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

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

(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.


( a ) WB shows accumulation of p62 in liver of NRBF2 KO mice . Liver lysates were immunoprobed with antibodies indicated . p62 are upregulated in KO mouse liver . ( b ) result shows that p62 level increased more than threefold in NRBF2 KO miceliver compared with WT miceliver . ( Data are shown as mean ± s . e . m . , P values are indicated on figures , unpaired Student ' s t - ; WT , n = 5 mice ; KO , n = 4 mice ) .

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

(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 shows accumulation of high - molecular ( HMW ) ubiquitin in liver of NRBF2 KO mice . Insoluble fractions of liver lysates were immunoprobed with an anti - ubiquitin antibody , n = 4 mice per genotype .

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

(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 mice of NRBF2 KO mice . Atg14L - linked Vps34 is pulled down by an anti - Atg14L antibody is subjected to KA . IP products inputs were immunoblotted using antibodies indicated . ( e ) of IPed Vps34kinase activity versus IPed Atg14L shows significant difference between WT NRBF2 KO miceliver . ( Data are shown as mean ± s . e . m . , P values are indicated on figure , unpaired Student ' s t - , n = 4 mice each group ) .

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

(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 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 KO mice liver but not in WT mice liver . Scale bars , 100 μm or 20 μm ( amplified regions ) . This is a representative image from four mice per genotype .

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

(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 an anti - NQO1 antibody . This is a representative image from four mice per genotype . Scale bars , 20 μm .

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

(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.


WT NRBF2 KO MEFs were treated with DMSO , 1 μM thapsigargin ( TP ) or 1 μM tunicamycin ( TN ) for 24 or 48 h . ( a ) Then were either imaged under phase - contrast microscope or ( b ) stained with propidium iodide ( PI ) subjected to rate by flow cytometry . representative of after treatments quantitative of PI - positive rate show marked increase of in NRBF2 KO MEFs compared with WT MEFs . ( Data are shown as mean ± s . e . m . , P values are indicated on figure , unpaired Student ' s t - , n = 5 ) . Scale bars , 100 μm ; Con , control .

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 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

WT 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 vulnerability of to ER stress . NRBF2 - CFP stably expressing were established in NRBF2 KO background . were treated with DMSO or 1 μM TP for 24 h then stained with PI subjected to rate by flow cytometry . rate in CFP - positive ( + ) population ( NRBF2 - CFP expression ) negative ( − ) population ( no NRBF2 - CFP expression ) were recorded analysed , respectively . ( Data are shown as mean ± s . e . m . , P values are indicated on figure , unpaired Student ' s t - , n = 3 ) .

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

(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 caspase - 3 activation in NRBF2 KO MEFs . MEFs were treated with DMSO , 1 μM TP or 1 μM TN for 24 h , then immunoprobed with indicated antibodies . Marked accumulation of caspase - 8 ( C - Cas 8 ; p43 p18 units ) caspase - 3 ( C - Cas 3 ) are observed in NRBF2 KO MEFs but not WT MEFs treated with TP or TN . This is a representative image from three independent experiments .

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

(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 , then co - stained with an anti - p62 antibody an anti - caspase - 8 antibody . C - Cas 8 signals are triggered in NRBF2 KO MEFs treated with TP preferentially localize at p62 aggregation foci . Scale bars , 20 μm . This is a representative image from three independent experiments .

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

(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.


( a ) NRBF2 is critical for assembly of Vps34 - Vps15 Atg14L - Beclin 1 . Brain lysate from WT NRBF2 KO mice were subjected to co - IP by Vps34 antibody Atg14L antibody . IP products were subjected to in vitro lipid KA WB using indicated antibodies . ( b , c ) results shows that Vps34 - Vps15 Atg14L - Beclin 1 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 figures , unpaired Student ' s t - , n = 4 mice in each group ; NS , not significant ) .

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

(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 were co - transfected with Myc - Vps34 - Vps15 - His CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP . Total Vps34 was pulled down by an anti - myc antibody subjected to KA . IP products inputs are immunoblotted using antibodies indicated . ( e ) of IPed Vps34kinase activity , Vps15 normalized with IPed Vps34 shows that NRBF2 - CFP , but not CFP , dMIT - CFP or dCCD - CFP significantly enhance Vps34kinase activity Vps34 - Vps15 interaction . ( Data are shown as mean ± s . e . m . , P values are indicated on figures . IPed Vps15 / IPed Vps34 , one sample t - versus hypothetical mean 1 , Bonferroni correction , n = 3 ; IPed Vps34kinase activity / IPed Vps34 , multiple t - followed by Bonferroni correction , n = 3 ) .

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

(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).


A NHF , Lowe 1676 Lowe 3265 were grown in complete media or 5 h serum starvation , then immunostained with LAMP1 antibody . Dashed lines in outline boundaries . Scale bar , 10 μm . C Quantitative analyses of lysosome perinuclear distribution in experiments shown in A . intracellular distribution of LAMP1 positive vesicles was quantified as described in Methods . n = 3 per group in which 30 were counted per condition per experiment . Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - Student ' s t - .

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

A 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 ) Ocrl - / - MEF were grown in complete media or 5 h serum starvation , then immunostained with indicated antibodies . Dashed lines in outline boundaries . Scale bar , 10 μm .

O O O B-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

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 mTOR perinuclear distribution in experiments shown in B . intracellular distribution of mTOR positive vesicles was quantified as described in Methods . n = 3 per group in which 30 were counted per condition per experiment . Data information : , data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - Student ' s t - .

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

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 of WT ( Ocrl + / y ) , OCRL knockout ( Ocrl - / - ) IOB MEFs transfected with lysosome - GFP . After transfection , WT , OCRL knockout IOB MEFs were left in serum starvation for 8 h . Scale bar , 5 μm . J Quantitative analyses of tubulated lysosomes in experiments shown in H . n = 3 per group in which 10 were counted per condition per experiment . D Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - Student ' s t - .

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

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.


A Immunoblot of endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) total p70 S6K in NHF , Lowe 1676 Lowe 3265 grown in complete media or 5 h serum starvation . p - p70 S6K ( Thr389 ) over total p70 S6K ratios normalized to NHF , three independent experiments . B Immunoblot of endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) total p70 S6K in WT ( Ocrl + / y ) IOB MEFs grown in complete medium or 5 h serum starvation . p - p70 S6K ( Thr389 ) over total p70 S6K ratios normalized to NHF , three independent experiments . C Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - Student ' s t - unless otherwise stated .

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

A 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 Lowe 1676 were grown in complete media , - acid ( aa ) / FBS starved for 5 h , or starved then recovered in - acid / FBS - containing medium , then immunoblotted using antibodies against endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) total p70 S6K . Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - Student ' s t - unless otherwise stated .

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

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.


A Representative of NHF , Lowe 1676 Lowe 3265 immunostained with anti - α - tubulin ( red ) 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 experiments shown in A . n = 3 per group , three independent experiments . C Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons .

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

A Representative images of 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 - / - ) IOB MEFs derived from corresponding mice were costained with anti - α - tubulin 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 experiments shown in C . n = 3 per group in which 30 were counted per condition per experiment . E Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons .

O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 was performed on NHF , Lowe 1676 Lowe 3265 with 20 μM nocodazole for 2 h on ice , followed by rinse with PBS , addition of pre - warmed medium to induce microtubule regrowth . were then immunostained with anti - α - tubulin anti - γ - tubulin antibodies at indicated time point after addition of pre - warmed medium . magnified view shows microtubules organization center . Scale bar , 10 μm . F Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center , MTOC ) measured in experiments shown in E . n = 3 per group in which 30 were counted per condition per experiment . D Data information : data are presented as mean ± . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons .

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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.


E Super - resolution of centrosome stained with anti - CEP164 ( red , labeling distal appendages of mother centriole ) anti - OCRL ( blue ) were acquired using Centrin2 - GFP - RPE - 1 by DeltaVision OMX V4 BLAZE system . Scale bar , 0 . 5 μm . F Super - resolution of centrosome stained with anti - CNAP1 ( red , labeling proximal ends of centriole ) anti - OCRL ( blue ) were acquired using Centrin2 - GFP - RPE - 1 by DeltaVision OMX V4 BLAZE system . Scale bar , 0 . 5 μm . G

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E 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 of co - localization of OCRL - WT , GFP - OCRL - △ RhoGAP GFP - OCRL - △ ASH + RhoGAP with γ - tubulin . Insets , enlargements of outlined areas . Scale bars , 10 μm . I Quantitative of with without centrosomal OCRL ( centrosomal GFP signal ) in experiments shown in A . n = 3 per group in which 10 were counted per condition per experiment . Means ± , * * * * P < 0 . 0001 , n . s . , not significant , calculated by two - way ANOVA , multiple comparisons .

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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.


A RPE - 1 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 ) nuclei ( blue , DAPI ) . Scale bar , 10 μm . B Quantitative analyses of centrosomal PI ( 4 , 5 ) P2 in experiments shown in A . n = 3 per group in which 50 were counted per condition per experiment . C Data information : data are presented as mean ± . n . s . , not significant , calculated by calculated by two - Student ' s t - .

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A 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 Lowe 3265 were subjected to IF microscopy to visualize PI ( 4 , 5 ) P2 ( green ) , gamma - tubulin ( red ) nuclei ( blue , DAPI ) . Scale bar , 10 μm . D of PI ( 4 , 5 ) P2 on centrosomes in C was measured . n = 3 per group in which 50 were counted per condition per experiment . D Data information : data are presented as mean ± . n . s . , not significant , calculated by calculated by two - Student ' s t - .

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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.


A SSX2IP interacts with OCRL . Plasmids carrying GFP - OCRL - WT , GFP - OCRL - △ RhoGAP GFP - OCRL - △ ASH + RhoGAP were transfected into RPE - 1 . Immunoprecipitation was performed with GFP - Trap , followed by blotting with indicated antibodies .

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A 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 Lowe 3265 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 . centrosomal microtubule nucleation in GFP - PACT - SSX2IP transfected was indicated by arrowhead . Scale bar , 5 μm .

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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 of GFP , endogenous phospho - mTOR , total mTOR , in NHF Lowe 1676 . NHF Lowe 1676 were left untransfected , transfected with GFP - SSX2IP - WT or GFP - PACT - SSX2IP , then immunoblotted with indicated antibodies .

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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.


A Z score for AUC IC50 of 37 different drugs ( shown along 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 IC50 . Each dot in a column represents a sample screened for that drug . of dot corresponds to IC50 z score for that sample ( larger dot , smaller IC50 ) . Dots below black horizontal represent sample with AUC z score of less than - 2 . Dots are color coded for drug hit types . color dots below black represent a hit for corresponding drug .

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A 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.


Event - free survival ( EFS ) of human CD45 + leukocytes in peripheral blood in acute lymphoblastic leukemia ( ) orthotopic models .

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Event-free survival (EFS) and percentage of human CD45+ leukocytes in peripheral blood in acute lymphoblastic leukemia (ALL) orthotopic models.


EFS 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 to event ( defined by neurologic symptoms or loss ) .

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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).


D Treatment response by therapy tier . 14 of 29 with molecular - therapeutic options received treatment .

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


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . Serum SUA

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Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Serum SUA level


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 .

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Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. body weight gain


Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. fat


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . lean

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Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. lean mass


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . gastrocnemius index .

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Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. gastrocnemius index.


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . Gastrointestinal muscle fiber immunofluorescent laminin

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Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Gastrointestinal muscle fiber immunofluorescent laminin staining


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . frequency histogram of fiber cross - sectional .

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Male 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.


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . muscle grip

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. The muscle grip strength


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . running time in low

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. running time in low speed


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . four - limb handing

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. four-limb handing time


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . running time in high speed .

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male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. running time in high speed.


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . serum of

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. serum concentration of RBC


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . HGB in whole

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. HGB in whole blood


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . ex vivo gastrocnemius muscle

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. ex vivo gastrocnemius muscle force


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 .

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male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. fatigability


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . glucose

male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. glucose consumption


C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . lactate production were tested .

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male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. lactate production were tested.


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . mRNA expression of MyHC I , MyHC IIa , PGC - 1α , myoglobin , TnnT1 MyHC IIb , MyHC IIx TnnT3 in gastrocnemius muscle ( n = 5 - 6 ) .

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Male 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).


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . Immunoblots of MyHC - I , IIa IIb expression in gastrocnemius ( n = 3 - 4 ) .

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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).


C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 . Representative of Laminin ( green ) , MyHC - I IIb immunofluorescent ( red ) in gastrocnemius ( n = 3 ) . Scale bar in ( C ) represents 100 µm .

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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.


C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 . O2 consumption ( VO2 ) respiratory exchange ( RER )

Male 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)


C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 . Serum of ( E ) NEFA in whole blood .

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.


C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 . enzymes activity of ( F ) SDH , ( G ) HK , ( H ) LDH in gastrocnemius .

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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.


C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 . Immunoblots of of p - AMPK ， PGC - 1α myoglobin in gastrocnemius .

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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.


C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 . of mitochondrial 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. Quantification of mitochondrial and electron transport chain (ETC) related genes expression in gastrocnemius.


C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 . OCRs were measured under basal condition 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.


C2C12 were treated with 0 , 0 . 5 2 mM SUC for 48 h . Representative of MyHC - I IIb immunofluorescent ( green ) in C2C12 ( n = 16 ) .

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C2C12 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 were treated with 0 , 0 . 5 2 mM SUC for 48 h . of MyHC - I IIb immunofluorescent ( green ) in C2C12 ( n = 16 ) .

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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 were treated with 0 , 0 . 5 2 mM SUC for 48 h . enzymes activity of ( C ) SDH , ( D )

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C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. The enzymes activity of (C) SDH, (D) LDH


C2C12 were treated with 0 , 0 . 5 2 mM SUC for 48 h . lactate production in C2C12 .

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C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. lactate production in C2C12 cells.


C2C12 were treated with 0 , 0 . 5 2 mM SUC for 48 h . of mitochondrial contents in C2C12 .

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C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Quantification of mitochondrial DNA contents in C2C12 cells.


C2C12 were treated with 0 , 0 . 5 2 mM SUC for 48 h . Mitochondrial electron microscopy showed ( H ) mitochondrial density , ( I ) mitochondrial coverage , ( J ) average mitochondrial in C2C12 . Scale bar in ( A ) represents 50 μm , scale bar in ( G ) represents 0 . 5 μm .

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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.


SUNCR1 expression in gastrocnemius from sedentary or post - running mice ( n = 4 ) .

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SUNCR1 protein expression in the gastrocnemius from sedentary or post-running mice (n=4).


mRNA level of SUNCR1 in gastrocnemius soleus ( n = 7 - 8 ) .

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

The mRNA level of SUNCR1 in gastrocnemius and soleus (n=7-8).


[ Ca2 + ] i in C2C12 treated with 0 or 2 mM SUC ( n = 18 - 20 ) .

[Ca2+]i in C2C12 cells treated with 0 or 2 mM SUC (n=18-20).


NFAT expression in nucleus cytoplasm of gastrocnemius 0 . 5 h or 3 h after i . p . injection of 15 mg / kg succinate in C57BL / 6J mice ( n = 4 ) .

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

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 expression in C2C12 transfected with vector or siSUNCR1 ( n = 3 ) .

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

SUNCR1 protein expression in C2C12 cells transfected with vector or siSUNCR1 (n=3).


[ Ca2 + ] i , in vector or siSUNCR1 transfected C2C12 treated with 0 or 2 mM SUC ( n = 5 - 6 ) .

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

[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 , ( L ) SDH in vector or siSUNCR1 transfected C2C12 treated with 0 or 2 mM SUC ( n = 5 - 6 ) .

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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 of MyHC - I IIb immunofluorescent ( green ) in C2C12 ( n = 3 ) . Scale bar in ( M ) represents 50 μm .

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Representative images and quantification of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=3). Scale bar in (M) represents 50 μm.


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . O2 consumption ( VO2 ) ,

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

Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The O2 consumption (VO2), RER


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . muscle grip

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

Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. muscle grip strength


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . four - limb handing

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

Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. four-limb handing time


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . low speed running time .

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

Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. low speed running time.


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . enzymes activity of ( H ) HK , ( I ) LDH , ( J ) SDH in gastrocnemius .

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

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.


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . Immunoblots of MyHC - I , IIb , NFAT , PGC - 1α in gastrocnemius .

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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.


C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . Representative of Laminin ( green ) , or MyHC - I IIb ( red ) immunofluorescent in gastrocnemius muscle ( n = 3 ) . Scale bar in ( M ) represents 100 µm .

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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.


C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into gastrocnemius at 6 of . After two of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . SUNCR1 expression in gastrocnemius from mice transfected with shSUNCR1 lentivirus or LV - shScrambled ( n = 3 ) .

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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. SUNCR1 protein expression in gastrocnemius from mice transfected with shSUNCR1 lentivirus or LV-shScrambled (n=3).


C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into gastrocnemius at 6 of . After two of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . running time in low

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

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 speed


C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into gastrocnemius at 6 of . After two of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . four - limb handing

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. four-limb handing time


C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into gastrocnemius at 6 of . After two of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . muscle grip strength of both control gastrocnemius - specific SUNCR1 knockdown mice .

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

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. muscle grip strength of both control and gastrocnemius-specific SUNCR1 knockdown mice.


C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into gastrocnemius at 6 of . After two of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . enzymes activity of ( F ) HK , ( G ) LDH , ( H ) SDH in gastrocnemius .

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

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.


C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into gastrocnemius at 6 of . After two of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 . Immunoblots of MyHC - I , IIb , NFAT , PGC - 1α in gastrocnemius ( n = 3 ) .

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

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).


C Western blots of total of CHO transfected with ApCatSper 2 or 3 , non ‐ transfected control ( ‐ ) , A . punctulata sperm ( Sp ) . Western blots were probed with anti ‐ HA , anti ‐ ApCatSper 2 , or anti ‐ ApCatSper 3 antibodies . Arrows indicate bands representing ApCatSper 2 3 .

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C 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 of sperm stained with anti ‐ ApCatSper 3 , anti ‐ GC , or anti ‐ CNGK antibodies ; superposition of obtained by microscopy bright ‐ field microscopy ; scale bar = 10 μm . was stained with DAPI ( blue ) .

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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 of co ‐ immunoprecipitation ( IP ) of A . punctulata sperm . input ( I ) , flow through ( FT ) , washes ( W1 ‐ 5 ) , eluate ( E ) of IP using anti ‐ ApCatSper 2 antibody were probed with anti ‐ ApCatSper 3 antibody ( upper panel ) vice versa ( lower panel ) . ApCatSper 3 ApCatSper 2 were co ‐ immunoprecipitated with anti ‐ ApCatSper 2 ( upper panel ) anti ‐ ApCatSper 3 antibody ( lower panel ) , respectively .

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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 by mass spectrometry of immunoprecipitated . ApCatSper 1 ‐ 4 , β , γ , δ were identified in immunoprecipitates obtained with both anti ‐ ApCatSper 2 anti ‐ ApCatSper 3 antibodies . number of tryptic peptides identified respective coverage are given .

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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.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


A Changes in pHi evoked by mixing with pHi ‐ clamp solutions ( see explanation in text ) ; sperm were loaded with pHi indicator BCECF . ΔR / R ( % ) indicates change in BCECF emission ( ΔR = F494 / F540 ) with respect to basal ( R , mean of first 3 - 5 data points ) . B Steady ‐ state change ( at t = 14 s ) of BCECF for pHi signals shown in ( A ) . intercept of fitted straight with x ‐ axis yields resting pHi ; slope of straight yields ΔR / R ( % ) × ΔpH − 1 . Inset : calibrated changes in pHi evoked by various pHi ‐ clamp solutions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

C Ca2+ signals evoked by mixing of sperm with pHi‐clamp solutions.


D Dose - response relation for Ca2 + signals shown in ( C ) .

D Dose-response relation for the Ca2+ signals shown in (C).


E Calibrated pHi increase ( red ) respective Ca2 + response ( black ) evoked by mixing of sperm with a pHi 7 . 6 ‐ clamp solution ; depicted are , on an extended time scale , first 500 ms of respective pHi increase Ca2 + signal shown in ( B , inset ) ( C ) , respectively . threshold pHi for CatSper activation was deduced from latency of Ca2 + signal . F Threshold pHi latency of Ca2 + signals evoked by various pHi ‐ clamp solutions ( mean ± SD ; n ≥ 3 ) .

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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).


A Depolarization ‐ evoked Ca2 + signals in sperm mixed with ASW containing high KCl concentrations .

A 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 CatSper inhibitor MDL12330A .

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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 MDL12330A .

C Ca2+ signals evoked by mixing of sperm with 160 mM KCl and MDL12330A.


D Dose - response relation for Ca2 + signals shown in ( B , C ) at t = 1 - 2 s .

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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 ) , normal ( 9 mM ) KCl ( mean ± SD ; n ≥ 3 ) ; data for 9 mM KCl are from Fig F . F Resting pHi resting Vm in sperm bathed in ASW containing low ( 3 mM ) , high ( 191 mM ) , normal ( 9 mM ) KCl ( black ) ( mean ± SD ; n ≥ 3 ) . Mean threshold pHi for CatSper activation at different potentials ( red ) ; mean threshold pHi was derived from data shown in ( E ) .

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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).


A Ca2 + signals in sperm evoked by photorelease ( at t = 0 ) of resact from caged resact in presence of CatSper inhibitor MDL12330A .

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A 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 presence of CatSper inhibitor MDL12330A .

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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 resact ‐ cGMP ‐ induced Ca2 + signals shown in ( A , B ) ( Ki = 6 . 2 4 . 3 μM , respectively ) .

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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 resact ‐ cGMP ‐ induced Ca2 + signals shown in Supplementary Fig S4 by CatSper inhibitor mibefradil ( Ki = 7 . 7 20 . 9 μM , respectively ) .

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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).


A Calibrated Vm ( left ) pHi ( right ) changes Ca2 + signals ( middle ) evoked by resact .

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A Calibrated Vm (left) and pHi (right) changes and Ca2+ signals (middle) evoked by resact.


B Normalized Vm , pHi , Ca2 + signals evoked by 10 pM resact ; first 600 ms after mixing are shown . hyperpolarization precedes pHi increase , whereas pHi increase precedes Ca2 + increase .

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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 Vm , pHi , Ca2 + signals evoked by various resact concentrations ( mean ± SD ; n = 3 ) .

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C Latency of the Vm, the pHi, and the Ca2+ signals evoked by various resact concentrations (mean ± SD; n = 3).


D Normalized pHi Ca2 + signals evoked by photorelease of cGMP in sperm loaded with caged cGMP .

D Normalized pHi and Ca2+ signals evoked by photorelease of cGMP in sperm loaded with caged cGMP.


E Calibrated Vm Ca2 + signals evoked by 1 nM resact ; threshold voltage ( Vthr ) for Ca2 + influx was deduced from latency of Ca2 + signal .

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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 Ca2 + signals evoked by 1 nM resact ; threshold pHi ( pHthr ) at which Ca2 + influx commences was deduced from latency of Ca2 + signal .

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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 Vthr for activation of Ca2 + influx by various resact concentrations ( data derived from Supplementary Fig S6 ; mean ± SD ; n ≥ 3 ) .

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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).


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

A 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).


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

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


( A ) Bcl - xL was cotransfected into HeLa 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 , were detected with anti - Bcl - xL , anti - HA , or anti - Bad , anti - Flag ( Rabbit ) .

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(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 ) .

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

(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 .

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

(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 lysates were subjected to GFP antibody ( negative control ) anti - Bim antibody immunoprecipitation . were detected with anti - Beclin 1 anti - Bim . ∗ ∗ , antibody heavy chain ; ∗ , antibody chain .

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

(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 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 chain .

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

(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 were transfected with vector , wild - type HA - Bim , or HA - BimEE . Lysates were probed with indicated antibodies . HeLa treated with staurosporine ( STS ) for 5 hr were a positive control . Note that wild - type Bim causes apoptosis , which reduces its compared to BimEE .

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(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 . Transfection efficiency was > 90 % . After 20 hr , viability was determined by measurement pf ATP . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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

(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 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 also contain GFP ) . After 16 hr , were stained with Annexin V - APC analyzed by FACS . of APC GFP double - positive / GFP - positive are shown . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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

(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 with empty vector ( IP negative control ) or Beclin 1 - Flag ; Bim ( EL ) - EE / Bcl - xL were also cotransfected into HeLa with Beclin 1 - Flag . Anti - Flag ( M2 ) was used for immunoprecipitation . Data are shown as mean ± SD . NS , not significant .

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

(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 , BimS - EE ( S ) / Beclin 1 - Flag were transfected into HeLa . Anti - Flag was used for immunoprecipitation . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 ; NS , not significant .

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 B-MEASURED_VAR O O O O O O B-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

(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.


( A ) Bim − / − Bim + / + MEFs were cultured in 6 - well dishes . were treated with vehicle or Bafilomycin A1 ( Baf ) for 4 hr . Blots were probed with indicated antibodies . LC3 - II / tubulin in wild - type control MEFs is set as 1 . relative value of LC3 - II / tubulin in Bim - knockout MEFs is shown ( n = 5 ) . Data are mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O 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) 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 were transfected with control siRNA Bim siRNA . After 48 hr , were treated with vehicle or Baf for 4 hr . Blots were probed with indicated antibodies analyzed as described in ( A ) . ∗ ∗ ∗ p < 0 . 001 .

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

(B) 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 − / − Bim + / + MEFs were transfected with GFP - LC3 . percentages of with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

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

(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 were transfected with control plasmid ( pMKO ) / GFP - LC3 or pMKO - BimshRNA / GFP - LC3 ( to ensure that GFP - LC3 - positive also contain BimshRNA or control plasmid , of pMKO or pMKO - BimshRNA / GFP - LC3 is 3 : 1 ) . percentages of with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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

(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 were transfected with control plasmid ( pMKO ) or pMKO - BimshRNA . Blots were probed with indicated antibodies . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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

(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 were transfected with control plasmid ( pMKO ) / GFP - PX or pMKO - BimshRNA / GFP - PX ( 3 : 1 ) . percentages of with GFP - vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

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

(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 were transfected with control siRNA or Bim siRNA . numbers of GFP were assessed . Data are shown as mean ± SD . ∗ p < 0 . 05 . represent DFCP1 - GFP vesicles in transfected with control siRNA Bim siRNA . Arrows mark DFCP1 - GFP .

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

(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 were treated with control siRNA or Bim siRNA . Blots were probed with indicated antibodies . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

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

(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 , vector / Bim siRNA were transfected into HeLa . Blots were probed with indicated antibodies . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

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

(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 were transfected with control plasmid or Bim ( EL ) EE . Blots were probed with indicated antibodies . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

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

(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 . percentages of with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

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

(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 clones were transfected with GFP - LC3 respectively . percentages of with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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

(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 were transfected with control plasmid / GFP - PX or Bim ( EL ) EE / GFP - PX ( 3 : 1 ) . percentages of with GFP - vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . microscope show GFP - PX vesicles .

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

(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.


( A ) Lysates from spleen - derived T of wild - type ( Bim + / + ) Bim - knockout ( Bim − / − ) mice were immunoblotted probed with indicated antibodies . Statistics were from three knockout control mice . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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(A) Lysates from spleen-derived T cells of wild-type (Bim+/+) and Bim-knockout (Bim−/−) mice were immunoblotted and probed with indicated antibodies. Statistics were from three knockout and control mice. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( B ) T from Bim + / + Bim − / − mice were cultured with / without Baf for 4 hr preceding harvest , immunoblotted , probed with indicated antibodies .

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(B) T cells from Bim+/+ and Bim−/− mice were cultured with/without Baf for 4 hr preceding harvest, immunoblotted, and probed with the indicated antibodies.


( C ) Spleen - derived T from Bim + / + or Bim − / − mice were stained with rabbit anti - LC3 antibody . Representative confocal from triplicates ( n = 100 - 110 ) are shown . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 0001 .

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(C) Spleen-derived T cells from Bim+/+ or Bim−/− mice were stained with rabbit anti-LC3 antibody. Representative confocal images and quantification from triplicates (n = 100-110) are shown. Data are shown as mean ± SD. ∗∗∗p < 0.0001.


( D ) Bim + / + Bim − / − mouse liver lysates were immunoblotted probed with indicated antibodies ( n = 3 ) . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

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(D) Bim+/+ and Bim−/− mouse liver lysates were immunoblotted and probed with the indicated antibodies (n = 3). Data are shown as mean ± SD. ∗∗p < 0.01.


( E ) Bim + / + Bim − / − mouse liver lysates were immunoblotted probed with indicated antibodies ( n = 3 ) . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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(E) Bim+/+ and Bim−/− mouse liver lysates were immunoblotted and probed with the indicated antibodies (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001.


( F ) from Bim wild - type or Bim - knockout mouse livers was analyzed by qRT - PCR for p62 / beta - actin mRNA . mean ± SD from three mice per group is shown . NS , not significant .

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

(F) RNA from Bim wild-type or Bim-knockout mouse livers was analyzed by qRT-PCR for p62/beta-actin mRNA. The mean ± SD from three mice per group is shown. NS, not significant.


( A ) HeLa were treated with control siRNA , Bim siRNA + control siRNA , control siRNA + Beclin 1 siRNA , or Bim siRNA + Beclin 1 siRNA . Blots were probed as indicated . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

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

(A) HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA. Blots were probed as indicated. Data are shown as mean ± SD. ∗∗p < 0.01.


( B ) HeLa were treated with control siRNA , Bim siRNA + control siRNA , control siRNA + Beclin 1 siRNA , or Bim siRNA + Beclin 1 siRNA . After 24 hr , were transfected with GFP - PX for 24 hr . GFP - PX vesicles were assessed with a confocal microscope . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . Arrows label with increased number / of GFP - PX vesicles .

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

(B) HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA. After 24 hr, cells were transfected with GFP-PX for 24 hr. GFP-PX vesicles were assessed with a confocal microscope. Data are shown as mean ± SD. ∗∗p < 0.01. Arrows label cells with increased number/size of GFP-PX vesicles.


( C ) GFP - mRFP - LC3 HeLa were treated with control , Bim siRNA , or Bcl - 2 siRNA then analyzed with Cellomics microscope . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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(C) GFP-mRFP-LC3 HeLa cells were treated with control, Bim siRNA, or Bcl-2 siRNA and then analyzed with Cellomics microscope. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( D ) GFP - mRFP - LC3 stable HeLa were treated with control siRNA , Bim siRNA + control siRNA , control siRNA + Beclin 1 siRNA , or Bim siRNA + Beclin 1 siRNA then analyzed with Cellomics microscope matching confocal are shown . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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

(D) GFP-mRFP-LC3 stable HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA and then analyzed with Cellomics microscope and matching confocal images are shown. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( A ) Bim ( EL ) EE / empty vector ( IP negative control ) or Bim ( EL ) EE / Beclin 1 - Flag ( in duplicate ) were transfected into HeLa . After 20 hr , one set of with Bim ( EL ) EE / Beclin 1 - Flag were starved in HBSS for 2 hr . Anti - Flag antibody ( M2 ) was used for immunoprecipitation , blots were probed as indicated . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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

(A) Bim(EL)EE/empty vector (IP negative control) or Bim(EL)EE/Beclin 1-Flag (in duplicate) were transfected into HeLa cells. After 20 hr, one set of cells with Bim(EL)EE/Beclin 1-Flag were starved in HBSS for 2 hr. Anti-Flag antibody (M2) was used for immunoprecipitation, and blots were probed as indicated. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( B ) Panel i : HeLa were treated with control siRNA or Bim siRNA . After 48 hr , one set of transfections were starved in HBSS for 2 hr . Blots were probed as indicated . LC3 - II / tubulin of siRNA - transfected is set as 1 ( n = 3 ) . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 ; NS , not significant . Panel ii : HeLa were treated with control siRNA or Bim siRNA . After 48 hr , one set of transfections were treated with Baf for 2 hr ; one set of transfections were starved treated with Baf for 2 hr . Blots were probed as indicated analyzed as in ( Bi ) . ∗ ∗ p < 0 . 01 ; NS , not significant .

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

(B) Panel i: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were starved in HBSS for 2 hr. Blots were probed as indicated. LC3-II/tubulin ratio of siRNA-transfected cells is set as 1 (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant. Panel ii: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were treated with Baf for 2 hr; one set of transfections were starved and treated with Baf for 2 hr. Blots were probed as indicated and analyzed as in (Bi). ∗∗p < 0.01; NS, not significant.


( C ) HeLa were transfected with control plasmid / GFP - LC3 ( 3 : 1 ) , or Bim ( EL ) EE / GFP - LC3 ( 3 : 1 ) . After 20 hr , one set of transfected were starved in HBSS for 2 hr then fixed . percentages of with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . NS , not significant .

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

(C) HeLa cells were transfected with control plasmid /GFP-LC3 (3:1), or Bim(EL)EE/GFP-LC3 (3:1). After 20 hr, one set of transfected cells were starved in HBSS for 2 hr and then fixed. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. NS, not significant.


( D ) BimS - Beclin 1 binding is not sensitive to HBSS starvation . BimEL - EE ( EL ) / vector ( IP negative control ) , BimEL - EE ( EL ) / Beclin 1 - Flag ( two replicates ) , BimL - EE ( L ) / Beclin 1 - Flag ( two replicates ) or BimS - EE ( S ) / Beclin 1 - Flag ( two replicates ) were transfected into HeLa . After 20 hr , one of EL - Beclin 1 - Flag transfections , one of L - Beclin - Flag transfections one of S - Beclin 1 - Flag transfections were subjected to 2 hr HBSS starvation . Anti - Flag ( M2 ) was used for immunoprecipitation .

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

(D) BimS-Beclin 1 binding is not sensitive to HBSS starvation. BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Beclin 1-Flag (two replicates), BimL-EE (L)/Beclin 1-Flag (two replicates) or BimS-EE (S)/Beclin 1-Flag (two replicates) were transfected into HeLa cells. After 20 hr, one of EL-Beclin 1-Flag transfections, one of L-Beclin-Flag transfections and one of S-Beclin 1-Flag transfections were subjected to 2 hr HBSS starvation. Anti-Flag (M2) was used for immunoprecipitation.


( E ) HBSS starvation increases Bim T116 phosphorylation . Bim ( EL ) EE or BimEE ( EL ) - T116A were transfected into HeLa . After 20 hr , one set of Bim ( EL ) EE transfections one of Bim ( EL ) EE - T116A were starved for 2 hr in HBSS . Blots were probed with indicated antibodies .

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

(E) HBSS starvation increases Bim T116 phosphorylation. Bim(EL)EE or BimEE(EL)-T116A were transfected into HeLa cells. After 20 hr, one set of Bim(EL)EE transfections and one of Bim(EL)EE-T116A were starved for 2 hr in HBSS. Blots were probed with the indicated antibodies.