours in three technical replicates . Data information : All data are shown as mean ± SEM .

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(B and C) IC50 values for MPA-response curves using cell viability assay were shown. Human AML cell lines (with MLL-fusions: MV4;11, MOLM13, NOMO1 and THP1, without MLL-fusions: HL60, Kasumi-1, OCI-AML3 and U937), human cord blood (CB) CD34+ cells, and those expressing MLL-AF9 (CB-MLL-AF9#1, 2, 3) or MLL-ENL (CB#MLL-ENL-1, 2), and patient cells with or without MLL-fusions (AML#1, 2, 3 or AML#4, 5, 6) were treated with titrating doses of MPA (1-100 μM) for 72 hours in three technical replicates. Data information: All data are shown as mean ± SEM.


( D ) Response curves of FF - 10501 - 01 ( 1 - 100 μM ) for 72 hours in three technical replicates .

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(D) Response curves of FF-10501-01 (1-100 μM) for 72 hours in three technical replicates.


( E ) FCM plots of the cell cycle and apoptosis analyses in MLL - AF9 expressing CB cells and MV4 ; 11 cells . Cells were incubated with / without 1 μM MPA and 100 mM Guanosine for 36 hours . Numbers indicate the frequency of S / G2 / M - phase cells ( left ) and Annexin V + cells ( right ) .

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(E) FCM plots of the cell cycle and apoptosis analyses in MLL-AF9 expressing CB cells and MV4;11 cells. Cells were incubated with/without 1 μM MPA and 100 mM Guanosine for 36 hours. Numbers indicate the frequency of S/G2/M-phase cells (left) and Annexin V+ cells (right).


( F and G ) Immunophenotypes and Wright - Giemsa staining of MLL - AF9 expressing CB cells after four days of culture with / without 3 . 3 μM MPA and 100 μM Guanosine ( Scale bar : 20 mm ) .

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(F and G) Immunophenotypes and Wright-Giemsa staining of MLL-AF9 expressing CB cells after four days of culture with/without 3.3 μM MPA and 100 μM Guanosine (Scale bar: 20 mm).


( H ) MLL - ENL - expressing CB cells and MOLM13 cells were transduced with Cas9 together with non - targeting ( NT ) sgRNA or IMPDH2 - targeting sgRNAs ( IMPDH2 - KO1 and KO2 ) co - expressing tRFP657 . Changes in the frequency of tRFP657 + cells ( sgRNA - transduced cells ) in cell cultures are shown . Results are normalized to the frequency of tRFP657 + cells at day 4 , set to 1 .

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(H) MLL-ENL-expressing CB cells and MOLM13 cells were transduced with Cas9 together with non-targeting (NT) sgRNA or IMPDH2-targeting sgRNAs (IMPDH2-KO1 and KO2) co-expressing tRFP657. Changes in the frequency of tRFP657+ cells (sgRNA-transduced cells) in cell cultures are shown. Results are normalized to the frequency of tRFP657+ cells at day 4, set to 1.


( I ) The tRFP657 + cells in ( H ) were sorted and subjected to western blotting . The depletion of IMPDH2 protein in MLL - ENL cells and MOLM13 cells was confirmed by western blotting .

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(I) The tRFP657+ cells in (H) were sorted and subjected to western blotting. The depletion of IMPDH2 protein in MLL-ENL cells and MOLM13 cells was confirmed by western blotting.


( A ) Immunoblotting of the cord blood cells and those expressing RUNX1 - ETO or MLL - AF9 . The cells were incubated with / without 1 μM MPA ( M ) and 100 μM Guanosine ( G ) for 24 h .

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(A) Immunoblotting of the cord blood cells and those expressing RUNX1-ETO or MLL-AF9. The cells were incubated with/without 1 μM MPA (M) and 100 μM Guanosine (G) for 24 h.


( B ) CB - MLL - AF9 # 2 cells were transduced with a Non - targeting ( NT ) control and an shRNA targeting p21 ( sh - p21 ) . Cells were incubated with / without 1 μM MPA ( M ) and 100 μM guanosine ( G ) for 24 hours . Total cell lysates were analyzed by western blotting using the antibodies for p21 and Tubulin .

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(B) CB-MLL-AF9#2 cells were transduced with a Non-targeting (NT) control and an shRNA targeting p21 (sh-p21). Cells were incubated with/without 1 μM MPA (M) and 100 μM guanosine (G) for 24 hours. Total cell lysates were analyzed by western blotting using the antibodies for p21 and Tubulin.


( C ) CB - MLL - AF9 # 2 cells transduced with NT or sh - p21 shRNAs were incubated with MPA at the indicated concentration for 72 hours . Cell viability assays were performed using WST - 1 in three technical replicates .

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(C) CB-MLL-AF9#2 cells transduced with NT or sh-p21 shRNAs were incubated with MPA at the indicated concentration for 72 hours. Cell viability assays were performed using WST-1 in three technical replicates.


( D ) CB - MLL - AF9 # 2 cells were transduced by vector control or p53DD ( dominant - negative ) and were incubated with / without 1 μM MPA ( M ) and 100 μM guanosine ( G ) for 24 h . Total cell lysates were analyzed by western blotting using the antibodies for p53 and Tubulin . Note the elevated levels of endogenous p53 protein ( upper arrow ) in p53DD ( lower arrow ) - expressing cells .

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(D) CB-MLL-AF9#2 cells were transduced by vector control or p53DD (dominant-negative) and were incubated with/without 1 μM MPA (M) and 100 μM guanosine (G) for 24 h. Total cell lysates were analyzed by western blotting using the antibodies for p53 and Tubulin. Note the elevated levels of endogenous p53 protein (upper arrow) in p53DD (lower arrow)-expressing cells.


( E ) Cells were incubated with MPA at the indicated concentration with / without 100 μM guanosine for 72 hours then measured with WST - 1 in three technical replicates .

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(E) Cells were incubated with MPA at the indicated concentration with/without 100 μM guanosine for 72 hours then measured with WST-1 in three technical replicates.


( F ) Mouse bone marrow c - Kit + cells derived from Trp53 - / - mice were transduced with MLL - AF9 - GFP and were transplanted into mice to generate p53 - deficient ( p53 - / - ) leukemia cells . GFP + MLL - AF9 leukemia cells were collected from bone marrows of vehicle - or FF - 10501 - 01 - treated wild - type and p53 - / - leukemic mice . Expression levels of p53 and Tubulin were assessed by western blotting 24 hours after the treatment .

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(F) Mouse bone marrow c-Kit+ cells derived from Trp53-/- mice were transduced with MLL-AF9-GFP and were transplanted into mice to generate p53-deficient (p53-/-) leukemia cells. GFP+ MLL-AF9 leukemia cells were collected from bone marrows of vehicle- or FF-10501-01-treated wild-type and p53-/- leukemic mice. Expression levels of p53 and Tubulin were assessed by western blotting 24 hours after the treatment.


( H ) Kaplan - Meier survival curves of p53 - / - MLL - AF9 leukemia mice treated with vehicle or FF - 10501 - 01or DS - 5272 ( n = 6 per group ) . Statistical significance was evaluated by the log - rank test ( left ) . Frequency of GFP + leukemia cells in peripheral blood at day 19 ( right , n = 6 per group ) . A two - tailed unpaired t - test was used for the comparison . Data information : All data are shown as mean ± SEM .

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(H) Kaplan-Meier survival curves of p53-/- MLL-AF9 leukemia mice treated with vehicle or FF-10501-01or DS-5272 (n = 6 per group). Statistical significance was evaluated by the log-rank test (left). Frequency of GFP+ leukemia cells in peripheral blood at day 19 (right, n = 6 per group). A two-tailed unpaired t-test was used for the comparison. Data information: All data are shown as mean ± SEM.


( B ) The TLR - expressing BaκB cells were stimulated with the corresponding TLR ligands ( 100 ng / mL Pam3CSK4 for TLR2 , 100 ng / mL LPS for TLR4 , 10 ng / mL Flagellin for TLR5 , 100 ng / ml R848 for TLR7 ) with titrating doses of MPA ( 1 - 10 μM ) . GFP expression was assessed 24 hours after the stimulation .

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(B) The TLR-expressing BaκB cells were stimulated with the corresponding TLR ligands (100 ng/mL Pam3CSK4 for TLR2, 100 ng/mL LPS for TLR4, 10 ng/mL Flagellin for TLR5, 100 ng/ml R848 for TLR7) with titrating doses of MPA (1 - 10 μM). GFP expression was assessed 24 hours after the stimulation.


( C ) 293T cells were transfected with FLAG - tagged TRAF6 and HA - tagged K63 - ubiquitin . After 24 hours , these cells were treated with 3 or 10 μM MPA ( M - 3 μM or M - 10 μM ) with / without 100 μM guanosine ( G ) . IRAK1 / 4 inhibitor ( IRAK1 / 4 i ) was also used as a control . Whole - cell extracts were immunoprecipitated with anti - FLAG antibody , and ubiquitinated TRAF6 was detected with anti - HA antibody .

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(C) 293T cells were transfected with FLAG-tagged TRAF6 and HA-tagged K63-ubiquitin. After 24 hours, these cells were treated with 3 or 10 μM MPA (M-3 μM or M-10 μM) with/without 100 μM guanosine (G). IRAK1/4 inhibitor (IRAK1/4 i) was also used as a control. Whole-cell extracts were immunoprecipitated with anti-FLAG antibody, and ubiquitinated TRAF6 was detected with anti-HA antibody.


( D , Murine bone marrow - derived macrophages ( BMDMs ) were treated with vehicle , 10 μM MPA alone ( D ) N = 3 ( technical replicates ) per group . mRNA levels of TNF - α and IL - 1β were measured by qPCR . Two - tailed unpaired t - tests were used for the comparison in ( D ) . Data information : All data are shown as mean ± SEM .

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(D, Murine bone marrow-derived macrophages (BMDMs) were treated with vehicle, 10 μM MPA alone (D) N=3 (technical replicates) per group. mRNA levels of TNF-α and IL-1β were measured by qPCR. Two-tailed unpaired t-tests were used for the comparison in (D). Data information: All data are shown as mean ± SEM.


E ) Murine bone marrow - derived macrophages ( BMDMs ) were treated with vehicle , 10 μM MPA alone or co - treated with vehicle , 10 μM MPA ( M ) , 100 ng / mL Pam3CSK4 and 100 μM Guanosine ( G ) for 4 hours ( E ) , as indicated . N = 3 ( technical replicates ) per group . mRNA levels of TNF - α and IL - 1β were measured by qPCR . Ordinary one - way ANOVA was used for the comparison in ( E ) . Data information : All data are shown as mean ± SEM .

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E) Murine bone marrow-derived macrophages (BMDMs) were treated with vehicle, 10 μM MPA alone or co-treated with vehicle, 10 μM MPA (M), 100 ng/mL Pam3CSK4 and 100 μM Guanosine (G) for 4 hours (E), as indicated. N=3 (technical replicates) per group. mRNA levels of TNF-α and IL-1β were measured by qPCR. Ordinary one-way ANOVA was used for the comparison in (E). Data information: All data are shown as mean ± SEM.


( F ) THP1 cells were pretreated with 10 μM MPA for 2 hours and then treated with 1000 ng / mL Pam3CSK4 for 0 , 30 , 60 , and ( 120 ) minutes . Cells were lysed and subjected to SDS - PAGE . Levels of total IκBα , phosphate - p38 , total p38 , and GAPDH were evaluated by western blotting .

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(F) THP1 cells were pretreated with 10 μM MPA for 2 hours and then treated with 1000 ng/mL Pam3CSK4 for 0, 30, 60, and (120) minutes. Cells were lysed and subjected to SDS-PAGE. Levels of total IκBα, phosphate-p38, total p38, and GAPDH were evaluated by western blotting.


( G ) MLL - AF9 - expressing CB cells ( CB - MLL - AF9 # 1 ) were pretreated with 10 μM MPA for 2 hours and then treated with 1000 ng / mL Pam3CSK4 for 0 , 30 , 60 Cells were lysed and subjected to SDS - PAGE . Levels of total IκBα , phosphate - p38 , total p38 were evaluated by western blotting .

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(G) MLL-AF9-expressing CB cells (CB-MLL-AF9#1) were pretreated with 10 μM MPA for 2 hours and then treated with 1000 ng/mL Pam3CSK4 for 0, 30, 60 Cells were lysed and subjected to SDS-PAGE. Levels of total IκBα, phosphate-p38, total p38 were evaluated by western blotting.


A . SDS - PAGE analysis of purified T4SS3 - 10 + D4 complex . Left panel : Coomassie - stained SDS - PAGE gel of the T4SS3 - 10 + D4 complex . Right panel : Sypro Ruby - stained SDS - PAGE gel of the T4SS3 - 10m and T4SS3 - 10 + D4 complexes . Molecular weights of MW markers are shown on the left . Identification of bands is shown in the middle . " * " indicates TrwK / VirB4 degradation products . " * * " indicates minor contaminants OmpA and OmpC .

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A. SDS-PAGE analysis of purified T4SS3-10+D4 complex. Left panel: Coomassie-stained SDS-PAGE gel of the T4SS3-10+D4 complex. Right panel: Sypro Ruby-stained SDS-PAGE gel of the T4SS3-10m and T4SS3-10+D4 complexes. Molecular weights of MW markers are shown on the left. Identification of bands is shown in the middle. "*" indicates TrwK/VirB4 degradation products. "**" indicates minor contaminants OmpA and OmpC.


B . Stoichiometry measurement . Fluorescence scan of an SDS - PAGE gel analysis of the T4SS3 - 10 + D4 complex where cysteine residues were reacted with AlexaFluor 633 C5 maleimide . Signals corresponding to the proteins coupled to AlexaFluor 633 were detected at a wavelength of 633 nm . Fluorescent Trw protein bands are labelled and the derived stoichiometry for TrwB / VirD4 is indicated .

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B. Stoichiometry measurement. Fluorescence scan of an SDS-PAGE gel analysis of the T4SS3-10+D4 complex where cysteine residues were reacted with AlexaFluor 633 C5 maleimide. Signals corresponding to the proteins coupled to AlexaFluor 633 were detected at a wavelength of 633 nm. Fluorescent Trw protein bands are labelled and the derived stoichiometry for TrwB/VirD4 is indicated.


C . SDS - PAGE analysis of complexes purified from cell expressing deletion mutants of the pBADM11 _ trwN / virB1 - trwE / virB10Strep _ trwD / virB11 _ HistrwB / virD4 constructs termed pBADM11 _ ΔtrwX / ΔvirBY ( see main text ) . The same molecular weights marker has been used on this gel as in the gel presented in Figure 1A . * indicates degradation products of TrwK / VirB4 .

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C. SDS-PAGE analysis of complexes purified from cell expressing deletion mutants of the pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11_HistrwB/virD4 constructs termed pBADM11_ΔtrwX/ΔvirBY (see main text). The same molecular weights marker has been used on this gel as in the gel presented in Figure 1A. * indicates degradation products of TrwK/VirB4.


Figure 2 . Interaction between TrwB / VirD4 and TrwE / VirB10 . Left panel : Coomassie - stained SDS - PAGE gel of the T4SS3 - 10 + D4 complex ( lane 1 ) , T4SS3 - 10 + D4 : TrwEΔN42 complex ( lane 2 ) and T4SS3 - 10 + D4 : TrwEΔN64 complex ( lane 3 ) . Middle panel : Western Blot analysis using αStrep antibodies to detect the TrwE / VirB10 protein within the T4SS3 - 10 + D4 complex and its variants . Right panel : Western Blot analysis using αHis antibodies to detect the TrwB / VirD4 protein within the T4SS3 - 10 + D4 complex and its variants .

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Figure 2. Interaction between TrwB/VirD4 and TrwE/VirB10. Left panel: Coomassie-stained SDS-PAGE gel of the T4SS3-10+D4 complex (lane 1), T4SS3-10+D4:TrwEΔN42 complex (lane 2) and T4SS3-10+D4:TrwEΔN64 complex (lane 3). Middle panel: Western Blot analysis using αStrep antibodies to detect the TrwE/VirB10 protein within the T4SS3-10+D4 complex and its variants. Right panel: Western Blot analysis using αHis antibodies to detect the TrwB/VirD4 protein within the T4SS3-10+D4 complex and its variants.


A . NS - EM micrograph of the T4SS3 - 10 + D4 complex . A few representative particles of the T4SS3 - 10 + D4 complex are circled in yellow . Scale bar 50nm . B . Representative class averages of T4SS3 - 10 + D4 complex obtained after initial alignment and classification .

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A. NS-EM micrograph of the T4SS3-10+D4 complex. A few representative particles of the T4SS3-10+D4 complex are circled in yellow. Scale bar 50nm. B. Representative class averages of T4SS3-10+D4 complex obtained after initial alignment and classification.