f4 ( a ) NRBF2 KO MEFs display impaired autophagosome acidification . WT and NRBF2 KO MEFs are transiently transfected with mCherry - GFP - LC3B . WT MEFs expressing mCherry - GFP - LC3B contain many red - only puncta along with yellow ( presence of both red and green ) puncta . The number of both red - only puncta and yellow puncta markedly increases after rapamycin ( Rap ; 50 μg ml − 1 , 4 h ) or starvation ( HBSS , 4 h ) treatment , suggesting the increased autophagolysosomes and nascent autophagosomes . In contrast , NRBF2 KO MEFs expressing mCherry - GFP - LC3B contain quite fewer red - only puncta and yellow puncta , even after Rap or starvation treatment . ( b ) The quantification results show that both the numbers of red - only puncta and percentage of red - only puncta ( verses total puncta ) in KO MEFs were markedly reduced . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , Mann - Whitney U - test , n = 20 randomly selected cells , a representative result from three independent repeats ) , indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells . Scale bars , 20 μm or 10 μm ( amplified regions ) . ( c ) Autophagolysome formation is impaired in NRBF2 KO MEFs . WT and NRBF2 KO MEFs were transiently transfected with RFP - LC3 then stained with LAMP2 antibody . The co - localization between RFP - LC3 and LAMP2 are illustrated by line profile . RFP - LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs ( as indicated by white arrows ) , suggesting the impairment of autophagolysosome formation in KO cells . Scale bars , 20 μm or 10 μm ( amplified regions ) . This is a representative image from three independent experiments . ( d ) Overexpression of NRBF2 enhances UVRAG - linked Vps34 kinase activity . HEK293T cells were co - transfected with Myc - Vps34 - Vps15 - His and FLAG - UVRAG , in the presence of CFP or NRBF2 - CFP . UVRAG - linked Vps34 was pulled down by an anti - FLAG antibody and subjected to KA . IP products and inputs are immunoblotted using the antibodies indicated . ( e ) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between two groups . ( Data are shown as mean ± s . e . m . , P value is indicated on the figures , one sample t - test versus hypothetical mean 1 , n = 5 ) .

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

f4(a) NRBF2 KO MEFs display impaired autophagosome acidification. WT and NRBF2 KO MEFs are transiently transfected with mCherry-GFP-LC3B. WT MEFs expressing mCherry-GFP-LC3B contain many red-only puncta along with yellow (presence of both red and green) puncta. The number of both red-only puncta and yellow puncta markedly increases after rapamycin (Rap; 50 μg ml−1, 4 h) or starvation (HBSS, 4 h) treatment, suggesting the increased autophagolysosomes and nascent autophagosomes. In contrast, NRBF2 KO MEFs expressing mCherry-GFP-LC3B contain quite fewer red-only puncta and yellow puncta, even after Rap or starvation treatment. (b) The quantification results show that both the numbers of red-only puncta and percentage of red-only puncta (verses total puncta) in KO MEFs were markedly reduced. (Data are shown as mean±s.e.m., P values are indicated on the figures, Mann-Whitney U-test, n=20 randomly selected cells, a representative result from three independent repeats), indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells. Scale bars, 20 μm or 10 μm (amplified regions).(c) Autophagolysome formation is impaired in NRBF2 KO MEFs. WT and NRBF2 KO MEFs were transiently transfected with RFP-LC3 then stained with LAMP2 antibody. The co-localization between RFP-LC3 and LAMP2 are illustrated by line profile. RFP-LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs (as indicated by white arrows), suggesting the impairment of autophagolysosome formation in KO cells. Scale bars, 20 μm or 10 μm (amplified regions). This is a representative image from three independent experiments.(d) Overexpression of NRBF2 enhances UVRAG-linked Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and FLAG-UVRAG, in the presence of CFP or NRBF2-CFP. UVRAG-linked Vps34 was pulled down by an anti-FLAG antibody and subjected to KA. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between two groups. (Data are shown as mean±s.e.m., P value is indicated on the figures, one sample t-test versus hypothetical mean 1, n=5).


f5 ( a ) The gross anatomical views of representative 4 % paraformaldehyde - fixed livers and quantification of liver index . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , unpaired Student ' s t - test , n = 3 mice each group ) . Scale bar , 1 cm . ( b ) The haematoxylin and eosin ( HE ) and CD45 staining of 10 - month - old WT and NRBF2 KO mice livers . The NRBF2 KO liver shows mild increase of ductular reaction ( yellow arrow ) and isolated necrosis ( red arrows ) . The necrotic foci were further confirmed by CD45 staining ( black arrow ) . This is a representative image from four mice per genotype . Scale bar , 50 μm . ( c ) The number of necrotic foci and ductular reaction scores were recorded under bright - field microscope . At least 10 randomly selected fields ( 10 × ) from each mouse and four mice per genotype were analysed . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures . Ductular reaction scores , unpaired Student ' s t - test ; necrotic foci , Mann - Whitney U - test , n = 4 mice each group ) . BD , bile duct ; CV , central vein ; PV , portal vein .

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

f5(a) The gross anatomical views of representative 4% paraformaldehyde-fixed livers and quantification of liver index. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test, n=3 mice each group). Scale bar, 1 cm.(b) The haematoxylin and eosin (HE) and CD45 staining of 10-month-old WT and NRBF2 KO mice livers. The NRBF2 KO liver shows mild increase of ductular reaction (yellow arrow) and isolated necrosis (red arrows). The necrotic foci were further confirmed by CD45 staining (black arrow). This is a representative image from four mice per genotype. Scale bar, 50 μm. (c) The number of necrotic foci and ductular reaction scores were recorded under bright-field microscope. At least 10 randomly selected fields (10 × ) from each mouse and four mice per genotype were analysed. (Data are shown as mean±s.e.m., P values are indicated on the figures. Ductular reaction scores, unpaired Student's t-test; necrotic foci, Mann-Whitney U-test, n=4 mice each group). BD, bile duct; CV, central vein; PV, portal vein.


f6 ( a ) WB analysis shows accumulation of p62 in the liver of NRBF2 KO mice . Liver lysates were immunoprobed with antibodies indicated . p62 protein levels are upregulated in the KO mouse liver . ( b ) The quantification result shows that the p62 level increased more than threefold in the NRBF2 KO miceliver compared with WT miceliver . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , unpaired Student ' s t - test ; WT , n = 5 mice ; KO , n = 4 mice ) . ( c ) WB analysis shows accumulation of high - molecular weight ( HMW ) ubiquitin in the liver of NRBF2 KO mice . Insoluble fractions of liver lysates were immunoprobed with an anti - ubiquitin antibody , n = 4 mice per genotype . ( d ) Atg14L - linked Vps34kinase activity is impaired in the mice of NRBF2 KO mice . Atg14L - linked Vps34 is pulled down by an anti - Atg14L antibody and is subjected to KA . IP products and inputs were immunoblotted using the antibodies indicated . ( e ) Quantification of IPed Vps34kinase activity versus IPed Atg14L shows the significant difference between WT and NRBF2 KO miceliver . ( Data are shown as mean ± s . e . m . , P values are indicated on the figure , unpaired Student ' s t - test , n = 4 mice each group ) . ( f ) Focal accumulation of p62 in the liver of NRBF2 KO mice . Liver frozen sections ( 15 μm in thickness ) were stained with an anti - p62 antibody . Focal accumulation of p62 is observed in the KO mice liver but not in the WT mice liver . Scale bars , 100 μm or 20 μm ( amplified regions ) . This is a representative image from four mice per genotype . ( g ) Accumulated p62 colocalizes with NQO1 . Liver frozen sections were double stained with an anti - p62 antibody and an anti - NQO1 antibody . This is a representative image from four mice per genotype . Scale bars , 20 μm .

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

f6(a) WB analysis shows accumulation of p62 in the liver of NRBF2 KO mice. Liver lysates were immunoprobed with antibodies indicated. p62 protein levels are upregulated in the KO mouse liver. (b) The quantification result shows that the p62 level increased more than threefold in the NRBF2 KO miceliver compared with WT miceliver. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test; WT, n=5 mice; KO, n=4 mice).(c) WB analysis shows accumulation of high-molecular weight (HMW) ubiquitin in the liver of NRBF2 KO mice. Insoluble fractions of liver lysates were immunoprobed with an anti-ubiquitin antibody, n=4 mice per genotype.(d) Atg14L-linked Vps34kinase activity is impaired in the mice of NRBF2 KO mice. Atg14L-linked Vps34 is pulled down by an anti-Atg14L antibody and is subjected to KA. IP products and inputs were immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34kinase activity versus IPed Atg14L shows the significant difference between WT and NRBF2 KO miceliver. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=4 mice each group).(f) Focal accumulation of p62 in the liver of NRBF2 KO mice. Liver frozen sections (15 μm in thickness) were stained with an anti-p62 antibody. Focal accumulation of p62 is observed in the KO mice liver but not in the WT mice liver. Scale bars, 100 μm or 20 μm (amplified regions). This is a representative image from four mice per genotype.(g) Accumulated p62 colocalizes with NQO1. Liver frozen sections were double stained with an anti-p62 antibody and an anti-NQO1 antibody. This is a representative image from four mice per genotype. Scale bars, 20 μm.


f7WT and NRBF2 KO MEFs were treated with DMSO , 1 μM thapsigargin ( TP ) or 1 μM tunicamycin ( TN ) for 24 or 48 h . ( a ) Then cells were either imaged under phase - contrast microscope or ( b ) stained with propidium iodide ( PI ) and subjected to cell death rate analysis by flow cytometry . The representative images of cell morphology after treatments and quantitative analysis of PI - positive cells rate show marked increase of cell death in the NRBF2 KO MEFs compared with WT MEFs . ( Data are shown as mean ± s . e . m . , P values are indicated on the figure , unpaired Student ' s t - test , n = 5 ) . Scale bars , 100 μm ; Con , control . ( c ) Re - introducing NRBF2 to NRBF2 KO MEFs reverses the vulnerability of cells to ER stress . NRBF2 - CFP stably expressing cells were established in the NRBF2 KO background . Cells were treated with DMSO or 1 μM TP for 24 h then stained with PI and subjected to cell death rate analysis by flow cytometry . The cell death rate in CFP - positive ( + ) population ( NRBF2 - CFP expression ) and negative ( − ) population ( no NRBF2 - CFP expression ) were recorded and analysed , respectively . ( Data are shown as mean ± s . e . m . , P values are indicated on the figure , unpaired Student ' s t - test , n = 3 ) . ( d ) ER stress inducers trigger caspase - 8 and caspase - 3 activation in NRBF2 KO MEFs . MEFs were treated with DMSO , 1 μM TP or 1 μM TN for 24 h , and then immunoprobed with indicated antibodies . Marked accumulation of cleaved caspase - 8 ( C - Cas 8 ; p43 and p18 units ) and cleaved caspase - 3 ( C - Cas 3 ) are observed in the NRBF2 KO MEFs but not WT MEFs treated with TP or TN . This is a representative image from three independent experiments . ( e ) C - Cas 8 preferentially localizes at p62 aggregation foci . MEFs were treated with DMSO or 1 μM TP for 24 h , and then co - stained with an anti - p62 antibody and an anti - cleaved caspase - 8 antibody . The C - Cas 8 fluorescence signals are triggered in NRBF2 KO MEFs treated with TP and preferentially localize at p62 aggregation foci . Scale bars , 20 μm . This is a representative image from three independent experiments .

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

f7WT and NRBF2 KO MEFs were treated with DMSO, 1 μM thapsigargin(TP) or 1 μM tunicamycin (TN) for 24 or 48 h. (a) Then cells were either imaged under phase-contrast microscope or (b) stained with propidium iodide (PI) and subjected to cell death rate analysis by flow cytometry. The representative images of cell morphology after treatments and quantitative analysis of PI-positive cells rate show marked increase of cell death in the NRBF2 KO MEFs compared with WT MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=5). Scale bars, 100 μm; Con, control.(c) Re-introducing NRBF2 to NRBF2 KO MEFs reverses the vulnerability of cells to ER stress. NRBF2-CFP stably expressing cells were established in the NRBF2 KO background. Cells were treated with DMSO or 1 μM TP for 24 h then stained with PI and subjected to cell death rate analysis by flow cytometry. The cell death rate in CFP-positive (+) population (NRBF2-CFP expression) and negative (−) population (no NRBF2-CFP expression) were recorded and analysed, respectively. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=3).(d) ER stress inducers trigger caspase-8 and caspase-3 activation in NRBF2 KO MEFs. MEFs were treated with DMSO, 1 μM TP or 1 μM TN for 24 h, and then immunoprobed with indicated antibodies. Marked accumulation of cleaved caspase-8 (C-Cas 8; p43 and p18 units) and cleaved caspase-3 (C-Cas 3) are observed in the NRBF2 KO MEFs but not WT MEFs treated with TP or TN. This is a representative image from three independent experiments.(e) C-Cas 8 preferentially localizes at p62 aggregation foci. MEFs were treated with DMSO or 1 μM TP for 24 h, and then co-stained with an anti-p62 antibody and an anti-cleaved caspase-8 antibody. The C-Cas 8 fluorescence signals are triggered in NRBF2 KO MEFs treated with TP and preferentially localize at p62 aggregation foci. Scale bars, 20 μm. This is a representative image from three independent experiments.


f8 ( a ) NRBF2 is critical for the assembly of Vps34 - Vps15 and Atg14L - Beclin 1 complex . Brain lysate from WT and NRBF2 KO mice were subjected to co - IP by Vps34 antibody and Atg14L antibody . The IP products were subjected to in vitro lipid KA and WB analysis using the indicated antibodies . ( b , c ) Quantification results shows that Vps34 - Vps15 and Atg14L - Beclin 1 complex interactions as well as Atg14L - linked Vps34 kinase activity are markedly impaired in NRBF2 KO mice . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , unpaired Student ' s t - test , n = 4 mice in each group ; NS , not significant ) . ( d ) Overexpression of NRBF2 enhances overall Vps34kinase activity . HEK293T cells were co - transfected with Myc - Vps34 - Vps15 - His and CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP . Total Vps34 was pulled down by an anti - myc antibody and subjected to KA . IP products and inputs are immunoblotted using the antibodies indicated . ( e ) Quantification of IPed Vps34kinase activity , Vps15 protein normalized with IPed Vps34 shows that NRBF2 - CFP , but not CFP , dMIT - CFP or dCCD - CFP significantly enhance Vps34kinase activity and Vps34 - Vps15 interaction . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures . IPed Vps15 / IPed Vps34 , one sample t - test versus hypothetical mean 1 , Bonferroni correction , n = 3 ; IPed Vps34kinase activity / IPed Vps34 , multiple t - test followed by Bonferroni correction , n = 3 ) .

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

f8(a) NRBF2 is critical for the assembly of Vps34-Vps15 and Atg14L-Beclin 1 complex. Brain lysate from WT and NRBF2 KO mice were subjected to co-IP by Vps34 antibody and Atg14L antibody. The IP products were subjected to in vitro lipid KA and WB analysis using the indicated antibodies. (b,c) Quantification results shows that Vps34-Vps15 and Atg14L-Beclin 1 complex interactions as well as Atg14L-linked Vps34 kinase activity are markedly impaired in NRBF2 KO mice. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test, n=4 mice in each group; NS, not significant).(d) Overexpression of NRBF2 enhances overall Vps34kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP. Total Vps34 was pulled down by an anti-myc antibody and subjected to KA. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34kinase activity, Vps15 protein normalized with IPed Vps34 shows that NRBF2-CFP, but not CFP, dMIT-CFP or dCCD-CFP significantly enhance Vps34kinase activity and Vps34-Vps15 interaction. (Data are shown as mean±s.e.m., P values are indicated on the figures. IPed Vps15/IPed Vps34, one sample t-test versus hypothetical mean 1, Bonferroni correction, n=3; IPed Vps34kinase activity/IPed Vps34, multiple t-test followed by Bonferroni correction, n=3).


Figure 1A NHF , Lowe 1676 and Lowe 3265 cells were grown in complete media or 5 h serum starvation , then immunostained with LAMP1 antibody . Dashed lines in images outline cell boundaries . Scale bar , 10 μm . C Quantitative analyses of lysosome perinuclear distribution in the experiments shown in A . The intracellular distribution of LAMP1 positive vesicles was quantified as described in the Methods . n = 3 per group in which 30 cells were counted per condition per experiment . Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test . B Wildtype ( Ocrl + / y ) and Ocrl - / - MEF cells were grown in complete media or 5 h serum starvation , then immunostained with indicated antibodies . Dashed lines in images outline cell boundaries . Scale bar , 10 μm . D , E Quantitative analyses of lysosome and mTOR perinuclear distribution in the experiments shown in B . The intracellular distribution of mTOR positive vesicles was quantified as described in the Methods . n = 3 per group in which 30 cells were counted per condition per experiment . Data information : , data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test . I Snap images of WT ( Ocrl + / y ) , OCRL knockout ( Ocrl - / - ) and IOB MEFs transfected with lysosome - GFP . After transfection , WT , OCRL knockout and IOB MEFs were left in serum starvation for 8 h . Scale bar , 5 μm . J Quantitative analyses of tubulated lysosomes in the experiments shown in H . n = 3 per group in which 10 cells were counted per condition per experiment . D Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test .

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

Figure 1A NHF, Lowe 1676 and Lowe 3265 cells were grown in complete media or 5 h serum starvation, then immunostained with LAMP1 antibody. Dashed lines in images outline cell boundaries. Scale bar, 10 μm. C Quantitative analyses of lysosome perinuclear distribution in the experiments shown in A. The intracellular distribution of LAMP1 positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.B Wildtype (Ocrl+/y) and Ocrl-/- MEF cells were grown in complete media or 5 h serum starvation, then immunostained with indicated antibodies. Dashed lines in images outline cell boundaries. Scale bar, 10 μm.D, E Quantitative analyses of lysosome and mTOR perinuclear distribution in the experiments shown in B. The intracellular distribution of mTOR positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment. Data information: , data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.I Snap images of WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs transfected with lysosome-GFP. After transfection, WT, OCRL knockout and IOB MEFs were left in serum starvation for 8 h. Scale bar, 5 μm. J Quantitative analyses of tubulated lysosomes in the experiments shown in H. n = 3 per group in which 10 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.


Figure 2A Immunoblot analysis of endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) and total p70 S6K in NHF , Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation . p - p70 S6K ( Thr389 ) over total p70 S6K ratios normalized to NHF cells , three independent experiments . B Immunoblot analysis of endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) and total p70 S6K in WT ( Ocrl + / y ) and IOB MEFs grown in complete medium or 5 h serum starvation . p - p70 S6K ( Thr389 ) over total p70 S6K ratios normalized to NHF cells , three independent experiments . C Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test unless otherwise stated . D NHF and Lowe 1676 cells were grown in complete media , amino - acid ( aa ) / FBS starved for 5 h , or starved and then recovered in amino - acid / FBS - containing medium , then immunoblotted using antibodies against endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) and total p70 S6K . Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test unless otherwise stated .

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

Figure 2A Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K in NHF, Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. B Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR , p-p70 S6K (Thr389) and total p70 S6K in WT (Ocrl+/y) and IOB MEFs grown in complete medium or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.D NHF and Lowe 1676 cells were grown in complete media, amino-acid (aa)/FBS starved for 5 h, or starved and then recovered in amino-acid/FBS-containing medium, then immunoblotted using antibodies against endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.


Figure 3A Representative images of NHF , Lowe 1676 and Lowe 3265 cells immunostained with anti - α - tubulin ( red ) and anti - γ - tubulin ( green ) antibodies . Scale bar , 10 μm . B Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center MTOC ) measured in the experiments shown in A . n = 3 cells per group , three independent experiments . C Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons . C WT ( Ocrl + / y ) , OCRL knockout ( Ocrl - / - ) and IOB MEFs derived from the corresponding mice were costained with anti - α - tubulin and anti - γ - tubulin antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm . D Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center MTOC ) measured in the experiments shown in C . n = 3 per group in which 30 cells were counted per condition per experiment . E Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons . E Microtubule regrowth assay was performed on NHF , Lowe 1676 and Lowe 3265 cells with 20 μM nocodazole for 2 h on ice , followed by rinse with PBS , and addition of pre - warmed medium to induce microtubule regrowth . Cells were then immunostained with anti - α - tubulin and anti - γ - tubulin antibodies at the indicated time point after addition of pre - warmed medium . The magnified view shows the cytoplasmic microtubules organization center . Scale bar , 10 μm . F Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center , MTOC ) measured in the experiments shown in E . n = 3 per group in which 30 cells were counted per condition per experiment . D Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons .

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

Figure 3A Representative images of NHF, Lowe 1676 and Lowe 3265 cells immunostained with anti-α-tubulin (red) and anti-γ-tubulin (green) antibodies. Scale bar, 10 μm. B Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in A. n = 3 cells per group, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.C WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs derived from the corresponding mice were costained with anti-α-tubulin and anti- γ-tubulin antibodies. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. D Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in C. n = 3 per group in which 30 cells were counted per condition per experiment. E Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.E Microtubule regrowth assay was performed on NHF, Lowe 1676 and Lowe 3265 cells with 20 μM nocodazole for 2 h on ice, followed by rinse with PBS, and addition of pre-warmed medium to induce microtubule regrowth. Cells were then immunostained with anti-α-tubulin and anti-γ-tubulin antibodies at the indicated time point after addition of pre-warmed medium. The magnified view shows the cytoplasmic microtubules organization center. Scale bar, 10 μm. F Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center, MTOC) measured in the experiments shown in E. n = 3 per group in which 30 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.


Figure 4E Super - resolution images of centrosome stained with anti - CEP164 ( red , labeling distal appendages of mother centriole ) and anti - OCRL ( blue ) were acquired using Centrin2 - GFP - RPE - 1 cells by DeltaVision OMX V4 BLAZE system . Scale bar , 0 . 5 μm . F Super - resolution images of centrosome stained with anti - CNAP1 ( red , labeling proximal ends of centriole ) and anti - OCRL ( blue ) were acquired using Centrin2 - GFP - RPE - 1 cells by DeltaVision OMX V4 BLAZE system . Scale bar , 0 . 5 μm . G H Representative images of the co - localization of OCRL - WT , GFP - OCRL - △ RhoGAP and GFP - OCRL - △ ASH + RhoGAP with γ - tubulin . Insets , enlargements of the outlined areas . Scale bars , 10 μm . I Quantitative analysis of cells with and without centrosomal OCRL ( centrosomal GFP signal ) in the experiments shown in A . n = 3 per group in which 10 cells were counted per condition per experiment . Means ± SEM , * * * * P < 0 . 0001 , n . s . , not significant , calculated by two - way ANOVA , multiple comparisons .

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

Figure 4E Super-resolution images of centrosome stained with anti-CEP164 (red, labeling distal appendages of mother centriole) and anti-OCRL (blue) were acquired using Centrin2-GFP-RPE-1 cells by DeltaVision OMX V4 BLAZE system. Scale bar, 0.5 μm. F Super-resolution images of centrosome stained with anti-CNAP1 (red, labeling proximal ends of centriole) and anti-OCRL (blue) were acquired using Centrin2-GFP-RPE-1 cells by DeltaVision OMX V4 BLAZE system. Scale bar, 0.5 μm. G H Representative images of the co-localization of OCRL-WT, GFP-OCRL-△RhoGAP and GFP-OCRL-△ASH+RhoGAP with γ-tubulin. Insets, enlargements of the outlined areas. Scale bars, 10 μm. I Quantitative analysis of cells with and without centrosomal OCRL (centrosomal GFP signal) in the experiments shown in A. n = 3 per group in which 10 cells were counted per condition per experiment. Means ±SEM, ****P < 0.0001, n.s., not significant, calculated by two-way ANOVA, multiple comparisons.


Figure 5A RPE - 1 cells transfected with nontargeting control siRNA or OCRL siRNA , were left for 48 h , then subjected to IF microscopy to visualize PI ( 4 , 5 ) P2 ( green ) , γ - tubulin ( red ) and nuclei ( blue , DAPI staining ) . Scale bar , 10 μm . B Quantitative analyses of centrosomal PI ( 4 , 5 ) P2 fluorescence intensity in the experiments shown in A . n = 3 per group in which 50 cells were counted per condition per experiment . C Data information : data are presented as mean ± SEM . n . s . , not significant , calculated by calculated by two - tailed Student ' s t - test . C NHF , Lowe 1676 and Lowe 3265 cells were subjected to IF microscopy to visualize PI ( 4 , 5 ) P2 ( green ) , gamma - tubulin ( red ) and nuclei ( blue , DAPI staining ) . Scale bar , 10 μm . D The fluorescence intensity of PI ( 4 , 5 ) P2 on centrosomes in C was measured . n = 3 per group in which 50 cells were counted per condition per experiment . D Data information : data are presented as mean ± SEM . n . s . , not significant , calculated by calculated by two - tailed Student ' s t - test .

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Figure 5A RPE-1 cells transfected with nontargeting control siRNA or OCRL siRNA, were left for 48 h, then subjected to IF microscopy to visualize PI(4,5)P2 (green), γ-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm. B Quantitative analyses of centrosomal PI(4,5)P2 fluorescence intensity in the experiments shown in A. n = 3 per group in which 50 cells were counted per condition per experiment. C Data information: data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test.C NHF, Lowe 1676 and Lowe 3265 cells were subjected to IF microscopy to visualize PI(4,5)P2 (green), gamma-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm. D The fluorescence intensity of PI (4,5)P2 on centrosomes in C was measured. n = 3 per group in which 50 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test.


Figure 6A SSX2IP interacts with OCRL . Plasmids carrying GFP - OCRL - WT , GFP - OCRL - △ RhoGAP and GFP - OCRL - △ ASH + RhoGAP were transfected into RPE - 1 cells . Immunoprecipitation was performed with GFP - Trap , followed by blotting with the indicated antibodies . D NHF , Lowe 1676 and Lowe 3265 cells were transfected with wildtype GFP - SSX2IP or GFP - PACT - SSX2IP for 48 h , then immunostained with anti - β - tubulin ( red ) antibody . Nuclei ( blue ) were stained with DAPI . The centrosomal microtubule nucleation in GFP - PACT - SSX2IP transfected cells was indicated by white arrowhead . Scale bar , 5 μm . F Western blot analysis of GFP , endogenous phospho - mTOR , total mTOR , in NHF and Lowe 1676 cells . NHF and Lowe 1676 cells were left untransfected , transfected with GFP - SSX2IP - WT or GFP - PACT - SSX2IP , then immunoblotted with indicated antibodies .

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

Figure 6A SSX2IP interacts with OCRL. Plasmids carrying GFP-OCRL-WT, GFP-OCRL-△RhoGAP and GFP-OCRL-△ASH+RhoGAP were transfected into RPE-1 cells. Immunoprecipitation was performed with GFP-Trap, followed by blotting with the indicated antibodies.D NHF, Lowe 1676 and Lowe 3265 cells were transfected with wildtype GFP-SSX2IP or GFP-PACT-SSX2IP for 48 h, then immunostained with anti-β-tubulin (red) antibody. Nuclei (blue) were stained with DAPI. The centrosomal microtubule nucleation in GFP-PACT-SSX2IP transfected cells was indicated by white arrowhead. Scale bar, 5 μm.F Western blot analysis of GFP, endogenous phospho-mTOR, total mTOR, in NHF and Lowe 1676 cells. NHF and Lowe 1676 cells were left untransfected, transfected with GFP-SSX2IP-WT or GFP-PACT-SSX2IP, then immunoblotted with indicated antibodies.


Figure 2A Z score for AUC and IC50 of 37 different drugs ( shown along the horizontal axis ) identified as hits in 13 of 17 samples screened . A drug hit is defined as z score of less than - 2 for both AUC and IC50 . Each dot in a column represents a sample screened for that drug . The size of the dot corresponds to the IC50 z score for that sample ( the larger the dot , the smaller the IC50 ) . Dots below the black horizontal line represent sample with AUC z score of less than - 2 . Dots are color coded for drug hit types . All color dots below the black line represent a hit for the corresponding drug .

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Figure 2A Z score for AUC and IC50 of 37 different drugs (shown along the horizontal axis) identified as hits in 13 of 17 samples screened. A drug hit is defined as z score of less than -2 for both AUC and IC50. Each dot in a column represents a sample screened for that drug. The size of the dot corresponds to the IC50 z score for that sample (the larger the dot, the smaller the IC50). Dots below the black horizontal line represent sample with AUC z score of less than -2. Dots are color coded for drug hit types. All color dots below the black line represent a hit for the corresponding drug.


Figure 3Event - free survival ( EFS ) and percentage of human CD45 + leukocytes in peripheral blood in acute lymphoblastic leukemia ( ALL ) orthotopic models . Event - free survival ( EFS ) and percentage of human CD45 + leukocytes in peripheral blood in acute lymphoblastic leukemia ( ALL ) orthotopic models . EFS and tumor volume in non - CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments . EFS and tumor volume in non - CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments . L EFS in a CNS orthotopic model in which drug sensitivity was observed . EFS is time of inoculation of tumor cells to event ( defined by neurologic symptoms or weight loss ) .

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Figure 3Event-free survival (EFS) and percentage of human CD45+ leukocytes in peripheral blood in acute lymphoblastic leukemia (ALL) orthotopic models.Event-free survival (EFS) and percentage of human CD45+ leukocytes in peripheral blood in acute lymphoblastic leukemia (ALL) orthotopic models.EFS and tumor volume in non-CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments.EFS and tumor volume in non-CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments.L EFS in a CNS orthotopic model in which drug sensitivity was observed. EFS is time of inoculation of tumor cells to event (defined by neurologic symptoms or weight loss).


Figure 4D Treatment response by therapy tier . 14 of 29 patients with molecular - based therapeutic options received the treatment .

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Figure 4D Treatment response by therapy tier. 14 of 29 patients with molecular-based therapeutic options received the treatment.


Figure 1Male C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . Serum SUA levelMale C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . body weight gainMale C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . fatMale C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . lean massMale C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . gastrocnemius index . Male C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . Gastrointestinal muscle fiber immunofluorescent laminin stainingMale C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . frequency histogram of fiber cross - sectional area .

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Figure 1Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Serum SUA levelMale C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. body weight gainMale C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. fatMale C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. lean massMale C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. gastrocnemius index.Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Gastrointestinal muscle fiber immunofluorescent laminin stainingMale C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. frequency histogram of fiber cross-sectional area.


Figure 2male C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . The muscle grip strengthmale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . running time in low speedmale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . four - limb handing timemale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . running time in high speed . male C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . serum concentration of RBCmale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . HGB in whole bloodmale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . ex vivo gastrocnemius muscle forcemale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . ex vivo gastrocnemius muscle forcemale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . fatigabilitymale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . glucose consumptionmale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . lactate production were tested .

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Figure 2male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. The muscle grip strengthmale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. running time in low speedmale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. four-limb handing timemale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. running time in high speed.male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. serum concentration of RBCmale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. HGB in whole bloodmale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. ex vivo gastrocnemius muscle forcemale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. ex vivo gastrocnemius muscle forcemale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. fatigabilitymale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. glucose consumptionmale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. lactate production were tested.


Figure 3Male C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . mRNA expression of MyHC I , MyHC IIa , PGC - 1α , myoglobin , TnnT1 MyHC IIb , MyHC IIx and TnnT3 in the gastrocnemius muscle ( n = 5 - 6 ) . Male C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . Immunoblots and quantification of MyHC - I , IIa and IIb protein expression in gastrocnemius ( n = 3 - 4 ) . Male C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . Representative images and quantification of Laminin ( green ) , MyHC - I and IIb immunofluorescent staining ( red ) in gastrocnemius ( n = 3 ) . Scale bar in ( C ) represents 100 µm .

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Figure 3Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. mRNA expression of MyHC I, MyHC IIa, PGC-1α, myoglobin, TnnT1 MyHC IIb, MyHC IIx and TnnT3 in the gastrocnemius muscle (n=5-6).Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Immunoblots and quantification of MyHC-I, IIa and IIb protein expression in gastrocnemius (n=3-4).Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Representative images and quantification of Laminin (green), MyHC-I and IIb immunofluorescent staining (red) in gastrocnemius (n=3). Scale bar in (C) represents 100 µm.


Figure 4Male C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 weeks . The O2 consumption ( VO2 ) and respiratory exchange ratio ( RER ) Male C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 weeks . Serum concentration of ( E ) NEFA in whole blood . Male C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 weeks . The enzymes activity of ( F ) SDH , ( G ) HK , and ( H ) LDH in gastrocnemius . Male C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 weeks . Immunoblots and quantification of of p - AMPK ， PGC - 1α and myoglobin in gastrocnemius . Male C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 weeks . Quantification of mitochondrial and electron transport chain ( ETC ) related genes expression in gastrocnemius . Male C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 weeks . OCRs were measured under basal condition in gastrocnemius .

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Figure 4Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. The O2 consumption (VO2) and respiratory exchange ratio (RER)Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Serum concentration of (E) NEFA in whole blood.Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. The enzymes activity of (F) SDH, (G) HK, and (H) LDH in gastrocnemius.Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Immunoblots and quantification of of p-AMPK，PGC-1α and myoglobin in gastrocnemius.Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Quantification of mitochondrial and electron transport chain (ETC) related genes expression in gastrocnemius.Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. OCRs were measured under basal condition in gastrocnemius.


Figure 5C2C12 cells were treated with 0 , 0 . 5 and 2 mM SUC for 48 h . Representative images of MyHC - I and IIb immunofluorescent staining ( green ) in C2C12 cells ( n = 16 ) . C2C12 cells were treated with 0 , 0 . 5 and 2 mM SUC for 48 h . quantification of MyHC - I and IIb immunofluorescent staining ( green ) in C2C12 cells ( n = 16 ) . C2C12 cells were treated with 0 , 0 . 5 and 2 mM SUC for 48 h . The enzymes activity of ( C ) SDH , ( D ) LDHC2C12 cells were treated with 0 , 0 . 5 and 2 mM SUC for 48 h . lactate production in C2C12 cells . C2C12 cells were treated with 0 , 0 . 5 and 2 mM SUC for 48 h . Quantification of mitochondrial DNA contents in C2C12 cells . C2C12 cells were treated with 0 , 0 . 5 and 2 mM SUC for 48 h . Mitochondrial electron microscopy showed the ( H ) mitochondrial density , ( I ) mitochondrial coverage , and ( J ) average mitochondrial area in C2C12 cell . Scale bar in ( A ) represents 50 μm , scale bar in ( G ) represents 0 . 5 μm .

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Figure 5C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Representative images of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=16).C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. quantification of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=16).C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. The enzymes activity of (C) SDH, (D) LDHC2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. lactate production in C2C12 cells.C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Quantification of mitochondrial DNA contents in C2C12 cells.C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Mitochondrial electron microscopy showed the (H) mitochondrial density, (I) mitochondrial coverage, and (J) average mitochondrial area in C2C12 cell. Scale bar in (A) represents 50 μm, scale bar in (G) represents 0.5 μm.


Figure 6SUNCR1 protein expression in the gastrocnemius from sedentary or post - running mice ( n = 4 ) . The mRNA level of SUNCR1 in gastrocnemius and soleus ( n = 7 - 8 ) . [ Ca2 + ] i in C2C12 cells treated with 0 or 2 mM SUC ( n = 18 - 20 ) . NFAT protein expression in nucleus and cytoplasm of gastrocnemius 0 . 5 h or 3 h after i . p . injection of 15 mg / kg succinate in C57BL / 6J mice ( n = 4 ) . SUNCR1 protein expression in C2C12 cells transfected with vector or siSUNCR1 ( n = 3 ) . [ Ca2 + ] i , in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC ( n = 5 - 6 ) . enzymes activity ( n = 9 - 10 ) of ( J ) HK , ( K ) LDH , and ( L ) SDH in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC ( n = 5 - 6 ) . Representative images and quantification of MyHC - I and IIb immunofluorescent staining ( green ) in C2C12 cells ( n = 3 ) . Scale bar in ( M ) represents 50 μm .

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Figure 6SUNCR1 protein expression in the gastrocnemius from sedentary or post-running mice (n=4).The mRNA level of SUNCR1 in gastrocnemius and soleus (n=7-8).[Ca2+]i in C2C12 cells treated with 0 or 2 mM SUC (n=18-20).NFAT protein expression in nucleus and cytoplasm of gastrocnemius 0.5 h or 3 h after i. p. injection of 15 mg/kg succinate in C57BL/6J mice (n=4).SUNCR1 protein expression in C2C12 cells transfected with vector or siSUNCR1 (n=3).[Ca2+]i, in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC (n=5-6).enzymes activity (n=9-10)of (J) HK, (K) LDH, and (L) SDH in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC (n=5-6).Representative images and quantification of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=3). Scale bar in (M) represents 50 μm.


Figure 7Male C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . The O2 consumption ( VO2 ) , RERMale C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . muscle grip strengthMale C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . four - limb handing timeMale C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . low speed running time . Male C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . The enzymes activity of ( H ) HK , ( I ) LDH , and ( J ) SDH in gastrocnemius . Male C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . Immunoblots and quantification of MyHC - I , IIb , NFAT , and PGC - 1α protein in gastrocnemius . Male C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . Representative images and quantification of Laminin ( green ) , or MyHC - I and IIb ( red ) immunofluorescent staining in gastrocnemius muscle ( n = 3 ) . Scale bar in ( M ) represents 100 µm .

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Figure 7Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The O2 consumption (VO2), RERMale C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. muscle grip strengthMale C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. four-limb handing timeMale C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. low speed running time.Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The enzymes activity of (H) HK, (I) LDH, and (J) SDH in gastrocnemius.Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Immunoblots and quantification of MyHC-I, IIb, NFAT, and PGC-1α protein in gastrocnemius.Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Representative images and quantification of Laminin (green), or MyHC-I and IIb (red) immunofluorescent staining in gastrocnemius muscle (n=3). Scale bar in (M) represents 100 µm.


Figure 8Male C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age . After two weeks of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . SUNCR1 protein expression in gastrocnemius from mice transfected with shSUNCR1 lentivirus or LV - shScrambled ( n = 3 ) . Male C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age . After two weeks of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . The running time in low speedMale C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age . After two weeks of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . four - limb handing timeMale C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age . After two weeks of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . muscle grip strength of both control and gastrocnemius - specific SUNCR1 knockdown mice . Male C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age . After two weeks of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . The enzymes activity of ( F ) HK , ( G ) LDH , ( H ) SDH in gastrocnemius . Male C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age . After two weeks of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . Immunoblots and quantification of MyHC - I , IIb , NFAT , and PGC - 1α protein in gastrocnemius ( n = 3 ) .

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

Figure 8Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. SUNCR1 protein expression in gastrocnemius from mice transfected with shSUNCR1 lentivirus or LV-shScrambled (n=3).Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The running time in low speedMale C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. four-limb handing timeMale C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. muscle grip strength of both control and gastrocnemius-specific SUNCR1 knockdown mice.Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The enzymes activity of (F) HK, (G) LDH, (H) SDH in gastrocnemius.Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Immunoblots and quantification of MyHC-I, IIb, NFAT, and PGC-1α protein in gastrocnemius (n=3).


Figure 1C Western blots of total protein of CHO cells transfected with ApCatSper 2 or 3 , non ‐ transfected control cells ( ‐ ) , and A . punctulata sperm ( Sp ) . The Western blots were probed with anti ‐ HA , anti ‐ ApCatSper 2 , or anti ‐ ApCatSper 3 antibodies . Arrows indicate bands representing ApCatSper 2 and 3 . D Immunocytochemistry analysis of sperm stained with anti ‐ ApCatSper 3 , anti ‐ GC , or anti ‐ CNGK antibodies ; superposition of images obtained by fluorescence microscopy and bright ‐ field light microscopy ; scale bar = 10 μm . The DNA was stained with DAPI ( blue ) . E Western blot analysis of co ‐ immunoprecipitation ( IP ) of A . punctulata sperm proteins . The input ( I ) , flow through ( FT ) , washes ( W1 ‐ 5 ) , and the eluate ( E ) of the IP using the anti ‐ ApCatSper 2 antibody were probed with the anti ‐ ApCatSper 3 antibody ( upper panel ) and vice versa ( lower panel ) . ApCatSper 3 and ApCatSper 2 were co ‐ immunoprecipitated with the anti ‐ ApCatSper 2 ( upper panel ) and anti ‐ ApCatSper 3 antibody ( lower panel ) , respectively . F Analysis by mass spectrometry of immunoprecipitated proteins . ApCatSper 1 ‐ 4 , β , γ , and δ were identified in the immunoprecipitates obtained with both the anti ‐ ApCatSper 2 and anti ‐ ApCatSper 3 antibodies . The number of tryptic peptides identified and the respective sequence coverage are given .

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Figure 1C Western blots of total protein of CHO cells transfected with ApCatSper 2 or 3, non‐transfected control cells (‐), and A. punctulata sperm (Sp). The Western blots were probed with anti‐HA, anti‐ApCatSper 2, or anti‐ApCatSper 3 antibodies. Arrows indicate bands representing ApCatSper 2 and 3.D Immunocytochemistry analysis of sperm stained with anti‐ApCatSper 3, anti‐GC, or anti‐CNGK antibodies; superposition of images obtained by fluorescence microscopy and bright‐field light microscopy; scale bar = 10 μm. The DNA was stained with DAPI (blue).E Western blot analysis of co‐immunoprecipitation (IP) of A. punctulata sperm proteins. The input (I), flow through (FT), washes (W1‐5), and the eluate (E) of the IP using the anti‐ApCatSper 2 antibody were probed with the anti‐ApCatSper 3 antibody (upper panel) and vice versa (lower panel). ApCatSper 3 and ApCatSper 2 were co‐immunoprecipitated with the anti‐ApCatSper 2 (upper panel) and anti‐ApCatSper 3 antibody (lower panel), respectively.F Analysis by mass spectrometry of immunoprecipitated proteins. ApCatSper 1‐4, β, γ, and δ were identified in the immunoprecipitates obtained with both the anti‐ApCatSper 2 and anti‐ApCatSper 3 antibodies. The number of tryptic peptides identified and the respective sequence coverage are given.


Figure 2A Alkaline ‐ evoked Ca2 + signals in sperm mixed with NH4Cl ; sperm were loaded with the Ca2 + indicator Fluo ‐ 4 . ΔF / F ( % ) indicates the change in Fluo ‐ 4 fluorescence ( ΔF ) with respect to the basal fluorescence ( F , mean of the first 3 - 5 data points ) . B Ca2 + signals evoked by mixing of sperm simultaneously with NH4Cl ( 30 mM ) and the CatSper inhibitor MDL12330A . C Dose - response relation of inhibition of the Ca2 + signals shown in panel ( B ) , Ki = 20 μM , amplitudes were determined at t = 4 s .

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Figure 2A Alkaline‐evoked Ca2+ signals in sperm mixed with NH4Cl; sperm were loaded with the Ca2+ indicator Fluo‐4. ΔF/F (%) indicates the change in Fluo‐4 fluorescence (ΔF) with respect to the basal fluorescence (F, mean of the first 3-5 data points).B Ca2+ signals evoked by mixing of sperm simultaneously with NH4Cl (30 mM) and the CatSper inhibitor MDL12330A.C Dose-response relation of inhibition of the Ca2+ signals shown in panel (B), Ki = 20 μM, amplitudes were determined at t = 4 s.


Figure 3A Changes in pHi evoked by mixing with pHi ‐ clamp solutions ( see explanation in the text ) ; sperm were loaded with the pHi indicator BCECF . ΔR / R ( % ) indicates the change in the BCECF fluorescence emission ratio ( ΔR = F494 / F540 ) with respect to the basal ratio ( R , mean of the first 3 - 5 data points ) . B Steady ‐ state change ( at t = 14 s ) of BCECF fluorescence for the pHi signals shown in ( A ) . The intercept of the fitted straight line with the x ‐ axis yields the resting pHi ; the slope of the straight line yields the ΔR / R ( % ) × ΔpH − 1 . Inset : calibrated changes in pHi evoked by various pHi ‐ clamp solutions . C Ca2 + signals evoked by mixing of sperm with pHi ‐ clamp solutions . D Dose - response relation for the Ca2 + signals shown in ( C ) . E Calibrated pHi increase ( red ) and respective Ca2 + response ( black ) evoked by mixing of sperm with a pHi 7 . 6 ‐ clamp solution ; depicted are , on an extended time scale , the first 500 ms of the respective pHi increase and Ca2 + signal shown in ( B , inset ) and ( C ) , respectively . The threshold pHi for CatSper activation was deduced from the latency of the Ca2 + signal . F Threshold pHi and latency of Ca2 + signals evoked by various pHi ‐ clamp solutions ( mean ± SD ; n ≥ 3 ) .

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

Figure 3A Changes in pHi evoked by mixing with pHi‐clamp solutions (see explanation in the text); sperm were loaded with the pHi indicator BCECF. ΔR/R (%) indicates the change in the BCECF fluorescence emission ratio (ΔR = F494/F540) with respect to the basal ratio (R, mean of the first 3-5 data points).B Steady‐state change (at t = 14 s) of BCECF fluorescence for the pHi signals shown in (A). The intercept of the fitted straight line with the x‐axis yields the resting pHi; the slope of the straight line yields the ΔR/R (%) × ΔpH−1. Inset: calibrated changes in pHi evoked by various pHi‐clamp solutions.C Ca2+ signals evoked by mixing of sperm with pHi‐clamp solutions.D Dose-response relation for the Ca2+ signals shown in (C).E Calibrated pHi increase (red) and respective Ca2+ response (black) evoked by mixing of sperm with a pHi 7.6‐clamp solution; depicted are, on an extended time scale, the first 500 ms of the respective pHi increase and Ca2+ signal shown in (B, inset) and (C), respectively. The threshold pHi for CatSper activation was deduced from the latency of the Ca2+ signal.F Threshold pHi and latency of Ca2+ signals evoked by various pHi‐clamp solutions (mean ± SD; n ≥ 3).


Figure 4A Depolarization ‐ evoked Ca2 + signals in sperm mixed with ASW containing high KCl concentrations . B Ca2 + signals evoked by mixing of sperm with 80 mM KCl and the CatSper inhibitor MDL12330A . C Ca2 + signals evoked by mixing of sperm with 160 mM KCl and MDL12330A . D Dose - response relation for the Ca2 + signals shown in ( B , C ) at t = 1 - 2 s . E Threshold pHi for Ca2 + signals evoked by pHi ‐ clamp solutions in sperm bathed in ASW containing low ( 3 mM ) , high ( 191 mM ) , and normal ( 9 mM ) KCl ( mean ± SD ; n ≥ 3 ) ; data for 9 mM KCl are from Fig F . F Resting pHi and resting Vm in sperm bathed in ASW containing low ( 3 mM ) , high ( 191 mM ) , and normal ( 9 mM ) KCl ( black ) ( mean ± SD ; n ≥ 3 ) . Mean threshold pHi for CatSper activation at different membrane potentials ( red ) ; mean threshold pHi was derived from data shown in ( E ) .

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Figure 4A Depolarization‐evoked Ca2+ signals in sperm mixed with ASW containing high KCl concentrations.B Ca2+ signals evoked by mixing of sperm with 80 mM KCl and the CatSper inhibitor MDL12330A.C Ca2+ signals evoked by mixing of sperm with 160 mM KCl and MDL12330A.D Dose-response relation for the Ca2+ signals shown in (B, C) at t = 1-2 s.E Threshold pHi for Ca2+ signals evoked by pHi‐clamp solutions in sperm bathed in ASW containing low (3 mM), high (191 mM), and normal (9 mM) KCl (mean ± SD; n ≥ 3); data for 9 mM KCl are from Fig F.F Resting pHi and resting Vm in sperm bathed in ASW containing low (3 mM), high (191 mM), and normal (9 mM) KCl (black) (mean ± SD; n ≥ 3). Mean threshold pHi for CatSper activation at different membrane potentials (red); mean threshold pHi was derived from data shown in (E).


Figure 5A Ca2 + signals in sperm evoked by photorelease ( at t = 0 ) of resact from caged resact in the presence of the CatSper inhibitor MDL12330A . B Ca2 + signals evoked by intracellular photorelease ( at t = 0 ) of cGMP in sperm loaded with caged cGMP in the presence of the CatSper inhibitor MDL12330A . C Normalized dose - response relation for inhibition of the resact ‐ and cGMP ‐ induced Ca2 + signals shown in ( A , B ) ( Ki = 6 . 2 and 4 . 3 μM , respectively ) . D Normalized dose ‐ response relation for inhibition of the resact ‐ and cGMP ‐ induced Ca2 + signals shown in Supplementary Fig S4 by the CatSper inhibitor mibefradil ( Ki = 7 . 7 and 20 . 9 μM , respectively ) .

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Figure 5A Ca2+ signals in sperm evoked by photorelease (at t = 0) of resact from caged resact in the presence of the CatSper inhibitor MDL12330A.B Ca2+ signals evoked by intracellular photorelease (at t = 0) of cGMP in sperm loaded with caged cGMP in the presence of the CatSper inhibitor MDL12330A.C Normalized dose-response relation for inhibition of the resact‐ and cGMP‐induced Ca2+ signals shown in (A, B) (Ki = 6.2 and 4.3 μM, respectively).D Normalized dose‐response relation for inhibition of the resact‐ and cGMP‐induced Ca2+ signals shown in Supplementary Fig S4 by the CatSper inhibitor mibefradil (Ki = 7.7 and 20.9 μM, respectively).


Figure 6A Calibrated Vm ( left ) and pHi ( right ) changes and Ca2 + signals ( middle ) evoked by resact . B Normalized Vm , pHi , and Ca2 + signals evoked by 10 pM resact ; the first 600 ms after mixing are shown . The hyperpolarization precedes the pHi increase , whereas the pHi increase precedes the Ca2 + increase . C Latency of the Vm , the pHi , and the Ca2 + signals evoked by various resact concentrations ( mean ± SD ; n = 3 ) . D Normalized pHi and Ca2 + signals evoked by photorelease of cGMP in sperm loaded with caged cGMP . E Calibrated Vm and Ca2 + signals evoked by 1 nM resact ; the threshold voltage ( Vthr ) for the Ca2 + influx was deduced from the latency of the Ca2 + signal . F Calibrated pHi and Ca2 + signals evoked by 1 nM resact ; the threshold pHi ( pHthr ) at which the Ca2 + influx commences was deduced from the latency of the Ca2 + signal . G Linear relationship between pHthr and Vthr for activation of Ca2 + influx by various resact concentrations ( data derived from Supplementary Fig S6 ; mean ± SD ; n ≥ 3 ) .

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

Figure 6A Calibrated Vm (left) and pHi (right) changes and Ca2+ signals (middle) evoked by resact.B Normalized Vm, pHi, and Ca2+ signals evoked by 10 pM resact; the first 600 ms after mixing are shown. The hyperpolarization precedes the pHi increase, whereas the pHi increase precedes the Ca2+ increase.C Latency of the Vm, the pHi, and the Ca2+ signals evoked by various resact concentrations (mean ± SD; n = 3).D Normalized pHi and Ca2+ signals evoked by photorelease of cGMP in sperm loaded with caged cGMP.E Calibrated Vm and Ca2+ signals evoked by 1 nM resact; the threshold voltage (Vthr) for the Ca2+ influx was deduced from the latency of the Ca2+ signal.F Calibrated pHi and Ca2+ signals evoked by 1 nM resact; the threshold pHi (pHthr) at which the Ca2+ influx commences was deduced from the latency of the Ca2+ signal.G Linear relationship between pHthr and Vthr for activation of Ca2+ influx by various resact concentrations (data derived from Supplementary Fig S6; mean ± SD; n ≥ 3).


Figure 7A Dark ‐ field light microscopy images of a sperm suspension before ( top ) and after ( bottom ) photorelease of a resact gradient ( middle ) in the absence ( control ; left ) or presence of the CatSper inhibitor MDL12330A ( 10 μM ; right ) . MDL12330A abolishes resact ‐ induced sperm accumulation . B Relative change of the sperm dispersion in the field of view evoked by photorelease of resact ( t = 0 , flash ) in the absence ( control ; blue ) or presence of MDL12330A ( red ) ; a decrease in dispersion indicates sperm accumulation in the irradiated area ( mean ± SD ; n = 4 ) .

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Figure 7A Dark‐field light microscopy images of a sperm suspension before (top) and after (bottom) photorelease of a resact gradient (middle) in the absence (control; left) or presence of the CatSper inhibitor MDL12330A (10 μM; right). MDL12330A abolishes resact‐induced sperm accumulation.B Relative change of the sperm dispersion in the field of view evoked by photorelease of resact (t = 0, flash) in the absence (control; blue) or presence of MDL12330A (red); a decrease in dispersion indicates sperm accumulation in the irradiated area (mean ± SD; n = 4).


Figure 8C Changes in pHi evoked by repetitive release ( arrows ) of cGMP from caged cGMP . The first UV flash was delivered at t = 0 .

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Figure 8C Changes in pHi evoked by repetitive release (arrows) of cGMP from caged cGMP. The first UV flash was delivered at t = 0.


Figure 1 ( A ) Bcl - xL was cotransfected into HeLa cells with empty vector ( IP negative control ) , Beclin 1 - Flag , Beclin 1 - Flag / HA - Bim ( EL ) , Beclin 1 - Flag / HA - Puma , Beclin 1 - Flag / HA - Noxa , or Beclin 1 - Flag / Bad . Anti - Flag ( M2 ) was used for immunoprecipitation , and proteins were detected with anti - Bcl - xL , anti - HA , or anti - Bad , anti - Flag ( Rabbit ) . ( B ) HA - Bim ( wild - type ) was cotransfected into Bax / Bak double - knockout ( DKO ) mouse embryonic fibroblasts ( MEFs ) with empty vector ( IP negative control ) or Beclin 1 - Flag . Immunoprecipitations were performed as in ( A ) . ( C ) Beclin 1 - Flag was cotransfected into Bax / Bak DKO MEFs with empty vector ( IP negative control ) or HA - Bim ( wild - type ) . Anti - HA was used for immunoprecipitation . ∗ , antibody heavy chain . ( D ) HeLa cell lysates were subjected to GFP antibody ( negative control ) and anti - Bim antibody immunoprecipitation . Proteins were detected with anti - Beclin 1 and anti - Bim . ∗ ∗ , antibody heavy chain ; ∗ , antibody light chain . ( E ) Bcl - xL was cotransfected into HeLa cells with empty vector ( IP negative control ) , wild - type HA - Bim ( EL ) , or HA - Bim L152E F159E [ Bim ( EL ) EE ] . Anti - Bim was used for immunoprecipitation . ∗ , antibody light chain . ( F ) HeLa cells were transfected with vector , wild - type HA - Bim , or HA - BimEE . Lysates were probed with the indicated antibodies . HeLa cells treated with staurosporine ( STS ) for 5 hr were a positive control . Note that wild - type Bim causes apoptosis , which reduces its levels compared to BimEE . ( G ) Vector , wild - type Bim ( EL ) , or Bim ( EL ) L152E F159E ( BimEE ) was transfected into HeLa cells . Transfection efficiency was > 90 % . After 20 hr , cell viability was determined by measurement pf ATP levels . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( H ) HeLa cells were transfected with vector / GFP ( 3 : 1 ) , wild - type HA - Bim / GFP ( 3 : 1 ) , or HA - BimEE / GFP ( 3 : 1 ) ( to ensure that Bim - transfected cells also contain GFP ) . After 16 hr , cells were stained with Annexin V - APC and analyzed by FACS . The percentage of APC and GFP double - positive cells / GFP - positive cells are shown . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( I ) Bim ( EL ) / Bcl - xL were cotransfected into HeLa cells with empty vector ( IP negative control ) or Beclin 1 - Flag ; Bim ( EL ) - EE / Bcl - xL were also cotransfected into HeLa cells with Beclin 1 - Flag . Anti - Flag ( M2 ) was used for immunoprecipitation . Data are shown as mean ± SD . NS , not significant . ( J ) BimEL - EE ( EL ) / vector ( IP negative control ) , BimEL - EE ( EL ) / Beclin 1 - Flag , BimL - EE ( L ) / Beclin 1 - Flag , and BimS - EE ( S ) / Beclin 1 - Flag were transfected into HeLa cells . Anti - Flag was used for immunoprecipitation . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 ; NS , not significant .

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

Figure 1(A) Bcl-xL was cotransfected into HeLa cells with empty vector (IP negative control), Beclin 1-Flag, Beclin 1-Flag/HA-Bim(EL), Beclin 1-Flag/HA-Puma, Beclin 1-Flag/HA-Noxa, or Beclin 1-Flag/Bad. Anti-Flag (M2) was used for immunoprecipitation, and proteins were detected with anti-Bcl-xL, anti-HA, or anti-Bad, anti-Flag (Rabbit).(B) HA-Bim (wild-type) was cotransfected into Bax/Bak double-knockout (DKO) mouse embryonic fibroblasts (MEFs) with empty vector (IP negative control) or Beclin 1-Flag. Immunoprecipitations were performed as in (A).(C) Beclin 1-Flag was cotransfected into Bax/Bak DKO MEFs with empty vector (IP negative control) or HA-Bim (wild-type). Anti-HA was used for immunoprecipitation. ∗, antibody heavy chain.(D) HeLa cell lysates were subjected to GFP antibody (negative control) and anti-Bim antibody immunoprecipitation. Proteins were detected with anti-Beclin 1 and anti-Bim. ∗∗, antibody heavy chain; ∗, antibody light chain.(E) Bcl-xL was cotransfected into HeLa cells with empty vector (IP negative control), wild-type HA-Bim(EL), or HA-Bim L152E F159E [Bim(EL)EE]. Anti-Bim was used for immunoprecipitation. ∗, antibody light chain.(F) HeLa cells were transfected with vector, wild-type HA-Bim, or HA-BimEE. Lysates were probed with the indicated antibodies. HeLa cells treated with staurosporine (STS) for 5 hr were a positive control. Note that wild-type Bim causes apoptosis, which reduces its levels compared to BimEE.(G) Vector, wild-type Bim(EL), or Bim(EL) L152E F159E (BimEE) was transfected into HeLa cells. Transfection efficiency was >90%. After 20 hr, cell viability was determined by measurement pf ATP levels. Data are shown as mean ± SD. ∗∗∗p < 0.001.(H) HeLa cells were transfected with vector/GFP (3:1), wild-type HA-Bim/GFP (3:1), or HA-BimEE/GFP (3:1) (to ensure that Bim-transfected cells also contain GFP). After 16 hr, cells were stained with Annexin V-APC and analyzed by FACS. The percentage of APC and GFP double-positive cells/GFP-positive cells are shown. Data are shown as mean ± SD. ∗∗∗p < 0.001.(I) Bim(EL)/Bcl-xL were cotransfected into HeLa cells with empty vector (IP negative control) or Beclin 1-Flag; Bim(EL)-EE/Bcl-xL were also cotransfected into HeLa cells with Beclin 1-Flag. Anti-Flag (M2) was used for immunoprecipitation. Data are shown as mean ± SD. NS, not significant.(J) BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Beclin 1-Flag, BimL-EE (L)/Beclin 1-Flag, and BimS-EE (S)/Beclin 1-Flag were transfected into HeLa cells. Anti-Flag was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant.


Figure 2 ( A ) Bim − / − and Bim + / + MEFs were cultured in 6 - well dishes . Cells were treated with vehicle or Bafilomycin A1 ( Baf ) for 4 hr . Blots were probed with the indicated antibodies . LC3 - II / tubulin in wild - type control MEFs is set as 1 . The relative value of LC3 - II / tubulin in Bim - knockout MEFs is shown ( n = 5 ) . Data are mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( B ) HeLa cells were transfected with control siRNA and Bim siRNA . After 48 hr , cells were treated with vehicle or Baf for 4 hr . Blots were probed with the indicated antibodies and analyzed as described in ( A ) . ∗ ∗ ∗ p < 0 . 001 . ( C ) Bim − / − and Bim + / + MEFs were transfected with GFP - LC3 . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . ( D ) HeLa cells were transfected with control plasmid ( pMKO ) / GFP - LC3 or pMKO - BimshRNA / GFP - LC3 ( to ensure that GFP - LC3 - positive cells also contain BimshRNA or control plasmid , the ratio of pMKO or pMKO - BimshRNA / GFP - LC3 is 3 : 1 ) . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( E ) HeLa cells were transfected with control plasmid ( pMKO ) or pMKO - BimshRNA . Blots were probed with the indicated antibodies . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( F ) HeLa cells were transfected with control plasmid ( pMKO ) / GFP - PX or pMKO - BimshRNA / GFP - PX ( 3 : 1 ) . The percentages of cells with GFP - vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . ( G ) DFCP1 - GFP cells were transfected with control siRNA or Bim siRNA . The numbers of GFP puncta were assessed . Data are shown as mean ± SD . ∗ p < 0 . 05 . Images represent DFCP1 - GFP vesicles in cells transfected with control siRNA and Bim siRNA . Arrows mark DFCP1 - GFP puncta . ( H ) HeLa cells were treated with control siRNA or Bim siRNA . Blots were probed with the indicated antibodies . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . ( I ) Bim ( EL ) / control siRNA , Bim ( EL ) / Bim siRNA , Bim ( EL ) EE / control siRNA , Bim ( EL ) EE / Bim siRNA , vector / control siRNA , and vector / Bim siRNA were transfected into HeLa cells . Blots were probed with the indicated antibodies . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . ( J ) HeLa cells were transfected with control plasmid or Bim ( EL ) EE . Blots were probed with the indicated antibodies . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . ( K ) Bax / Bak DKO MEFs were transfected with control plasmid / GFP - LC3 , wild - type Bim ( EL ) / GFP - LC3 ( 3 : 1 ) , or Bim ( EL ) EE / GFP - LC3 . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . ( L ) Two independent control vector ( pcDNA3 ) or Bim ( EL ) EE stably expressing HeLa cell clones were transfected with GFP - LC3 respectively . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( M ) HeLa cells were transfected with control plasmid / GFP - PX or Bim ( EL ) EE / GFP - PX ( 3 : 1 ) . The percentages of cells with GFP - vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . Fluorescent microscope images show GFP - PX vesicles .

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

Figure 2(A) Bim−/− and Bim+/+ MEFs were cultured in 6-well dishes. Cells were treated with vehicle or Bafilomycin A1 (Baf) for 4 hr. Blots were probed with the indicated antibodies. LC3-II/tubulin in wild-type control MEFs is set as 1. The relative value of LC3-II/tubulin in Bim-knockout MEFs is shown (n = 5). Data are mean ± SD. ∗∗∗p < 0.001.(B) HeLa cells were transfected with control siRNA and Bim siRNA. After 48 hr, cells were treated with vehicle or Baf for 4 hr. Blots were probed with the indicated antibodies and analyzed as described in (A). ∗∗∗p < 0.001.(C) Bim−/− and Bim+/+ MEFs were transfected with GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.(D) HeLa cells were transfected with control plasmid (pMKO)/GFP-LC3 or pMKO-BimshRNA/GFP-LC3 (to ensure that GFP-LC3-positive cells also contain BimshRNA or control plasmid, the ratio of pMKO or pMKO-BimshRNA/GFP-LC3 is 3:1). The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001.(E) HeLa cells were transfected with control plasmid (pMKO) or pMKO-BimshRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗∗p < 0.001.(F) HeLa cells were transfected with control plasmid (pMKO)/GFP-PX or pMKO-BimshRNA/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.(G) DFCP1-GFP cells were transfected with control siRNA or Bim siRNA. The numbers of GFP puncta were assessed. Data are shown as mean ± SD.∗p < 0.05.Images represent DFCP1-GFP vesicles in cells transfected with control siRNA and Bim siRNA. Arrows mark DFCP1-GFP puncta.(H) HeLa cells were treated with control siRNA or Bim siRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.(I) Bim(EL)/control siRNA, Bim(EL)/Bim siRNA, Bim(EL)EE/control siRNA, Bim(EL)EE/Bim siRNA, vector/control siRNA, and vector/Bim siRNA were transfected into HeLa cells. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.(J) HeLa cells were transfected with control plasmid or Bim(EL)EE. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.(K) Bax/Bak DKO MEFs were transfected with control plasmid /GFP-LC3, wild-type Bim(EL)/GFP-LC3 (3:1), or Bim (EL) EE/GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.(L) Two independent control vector (pcDNA3) or Bim(EL)EE stably expressing HeLa cell clones were transfected with GFP-LC3 respectively. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001.(M) HeLa cells were transfected with control plasmid/GFP-PX or Bim(EL)EE/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. Fluorescent microscope images show GFP-PX vesicles.