f2 ( b ) Expression levels of miR - 212 and miR - 132 in heart samples of individual wild - type ( WT ) and miR - 212 / 132 transgenic ( TG ) mice as assayed by RT - PCR analysis against the mature forms of corresponding microRNAs . Rnu6b was used as housekeeping control . ( c ) Survival rate of two different miR - 212 / 132 TG mouse families ( TG - Fam23 , TG - Fam43 ) versus WT controls was analysed by Kaplan - Meier survival assay ( n = 87 , 65 and 53 for WT , TG - Fam23 and TG - Fam43 , respectively ) . ( d ) Morphology of explanted hearts from TG and WT mice at 10 weeks after birth . Scale bar , 1 cm . ( e , f ) Heart - to - body - weight ratios ( e ) and cardiomyocyte diameter ( f ) in 8 - week - old α - MHC - miR - 212 / 132 transgenic mice compared with their wild - type littermates ( n = 5 - 7 ) . Scale bar , 50 μm . ( g - i ) Echocardiography analysis of cardiac dimensions and function for α - MHC - miR - 212 / 132 TG mice and WT controls ( n = 16 - 18 ) ( g ) end - systolic area , ( h ) end - diastolic area , ( i ) fractional shortening . All values in e - i represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; WGA , wheat germ agglutinin ( membrane stain ) .

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f2(b) Expression levels of miR-212 and miR-132 in heart samples of individual wild-type (WT) and miR-212/132 transgenic (TG) mice as assayed by RT-PCR analysis against the mature forms of corresponding microRNAs. Rnu6b was used as housekeeping control.(c) Survival rate of two different miR-212/132 TG mouse families (TG-Fam23, TG-Fam43) versus WT controls was analysed by Kaplan-Meier survival assay (n=87, 65 and 53 for WT, TG-Fam23 and TG-Fam43, respectively).(d) Morphology of explanted hearts from TG and WT mice at 10 weeks after birth. Scale bar, 1 cm.(e,f) Heart-to-body-weight ratios (e) and cardiomyocyte diameter (f) in 8-week-old α-MHC-miR-212/132 transgenic mice compared with their wild-type littermates (n=5-7). Scale bar, 50 μm.(g-i) Echocardiography analysis of cardiac dimensions and function for α-MHC-miR-212/132 TG mice and WT controls (n=16-18) (g) end-systolic area, (h) end-diastolic area, (i) fractional shortening. All values in e-i represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. DAPI, 4′,6-diamidino-2-phenylindole; WGA, wheat germ agglutinin (membrane stain).


f3 ( a ) Heart - to - body - weight ratios for 12 - week - old miR - 212 / 132 null ( KO ) and wild - type ( WT ) mice ( n = 5 - 6 ) . ( b ) Cardiomyocyte cell size of neonatal miR - 212 / 132 null and WT mice ( n = 5 - 6 isolations ) . ( c - f ) Heart - to - body - weight ratios ( c ) in Sham - operated WT mice and miR - 212 / 132 null and WT mice3 weeks after TAC surgery ( n = 4 - 7 ) . Scale bar , 50 μm . ( c - f ) cardiomyocyte diameter ( d ) , cardiac fibrosis ( e ) in Sham - operated WT mice and miR - 212 / 132 null and WT mice3 weeks after TAC surgery ( n = 4 - 7 ) . Scale bar , 50 μm . ( c - f ) lung wet weight ( f ) in Sham - operated WT mice and miR - 212 / 132 null and WT mice 3 weeks after TAC surgery ( n = 4 - 7 ) . Scale bar , 50 μm . ( g - i ) Echocardiography analysis of cardiac dimensions and function in Sham - operated WT mice and miR - 212 / 132 null and WT mice 3 weeks after TAC ( n = 4 - 11 ) . ( g ) End - diastolic area , ( h ) end - systolic area and ( i ) fractional shortening . All values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 ; # P0 . 05 compared with WT TAC ; # # # P0 . 005 compared with WT TAC ; PSR , picrosirius red ( collagen stain ) . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; WGA , wheat germ agglutinin .

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

f3(a) Heart-to-body-weight ratios for 12-week-old miR-212/132 null (KO) and wild-type (WT) mice (n=5-6).(b) Cardiomyocyte cell size of neonatal miR-212/132 null and WT mice (n=5-6 isolations).(c-f) Heart-to-body-weight ratios (c) in Sham-operated WT mice and miR-212/132 null and WT mice3 weeks after TAC surgery (n=4-7). Scale bar, 50 μm.(c-f) cardiomyocyte diameter (d), cardiac fibrosis (e) in Sham-operated WT mice and miR-212/132 null and WT mice3 weeks after TAC surgery (n=4-7). Scale bar, 50 μm.(c-f) lung wet weight (f) in Sham-operated WT mice and miR-212/132 null and WT mice 3 weeks after TAC surgery (n=4-7). Scale bar, 50 μm.(g-i) Echocardiography analysis of cardiac dimensions and function in Sham-operated WT mice and miR-212/132 null and WT mice 3 weeks after TAC (n=4-11). (g) End-diastolic area, (h) end-systolic area and (i) fractional shortening. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 compared with WT TAC; ###P0.005 compared with WT TAC; PSR, picrosirius red (collagen stain). DAPI, 4′,6-diamidino-2-phenylindole; WGA, wheat germ agglutinin.


f4 ( a ) Luciferase activity levels upon cotransfection of a luciferase construct containing wild - type ( WT ) or mutated ( Mut . ) 3 ′ UTR of FoxO3 with scrambled ( Scr ) control ( Ctrl ) , pre - miR - 132 or pre - miR - 212 ( n = 9 ) . ( b , c ) Expression levels of FoxO3 on mRNA ( b ) and protein levels ( c ) in hearts of WT and α - MHC - miR - 212 / - 132 transgenic ( TG ) mice . M : Size marker ( n = 9 - 13 ) . ( d ) FoxO3 mRNA levels in neonatal rat cardiomyocytes transfected with Scr Ctrl , anti - miR - 212 and anti - miR - 132 after phenylephrine ( PE , 10 μM ) treatment ( n = 6 - 9 ; P - values against Scr + PE ) . ( e - g ) Expression levels of atrogin - 1 ( e ) and Mcip1 ( f ) and calcineurin phosphatase activity levels ( g ) in hearts of WT and α - MHC - miR - 212 / 132 TG mice ( n = 5 - 9 ) . ( h ) Luciferase activity levels showing the NFAT transcriptional activity in cardiomyocytes transfected with Scr Ctrl , pre - miR - 132 or pre - miR - 212 ( n = 5 ) . ( i , j ) Mcip1 . 4 ( i ) and atrogin - 1 ( j ) mRNA levels in WT and miR - 212 / 132 null ( KO ) mice 3 weeks after transaortic constriction ( TAC ) or Sham operation ( n = 5 - 7 per group ) . All values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 . # P0 . 05 compared with WT TAC . FC , fold change .

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

f4(a) Luciferase activity levels upon cotransfection of a luciferase construct containing wild-type (WT) or mutated (Mut.) 3′UTR of FoxO3 with scrambled (Scr) control (Ctrl), pre-miR-132 or pre-miR-212 (n=9).(b,c) Expression levels of FoxO3 on mRNA (b) and protein levels (c) in hearts of WT and α-MHC-miR-212/-132 transgenic (TG) mice. M: Size marker (n=9-13).(d) FoxO3 mRNA levels in neonatal rat cardiomyocytes transfected with Scr Ctrl, anti-miR-212 and anti-miR-132 after phenylephrine (PE, 10 μM) treatment (n=6-9; P-values against Scr+PE).(e-g) Expression levels of atrogin-1 (e) and Mcip1 (f) and calcineurin phosphatase activity levels (g) in hearts of WT and α-MHC-miR-212/132 TG mice (n=5-9).(h) Luciferase activity levels showing the NFAT transcriptional activity in cardiomyocytes transfected with Scr Ctrl, pre-miR-132 or pre-miR-212 (n=5).(i,j) Mcip1.4 (i) and atrogin-1 (j) mRNA levels in WT and miR-212/132 null (KO) mice 3 weeks after transaortic constriction (TAC) or Sham operation (n=5-7 per group). All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. #P0.05 compared with WT TAC. FC, fold change.


f5 ( a ) mRNA expression levels of autophagic marker genes in hearts of wild - type ( WT ) and α - MHC - miR - 212 / 132 transgenic ( TG ) mice ( n = 9 - 10 ) . ( b , c ) Ratio of LC3II to LC3I ( b ) and p62 protein levels ( c ) in WT , miR - 212 / 132 null ( KO ) and α - MHC miR - 212 / 132 TG mice . M : Size marker ( n = 4 - 12 ) . ( d , e ) Representative images ( d ) and quantification ( e ) of LC3 : mCherry puncta ( in red ) in control and miR - 212 / 132 - overexpressing TG H9c2 cells under normal and serum / glucose - deprivation conditions ( n = 30 ) . Nuclei are stained in blue with Hoechst33342 . ( f , g ) Representative electron microscopy images ( f ) and quantification ( g ) of autophagic vacuoles in control and miR - 212 / 132 - overexpressing TG H9c2 cells under normal and serum / glucose - deprivation conditions ( n = 20 ) . ( h , i ) Representative FACS plots ( h ) and quantification ( i ) of percent increase of autophagic flux in control and miR - 212 / 132 - overexpressing TG H9c2 cells under normal and serum / glucose - deprivation conditions ( n = 3 - 4 experiments ) . All values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 . Scale bars , 50 μm in d and 2 μm in f . FC , fold change .

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

f5(a) mRNA expression levels of autophagic marker genes in hearts of wild-type (WT) and α-MHC-miR-212/132 transgenic (TG) mice (n=9-10).(b,c) Ratio of LC3II to LC3I (b) and p62 protein levels (c) in WT, miR-212/132 null (KO) and α-MHC miR-212/132 TG mice. M: Size marker (n=4-12).(d,e) Representative images (d) and quantification (e) of LC3:mCherry puncta (in red) in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=30). Nuclei are stained in blue with Hoechst33342.(f,g) Representative electron microscopy images (f) and quantification (g) of autophagic vacuoles in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=20).(h,i) Representative FACS plots (h) and quantification (i) of percent increase of autophagic flux in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=3-4 experiments). All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. Scale bars, 50 μm in d and 2 μm in f. FC, fold change.


f6 ( a ) Schematic representation of starvation experiment in mice . The white and grey parts on the time line represent the day and night phases , respectively . The arrow shows the start and duration of the starvation and the indicated time points of starvation are when animals are scored for body conditioning index . ( b ) LC3II to LC3I ratios in wild - type ( WT ) mice fed with normal diet and WT , miR - 212 / 132 − / − null and α - MHC miR - 212 / 132 transgenic ( TG ) mice under starvation for 31 h ( n = 4 ) . ( c ) Electron microscopy from ultrathin sections of resin - embedded heart biopsies of fed and starved WT , miR - 212 / 132 null ( KO ) and cardiomyocyte - specific miR - 212 / 132 - overexpressing ( TG ) mice . White spots around the mitochondria ( dark grey structures ) are autophagic vacuoles . The electron - dense black spots shown with white arrows are autophagosomes . Scale bars , 4 μm . ( d ) p - mTOR / mTOR ratios in WT and miR - 212 / 132 TG H9c2 cells 24 h after normal and starvation ( serum / glucose - deprived ) conditions ( n = 6 ) . ( e ) p - mTOR / mTOR ratios in hearts of WT and α - MHC miR - 212 / 132 TG mice fed with normal diet or 31 h after starvation ( n = 4 ) . M , size marker . All values represent mean ± s . e . m . * * P0 . 01 ; * * * P0 . 005 ; # P = 0 . 11 .

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

f6(a) Schematic representation of starvation experiment in mice. The white and grey parts on the time line represent the day and night phases, respectively. The arrow shows the start and duration of the starvation and the indicated time points of starvation are when animals are scored for body conditioning index.(b) LC3II to LC3I ratios in wild-type (WT) mice fed with normal diet and WT, miR-212/132−/− null and α-MHC miR-212/132 transgenic (TG) mice under starvation for 31 h (n=4).(c) Electron microscopy from ultrathin sections of resin-embedded heart biopsies of fed and starved WT, miR-212/132 null (KO) and cardiomyocyte-specific miR-212/132-overexpressing (TG) mice. White spots around the mitochondria (dark grey structures) are autophagic vacuoles. The electron-dense black spots shown with white arrows are autophagosomes. Scale bars, 4 μm.(d) p-mTOR/mTOR ratios in WT and miR-212/132 TG H9c2 cells 24 h after normal and starvation (serum/glucose-deprived) conditions (n=6).(e) p-mTOR/mTOR ratios in hearts of WT and α-MHC miR-212/132 TG mice fed with normal diet or 31 h after starvation (n=4). M, size marker. All values represent mean±s.e.m. **P0.01; ***P0.005; #P=0.11.


f7 ( a - c ) Heart - to - body - weight ratios ( a ) , cardiomyocyte diameters ( b ) and cardiac fibrosis ( c ) in Sham - operated mice and mice treated with intravenous injection of either scrambled control ( Scr ) or miR - 132 inhibitors ( Ant - 132 ) after TAC . These mice were analysed 3 weeks after TAC ( n = 4 - 11 ) . PSR , picrosirius red staining . ( d - f ) Echocardiography analysis of cardiac dimensions and function in Sham - operated mice and mice treated with intravenous injection of either control ( Scr ) or miR - 132 inhibitors ( Ant - 132 ) after TAC . These mice were analysed 3 weeks after TAC for ( d ) fractional shortening , ( e ) end - diastolic area and ( f ) end - systolic area ( n = 4 - 9 ) . ( g - i ) Cardiac FoxO3 protein levels in mice treated with intravenous injection of either control ( Scr ) or miR - 132 inhibitors ( Ant - 132 ) 3 weeks after TAC and treatment ( n = 4 - 8 ) . M : size marker . All values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 ; # P0 . 05 against TAC - control ; # # P0 . 01 against TAC - control ; # # # P0 . 005 against TAC - control . Scale bars , 50 μm . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; FC , fold change ; WGA , wheat germ agglutinin . ( g - i ) calcineurin activity ( h ) and Mcip1 . 4 mRNA levels ( i ) in mice treated with intravenous injection of either control ( Scr ) or miR - 132 inhibitors ( Ant - 132 ) 3 weeks after TAC and treatment ( n = 4 - 8 ) . M : size marker . All values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 ; # P0 . 05 against TAC - control ; # # P0 . 01 against TAC - control ; # # # P0 . 005 against TAC - control . Scale bars , 50 μm . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; FC , fold change ; WGA , wheat germ agglutinin .

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

f7(a-c) Heart-to-body-weight ratios (a), cardiomyocyte diameters (b) and cardiac fibrosis (c) in Sham-operated mice and mice treated with intravenous injection of either scrambled control (Scr) or miR-132 inhibitors (Ant-132) after TAC. These mice were analysed 3 weeks after TAC (n=4-11). PSR, picrosirius red staining.(d-f) Echocardiography analysis of cardiac dimensions and function in Sham-operated mice and mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) after TAC. These mice were analysed 3 weeks after TAC for (d) fractional shortening, (e) end-diastolic area and (f) end-systolic area (n=4-9).(g-i) Cardiac FoxO3 protein levels in mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) 3 weeks after TAC and treatment (n=4-8). M: size marker. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 against TAC-control; ##P0.01 against TAC-control; ###P0.005 against TAC-control. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; WGA, wheat germ agglutinin.(g-i) calcineurin activity (h) and Mcip1.4 mRNA levels (i) in mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) 3 weeks after TAC and treatment (n=4-8). M: size marker. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 against TAC-control; ##P0.01 against TAC-control; ###P0.005 against TAC-control. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; WGA, wheat germ agglutinin.


Figure 1A LOUCY cells were treated in vitro with the indicated SMs with or without vincristine ( VCR ) for 48h . Apoptosis was determined by flow cytometry upon staining with Annexin - V and Propidium Iodide ( PI ) . Mean ± SD of 3 independent experiments is shown ; * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 by unpaired Student ' s t - test . B Fresh primary leukemic cells from two pediatric patients , one with common B - ALL at relapse ( left ) and one with T - ALL at diagnosis ( right ) , both with poor response to second line chemotherapy , were treated with LBW242 and VCR at the indicated concentrations . Apoptosis was measured after 24 h as described in A . The SM - drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h . Cells were stained with respective fluorescent substrates for caspase - 8 ( C ) and - 9 ( D ) , and were subsequently analyzed by flow cytometry . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 by unpaired Student ' s t - test . The SM - drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h . Cells were stained with an antibody detecting the active caspase - 3 subunit ( E ) , and were subsequently analyzed by flow cytometry . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 by unpaired Student ' s t - test . The SM - drug combination induces PARP cleavage . LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h . Cleavage of PARP was measured by Western blot using Actin as loading control ( F ) . One representative immunoblot out 3 independent experiments is shown .

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Figure 1A LOUCY cells were treated in vitro with the indicated SMs with or without vincristine (VCR) for 48h. Apoptosis was determined by flow cytometry upon staining with Annexin-V and Propidium Iodide (PI). Mean ± SD of 3 independent experiments is shown; ** p <0.01, *** p <0.001 and **** p <0.0001 by unpaired Student's t-test.B Fresh primary leukemic cells from two pediatric patients, one with common B-ALL at relapse (left) and one with T-ALL at diagnosis (right), both with poor response to second line chemotherapy, were treated with LBW242 and VCR at the indicated concentrations. Apoptosis was measured after 24 h as described in A.The SM-drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cells were stained with respective fluorescent substrates for caspase-8 (C) and -9 (D) , and were subsequently analyzed by flow cytometry. Mean ± SD of 3 independent experiments is shown; * p <0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired Student's t-test.The SM-drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cells were stained with an antibody detecting the active caspase-3 subunit (E), and were subsequently analyzed by flow cytometry. Mean ± SD of 3 independent experiments is shown; * p <0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired Student's t-test.The SM-drug combination induces PARP cleavage. LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cleavage of PARP was measured by Western blot using Actin as loading control (F). One representative immunoblot out 3 independent experiments is shown.


Figure 2A Necrostatin - 1 ( Nec ) does not affect LBW - mediated sensitization to VCR - induced apoptosis . Apoptosis induction in LOUCY cells was determined by flow cytometry 48h after treatment with SM and / or VCR ( left ) and / or TNFα ( right ) in the presence or absence of the RIPK1 - inhibitor Nec . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 and * * p < 0 . 01 by unpaired Student ' s t - test . B RIPK1 is dispensable for apoptosis induction by LBW242 and VCR . LOUCY cells were transfected with a non - targeting siRNA ( neg - siRNA ) or a RIPK1 - specific siRNA . 6h post transfection , cells were treated with the indicated drugs and apoptosis was measured 40h thereafter . Mean ± SD of 3 independent experiments is shown ; * * * p < 0 . 001 by unpaired Student ' s t - test . Knockdown ( KD ) was verified by Western blot at the time of apoptosis measurement and KD efficiency calculated compared to the control . One representative immunoblot out of 3 biological replicates is shown . C The SM - drug combination does not activate the canonical NF - κB pathway . Phosphorylation and degradation of IκBα upon treatment with VCR or TNFα and / or LBW242 was determined in LOUCY cells by Western blot following treatment with the indicated drugs for 5 , 15 or 60 minutes using Actin as loading control . One representative immunoblot out of 3 independent biological replicates is shown . D KD of NEMO does not affect the SM - mediated sensitization to chemotherapy - induced apoptosis . Experiment was performed as described in B , except that an siRNA targeting NEMO was used . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 by unpaired Student ' s t - test . E The SM - drug combination activates the non - canonical NF - κB pathway . Cleavage of p100 NF - κB2 to the p52 fragment was determined by Western blot 24h after treatment of LOUCY cells with chemotherapy or TNFα in combination with SM . Asterisk indicates an unspecific signal . One representative immunoblot out of 3 biological replicates is shown . F KD of RelB does not inhibit apoptosis . Experiment was performed as described in B , except that an siRNA targeting RelB was used . Mean ± SD of 3 independent experiments is shown ; differences are not significant , by unpaired Student ' s t - test .

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

Figure 2A Necrostatin-1 (Nec) does not affect LBW-mediated sensitization to VCR-induced apoptosis. Apoptosis induction in LOUCY cells was determined by flow cytometry 48h after treatment with SM and/or VCR (left) and/or TNFα (right) in the presence or absence of the RIPK1-inhibitor Nec. Mean ± SD of 3 independent experiments is shown; * p <0.05 and ** p<0.01 by unpaired Student's t-test.B RIPK1 is dispensable for apoptosis induction by LBW242 and VCR. LOUCY cells were transfected with a non-targeting siRNA (neg-siRNA) or a RIPK1-specific siRNA. 6h post transfection, cells were treated with the indicated drugs and apoptosis was measured 40h thereafter. Mean ± SD of 3 independent experiments is shown; *** p<0.001 by unpaired Student's t-test. Knockdown (KD) was verified by Western blot at the time of apoptosis measurement and KD efficiency calculated compared to the control. One representative immunoblot out of 3 biological replicates is shown.C The SM-drug combination does not activate the canonical NF-κB pathway. Phosphorylation and degradation of IκBα upon treatment with VCR or TNFα and/or LBW242 was determined in LOUCY cells by Western blot following treatment with the indicated drugs for 5, 15 or 60 minutes using Actin as loading control. One representative immunoblot out of 3 independent biological replicates is shown.D KD of NEMO does not affect the SM-mediated sensitization to chemotherapy-induced apoptosis. Experiment was performed as described in B, except that an siRNA targeting NEMO was used. Mean ± SD of 3 independent experiments is shown; * p<0.05 by unpaired Student's t-test.E The SM-drug combination activates the non-canonical NF-κB pathway. Cleavage of p100 NF-κB2 to the p52 fragment was determined by Western blot 24h after treatment of LOUCY cells with chemotherapy or TNFα in combination with SM. Asterisk indicates an unspecific signal. One representative immunoblot out of 3 biological replicates is shown.F KD of RelB does not inhibit apoptosis. Experiment was performed as described in B, except that an siRNA targeting RelB was used. Mean ± SD of 3 independent experiments is shown; differences are not significant, by unpaired Student's t-test.


Figure 3A Expression of XIAP mRNA in samples obtained from BM of healthy individuals ( Ctrl , n = 10 ) or patients with AML ( n = 517 ) or ALL ( n = 306 ) . Data are expressed as median , 25th and 75th percentile , whiskers indicate min / max ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by Games - Howell post - hoc test . B Protein levels of XIAP normalized to loading control in PDX ALL samples from pediatric and adult patients , analyzed by protein immunoassay ( Simple Western ) and depicted as mean ( n = 2 , biological replicates ) ; PBMC = peripheral blood mononuclear cellsParental and XIAP over - expressing NALM - 6 cells ( C ) were treated with VCR apoptosis ( Annexin - V / PI ) was measured at the indicated time points ( C ) by flow cytometry . Mean ± SEM of 3 independent experiments is shown ; * * * * p ≤ 0 . 0001 by unpaired Student ' s t - test . XIAP protein level was analyzed in parental and XIAP - overexpressing NALM - 6 cells by protein immunoassay ( Simple Western ) ; Actin was used as loading control . One representative immunoassay out of 3 independent experiments is shown . D Parental and XIAP over - expressing REH cells ( D ) were treated with VCR or LBW242 ; apoptosis ( Annexin - V / PI ) was measured after 72h ( D ) by flow cytometry . Mean ± SEM of 3 independent experiments is shown ; * * * * p ≤ 0 . 0001 by unpaired Student ' s t - test . XIAP protein level was analyzed by protein immunoassay ( Simple Western ) ; Actin was used as loading control . One representative immunoassay out of 3 independent experiments is shown .

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Figure 3A Expression of XIAP mRNA in samples obtained from BM of healthy individuals (Ctrl, n=10) or patients with AML (n=517) or ALL (n=306). Data are expressed as median, 25th and 75th percentile, whiskers indicate min/max; ** p<0.01; **** p<0.0001 by Games-Howell post-hoc test.B Protein levels of XIAP normalized to loading control in PDX ALL samples from pediatric and adult patients, analyzed by protein immunoassay (Simple Western) and depicted as mean (n=2, biological replicates); PBMC = peripheral blood mononuclear cellsParental and XIAP over-expressing NALM-6 cells (C) were treated with VCR apoptosis (Annexin-V / PI) was measured at the indicated time points (C) by flow cytometry. Mean ± SEM of 3 independent experiments is shown; **** p ≤0.0001 by unpaired Student's t-test. XIAP protein level was analyzed in parental and XIAP-overexpressing NALM-6 cells by protein immunoassay (Simple Western); Actin was used as loading control. One representative immunoassay out of 3 independent experiments is shown.D Parental and XIAP over-expressing REH cells (D) were treated with VCR or LBW242; apoptosis (Annexin-V / PI) was measured after 72h (D) by flow cytometry. Mean ± SEM of 3 independent experiments is shown; **** p ≤0.0001 by unpaired Student's t-test. XIAP protein level was analyzed by protein immunoassay (Simple Western); Actin was used as loading control. One representative immunoassay out of 3 independent experiments is shown.


Figure 4B shRNA expression control . Equal expression levels of ds - RED in the sh - CTRL / mTagBFP and sh - XIAP / eGFP subpopulation indicates identical expression levels of control and XIAP shRNA . One representative histogram out of 3 independent experiments is shown . C Expression of IAP . Western blot analysis of XIAP in NALM - 6 cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP under the SFFV promoter ; GAPDH was used as loading control . One representative immunoblot out of 3 independent experiments is shown . D Western blot analysis of NALM - 6 cells with moderate ( 69 % , EF1α promoter ) or strong ( 92 % , SFFV promoter ) KD of XIAP ; GAPDH was used as loading control . The XIAP KD levels ( % KD ) were quantified relative to sh - CTRL . One representative immunoblot out of 3 independent experiments is shown . F Moderate knockdown of XIAP sensitizes towards chemotherapy . NALM - 6 cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP were mixed at a 1 : 1 ratio and injected into groups of mice . 4 days after injection , mice were either treated with PBS ( n = 4 ) , or with Vincristin ( VCR , n = 8 ) , 0 . 3mg / kg i . v . once a week for 3 consecutive weeks . 7 days after last treatment , mice were sacrificed , and relative proportion of sh - CTRL and sh - XIAP subpopulations re - isolated from the BM was quantified by flow cytometry . Data are depicted as mean ± SEM of all mice analyzed with * * * * p ≤ 0 . 0001 by unpaired Student ' s t - test with Welch ' s correction . G Strong knockdown of XIAP impairs NALM - 6 growth in vivo . NALM - 6 cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP were mixed at a 1 : 1 ratio and injected into groups of mice ( n = 8 ) . 38 or 46 days after injection , mice were sacrificed , and relative proportion of sh - CTRL and sh - XIAP subpopulations re - isolated from the BM was quantified by flow cytometry . Data are depicted as mean ± SEM of all mice analyzed with * * * * p ≤ 0 . 0001 by unpaired Student ' s t - test . H PARP cleavage in NALM - 6 cells with strong XIAP inhibition . NALM - 6 cells were lentivirally transduces with sh - CTRL or sh - XIAP ( SFFV ) . 4 days later , cells were lysed and relative cleavage level of PARP ( % ) was analyzed by protein immunoassay ( Simple Western ) . Mean ± SD of 3 independent experiments is shown ; * * p < 0 . 01 by unpaired Student ' s t - test . I XIAP reconstitution in NALM - 6 cells . NALM - 6 cells were transduced with shCTRL , shXIAP . 1 alone or shXIAP . 1 together with the XIAP overexpression vector . At day 0 , transduced cells were mixed in a 1 : 1 ratio with parental ( untransduced ) NALM - 6 cells . Distribution of the fluorochrome marker was used as a readout to determine growth variations of the respective subpopulation over time . Mean ± SD of 3 independent experiments is shown ; * * p < 0 . 01 by unpaired Student ' s t - test .

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

Figure 4B shRNA expression control. Equal expression levels of ds-RED in the sh-CTRL/mTagBFP and sh-XIAP/eGFP subpopulation indicates identical expression levels of control and XIAP shRNA. One representative histogram out of 3 independent experiments is shown.C Expression of IAP. Western blot analysis of XIAP in NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP under the SFFV promoter; GAPDH was used as loading control. One representative immunoblot out of 3 independent experiments is shown.D Western blot analysis of NALM-6 cells with moderate (69%, EF1α promoter) or strong (92%, SFFV promoter) KD of XIAP; GAPDH was used as loading control. The XIAP KD levels (% KD) were quantified relative to sh-CTRL. One representative immunoblot out of 3 independent experiments is shown.F Moderate knockdown of XIAP sensitizes towards chemotherapy. NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice. 4 days after injection, mice were either treated with PBS (n=4), or with Vincristin (VCR, n=8), 0.3mg/kg i.v. once a week for 3 consecutive weeks. 7 days after last treatment, mice were sacrificed, and relative proportion of sh-CTRL and sh-XIAP subpopulations re-isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** p ≤0.0001 by unpaired Student's t-test with Welch's correction.G Strong knockdown of XIAP impairs NALM-6 growth in vivo. NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice (n=8). 38 or 46 days after injection, mice were sacrificed, and relative proportion of sh-CTRL and sh-XIAP subpopulations re-isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** p ≤0.0001 by unpaired Student's t-test.H PARP cleavage in NALM-6 cells with strong XIAP inhibition. NALM-6 cells were lentivirally transduces with sh-CTRL or sh-XIAP (SFFV). 4 days later, cells were lysed and relative cleavage level of PARP (%) was analyzed by protein immunoassay (Simple Western). Mean ± SD of 3 independent experiments is shown; ** p <0.01 by unpaired Student's t-test.I XIAP reconstitution in NALM-6 cells. NALM-6 cells were transduced with shCTRL, shXIAP.1 alone or shXIAP.1 together with the XIAP overexpression vector. At day 0, transduced cells were mixed in a 1:1 ratio with parental (untransduced) NALM-6 cells. Distribution of the fluorochrome marker was used as a readout to determine growth variations of the respective subpopulation over time. Mean ± SD of 3 independent experiments is shown; ** p <0.01 by unpaired Student's t-test.


Figure 5XIAP plays an essential role for ALL - 265 growing in mice . Experimental scheme and stability of XIAP knockdown over passaging . ALL - 265 PDX cells produced as described in A were mixed at a 1 : 1 ratio and injected into mice ( P1 , n = 4 ) . At clinical signs of illness , cells were isolated and re - injected into next recipient mice ( P2 , n = 4 ) . XIAP expression levels of P1 and P2 PDX cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP were analyzed by Western blot at the end of each passage ; GAPDH was used as loading control ( B ) . XIAP plays an essential role for ALL - 265 growing in mice . ALL - 265 PDX cells produced as described in A were mixed at a 1 : 1 ratio and injected into mice ( P1 , n = 4 ) . At clinical signs of illness , cells were isolated and re - injected into next recipient mice ( P2 , n = 4 ) . Relative proportion of ALL - 265 GEPDX cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP re - isolated from the BM at the end of each passage was quantified by flow cytometry ( C ) . Data are depicted as mean ± SEM of all mice analyzed ( n = 4 , each passage ) with * * * * p < 0 . 0001 by unpaired Student ' s t - test . D Dose - response relationship between strength of XIAP knockdown and growth disadvantage ; ALL - 265 PDX cells of a representative mouse were analyzed separately for dsRED / shRNA high and low expressing cells .

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Figure 5XIAP plays an essential role for ALL-265 growing in mice. Experimental scheme and stability of XIAP knockdown over passaging. ALL-265 PDX cells produced as described in A were mixed at a 1:1 ratio and injected into mice (P1, n=4). At clinical signs of illness, cells were isolated and re-injected into next recipient mice (P2, n=4). XIAP expression levels of P1 and P2 PDX cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were analyzed by Western blot at the end of each passage; GAPDH was used as loading control (B).XIAP plays an essential role for ALL-265 growing in mice. ALL-265 PDX cells produced as described in A were mixed at a 1:1 ratio and injected into mice (P1, n=4). At clinical signs of illness, cells were isolated and re-injected into next recipient mice (P2, n=4). Relative proportion of ALL-265 GEPDX cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP re-isolated from the BM at the end of each passage was quantified by flow cytometry (C). Data are depicted as mean ± SEM of all mice analyzed (n=4, each passage) with **** p<0.0001 by unpaired Student's t-test.D Dose-response relationship between strength of XIAP knockdown and growth disadvantage; ALL-265 PDX cells of a representative mouse were analyzed separately for dsRED/shRNA high and low expressing cells.


figf1 ( b ) Coimmuno - precipitation ( IP ) of Fbxo7 - HA and Flag - Parkin in whole - cell lysates from U2OS cells over - expressing both proteins . IB , immunoblot . Molecular mass markers are in kilodaltons . ( c ) Coimmunoprecipitation of endogenous Fbxo7 with Flag - Parkin in HEK293T cells transfected with Flag - Parkin or a control protein ( EGFP ) . ( d ) Flag immunoprecipitation was repeated in U2OS cells transfected with Flag - Parkin and either full - length ( 1 - 522 ) T7 - Fbxo7 or an N - terminal truncation lacking the Ubl domain ( 89 - 522 ) . ( e ) As in d , using lysates from U2OS cells expressing Flag - Parkin and T7 - Fbxo7 , either full length ( 1 - 522 ) or a C - terminal deletion of the proline - rich region ( 1 - 398 ) . * indicates IgG heavy chain . ( f ) In vitro - translated Flag - Parkin was incubated with bacterially expressed GST or GST fused to the Fbxo7 Ubl domain ( 1 - 88 ) immobilized on glutathione beads . Bead - bound proteins and inputs were analyzed by immunoblotting with anti - Parkin antibodies . ( g ) The disease - causing mutation T22M interferes with Fbxo7 ' s interaction with Parkin . Coimmunoprecipitation was performed as in b using lysates from U2OS cells expressing Flag - Parkin and WT or T22M Fbxo7 - HA . ( h ) Coimmunoprecipitation of Flag - Parkin and Fbxo7 in the mitochondrial and cytosolic fractions of HEK293T cells overexpressing both proteins . CxVα and GAPDH are markers for mitochondria and cytoplasm , respectively . All western blots are representative of experiments performed at least three times . Full - length blots are presented in Supplementary Figure 9 .

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figf1(b) Coimmuno-precipitation (IP) of Fbxo7-HA and Flag-Parkin in whole-cell lysates from U2OS cells over-expressing both proteins. IB, immunoblot. Molecular mass markers are in kilodaltons.(c) Coimmunoprecipitation of endogenous Fbxo7 with Flag-Parkin in HEK293T cells transfected with Flag-Parkin or a control protein (EGFP).(d) Flag immunoprecipitation was repeated in U2OS cells transfected with Flag-Parkin and either full-length (1-522) T7-Fbxo7 or an N-terminal truncation lacking the Ubl domain (89-522).(e) As in d, using lysates from U2OS cells expressing Flag-Parkin and T7-Fbxo7, either full length (1-522) or a C-terminal deletion of the proline-rich region (1-398). * indicates IgG heavy chain.(f) In vitro-translated Flag-Parkin was incubated with bacterially expressed GST or GST fused to the Fbxo7 Ubl domain (1-88) immobilized on glutathione beads. Bead-bound proteins and inputs were analyzed by immunoblotting with anti-Parkin antibodies.(g) The disease-causing mutation T22M interferes with Fbxo7's interaction with Parkin. Coimmunoprecipitation was performed as in b using lysates from U2OS cells expressing Flag-Parkin and WT or T22M Fbxo7-HA.(h) Coimmunoprecipitation of Flag-Parkin and Fbxo7 in the mitochondrial and cytosolic fractions of HEK293T cells overexpressing both proteins. CxVα and GAPDH are markers for mitochondria and cytoplasm, respectively. All western blots are representative of experiments performed at least three times. Full-length blots are presented in Supplementary Figure 9.


figf2 ( a ) Fbxo7 relocates from the cytosolic to the mitochondrial fractions of HEK293T cells treated with CCCP ( 10 μM ) . IB , immunoblot . ( b ) Fbxo7 levels are increased in Flag - Parkin complexes immunoprecipitated ( IP ) from the mitochondrial fraction of HEK293T cells transfected with Flag - Parkin and untagged Fbxo7 following 1 or 3 h treatment with CCCP ( 10 µM ) . ( c ) Parkin localization at the mitochondria was assessed by immunocytochemistry in SH - SY5Y cells transfected with Flag - Parkin plus scrambled ( scr ) or Fbxo7 siRNA , following 1 or 3 h treatment with CCCP ( 10 μM ) . Cells were scored visually for the colocalization of Flag - Parkin with HtrA2 , a mitochondrial marker . Images are displayed for cells transfected as indicated , following 0 or 3 h CCCP treatment . For corresponding images at 1 h treatment , see Supplementary Figure 2c . Nuclei ( blue ) were stained with DAPI . Scale bars , 10 μm . ( d ) Loss of Flag - Parkin translocation upon Fbxo7 silencing is rescued by WT and R378G Fbxo7 , but not by T22M Fbxo7 , by R498X Fbxo7 or by Fbxo7 in which the mitochondrial targeting sequence is mutated ( mt - MTS ) . For c , d , histograms indicate the percentage of cells in which Parkin localized to the mitochondria . Data are presented as mean of three experiments ± s . e . m . * P 0 . 05 , * * P 0 . 01 compared to cells transfected with Flag - Parkin plus Fbxo7 siRNA . ( e ) T7 - tagged WT Fbxo7 localizes to both cytosolic ( C ) and mitochondrial ( M ) fractions of transfected HEK293T cells , but MTS mutant ( mt - MTS ) Fbxo7 localizes only to the cytosolic fraction . CxVα and PDHE1α are mitochondrial markers ; GAPDH is a cytosolic marker . All western blots are representative of experiments performed at least three times . Full - length blots are presented in Supplementary Figure 9 .

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figf2(a) Fbxo7 relocates from the cytosolic to the mitochondrial fractions of HEK293T cells treated with CCCP (10 μM). IB, immunoblot.(b) Fbxo7 levels are increased in Flag-Parkin complexes immunoprecipitated (IP) from the mitochondrial fraction of HEK293T cells transfected with Flag-Parkin and untagged Fbxo7 following 1 or 3 h treatment with CCCP (10 µM).(c) Parkin localization at the mitochondria was assessed by immunocytochemistry in SH-SY5Y cells transfected with Flag-Parkin plus scrambled (scr) or Fbxo7 siRNA, following 1 or 3 h treatment with CCCP (10 μM). Cells were scored visually for the colocalization of Flag-Parkin with HtrA2, a mitochondrial marker. Images are displayed for cells transfected as indicated, following 0 or 3 h CCCP treatment. For corresponding images at 1 h treatment, see Supplementary Figure 2c. Nuclei (blue) were stained with DAPI. Scale bars, 10 μm.(d) Loss of Flag-Parkin translocation upon Fbxo7 silencing is rescued by WT and R378G Fbxo7, but not by T22M Fbxo7, by R498X Fbxo7 or by Fbxo7 in which the mitochondrial targeting sequence is mutated (mt-MTS). For c,d, histograms indicate the percentage of cells in which Parkin localized to the mitochondria. Data are presented as mean of three experiments ± s.e.m. *P 0.05, **P 0.01 compared to cells transfected with Flag-Parkin plus Fbxo7 siRNA.(e) T7-tagged WT Fbxo7 localizes to both cytosolic (C) and mitochondrial (M) fractions of transfected HEK293T cells, but MTS mutant (mt-MTS) Fbxo7 localizes only to the cytosolic fraction. CxVα and PDHE1α are mitochondrial markers; GAPDH is a cytosolic marker. All western blots are representative of experiments performed at least three times. Full-length blots are presented in Supplementary Figure 9.


figf3 ( a , b ) Overexpression of Fbxo7 by da - GAL4 ( da > Fbxo7 ) suppresses climbing ( a ) and flight ( b ) defects of parkin mutants . ( c ) Overexpression of Fbxo7 also suppresses dopaminergic ( DA ) neurodegeneration parkin mutants . ( d - f ) Top and middle panels , toluidine blue - stained sections of adult thorax . Bottom panels , transmission electron micrographs of muscle . Fbxo7 overexpression suppresses muscle degeneration and mitochondrial disruption in parkin mutants . Scale bars : 200 μm ( top ) , 20 μm ( middle ) , 2 μm ( bottom ) . Images are representative of three animals per genotype . ( g , h ) Overexpression of Fbxo7 pathogenic mutants , mt - MTS or isoform 2 by da - GAL4 fails to rescue climbing ( g ) and flight ( h ) deficits in parkin mutants . Control genotype is park25 / + ; da - GAL4 / + . All transgenic lines are site - directed integration except WT ( 4 ) , which is random integration line 4 ( see Supplementary Fig . 3 and Online Methods ) . Histograms indicate mean ± s . e . m . One - way ANOVA with Bonferroni correction ( * * * P 0 . 001 , * * P 0 . 01 ) . For climbing and flight assays , at least 50 flies were assessed ; number of flies is shown for each bar .

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figf3(a,b) Overexpression of Fbxo7 by da-GAL4 (da>Fbxo7) suppresses climbing (a) and flight (b) defects of parkin mutants.(c) Overexpression of Fbxo7 also suppresses dopaminergic (DA) neurodegeneration parkin mutants.(d-f) Top and middle panels, toluidine blue-stained sections of adult thorax. Bottom panels, transmission electron micrographs of muscle. Fbxo7 overexpression suppresses muscle degeneration and mitochondrial disruption in parkin mutants. Scale bars: 200 μm (top), 20 μm (middle), 2 μm (bottom). Images are representative of three animals per genotype.(g,h) Overexpression of Fbxo7 pathogenic mutants, mt-MTS or isoform 2 by da-GAL4 fails to rescue climbing (g) and flight (h) deficits in parkin mutants. Control genotype is park25/+; da-GAL4/+. All transgenic lines are site-directed integration except WT(4), which is random integration line 4 (see Supplementary Fig. 3 and Online Methods). Histograms indicate mean ± s.e.m. One-way ANOVA with Bonferroni correction (***P 0.001, **P 0.01). For climbing and flight assays, at least 50 flies were assessed; number of flies is shown for each bar.


figf4 ( a ) Coimmunoprecipitation ( IP ) of PINK1 - Myc and Flag - Fbxo7 in whole - cell lysates from U2OS cells overexpressing both proteins . IB , immunoblot . ( b ) Coimmunoprecipitation of PINK1 - Myc with full - length and two N - terminally deleted Flag - Fbxo7 forms . PINK1 - Myc is detected at low levels in complex with Flag - Fbxo7 ( 89 - 522 ) but not with Flag - Fbxo7 ( 129 - 522 ) . ( c ) As in b , using lysates from U2OS cells expressing PINK1 - Myc , and Flag - Fbxo7 containing either N - or C - terminal truncations . ( d ) In vitro coprecipitation experiments were performed using in vitro - translated ( IVT ) PINK1 - Myc and either GST or GST fusions of Fbxo7 ( 1 - 398 ) or Fbxo7 ( 129 - 398 ) immobilized on glutathione beads . ( e ) Competitive binding assays using immobilized GST - Fbxo7 ( 1 - 398 ) incubated with IVT Flag - Parkin and / or full length ( top panel ) or N - terminally truncated ( bottom panel ) PINK1 - Myc . Input and bead - bound proteins were analyzed by immunoblotting as indicated . ( f ) As in e , immobilized GST - ΔN - PINK1 ( top panel ) , GST - Parkin ( middle panel ) and immobilized GST alone ( bottom panel ) were incubated with combinations of IVT Flag - Parkin , T7 - ΔN - PINK1 and Fbxo7 - HA as indicated . Input and bead - bound proteins were analyzed by immunoblotting with the indicated antibodies . All western blots were performed a minimum of three times . Full - length blots are presented in Supplementary Figure 9 .

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figf4(a) Coimmunoprecipitation (IP) of PINK1-Myc and Flag-Fbxo7 in whole-cell lysates from U2OS cells overexpressing both proteins. IB, immunoblot.(b) Coimmunoprecipitation of PINK1-Myc with full-length and two N-terminally deleted Flag-Fbxo7 forms. PINK1-Myc is detected at low levels in complex with Flag-Fbxo7(89-522) but not with Flag-Fbxo7(129-522).(c) As in b, using lysates from U2OS cells expressing PINK1-Myc, and Flag-Fbxo7 containing either N- or C-terminal truncations.(d) In vitro coprecipitation experiments were performed using in vitro-translated (IVT) PINK1-Myc and either GST or GST fusions of Fbxo7(1-398) or Fbxo7(129-398) immobilized on glutathione beads.(e) Competitive binding assays using immobilized GST-Fbxo7(1-398) incubated with IVT Flag-Parkin and/or full length (top panel) or N-terminally truncated (bottom panel) PINK1-Myc. Input and bead-bound proteins were analyzed by immunoblotting as indicated.(f) As in e, immobilized GST-ΔN-PINK1 (top panel), GST-Parkin (middle panel) and immobilized GST alone (bottom panel) were incubated with combinations of IVT Flag-Parkin, T7-ΔN-PINK1 and Fbxo7-HA as indicated. Input and bead-bound proteins were analyzed by immunoblotting with the indicated antibodies. All western blots were performed a minimum of three times. Full-length blots are presented in Supplementary Figure 9.


figf5 ( a ) PINK1 localization at the mitochondria was assessed by immunocytochemistry in SH - SY5Y cells transfected with PINK1 - HA plus scrambled ( scr ) or Fbxo7 siRNA following 1 or 3 h treatment with CCCP ( 10 μM ) . Cells were scored visually for the colocalization of PINK1 - HA with complex Vβ subunit ( CxVβ ) , a mitochondrial marker . Nuclei ( blue ) are stained with DAPI . Histograms indicate the percentage of cells in which PINK1 - HA accumulated at the mitochondria . Data are presented as mean ± s . e . m . ; * P 0 . 05 . Representative images are displayed for cells transfected as indicated , following 0 or 3 h CCCP treatment . For corresponding images at 0 h and 1 h treatment , see Supplementary Figure 5a . Scale bars , 10 μm . ( b ) Fbxo7 accumulation in the mitochondrial fraction following treatment with CCCP ( 10 μM ) is impaired in SH - SY5Y cells transfected with PINK1 siRNA compared to scrambled siRNA ( scr ) . IB , immunoblot . Full - length blots are presented in Supplementary Figure 9 . ( c - f ) Overexpression of Fbxo7 does not rescue climbing ( c , e ) or flight ( d , f ) defects in PINK1 male mutants ( PINK1B9 ) or PINK1 : parkin double mutants ( PINK1B9 ; park25 , daG4 ) . Control genotypes are PINK1B9 / + ; da - GAL4 / + ( c , d ) and da - GAL4 / + ( e , f ) . Histograms indicate mean ± s . e . m . One - way ANOVA with Bonferroni correction ( * * * P 0 . 001 ) . For climbing and flight assays , at least 50 flies were assessed ; number of flies is shown for each bar for c - f .

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figf5(a) PINK1 localization at the mitochondria was assessed by immunocytochemistry in SH-SY5Y cells transfected with PINK1-HA plus scrambled (scr) or Fbxo7 siRNA following 1 or 3 h treatment with CCCP (10 μM). Cells were scored visually for the colocalization of PINK1-HA with complex Vβ subunit (CxVβ), a mitochondrial marker. Nuclei (blue) are stained with DAPI. Histograms indicate the percentage of cells in which PINK1-HA accumulated at the mitochondria. Data are presented as mean ± s.e.m.; *P 0.05. Representative images are displayed for cells transfected as indicated, following 0 or 3 h CCCP treatment. For corresponding images at 0 h and 1 h treatment, see Supplementary Figure 5a. Scale bars, 10 μm.(b) Fbxo7 accumulation in the mitochondrial fraction following treatment with CCCP (10 μM) is impaired in SH-SY5Y cells transfected with PINK1 siRNA compared to scrambled siRNA (scr). IB, immunoblot. Full-length blots are presented in Supplementary Figure 9.(c-f) Overexpression of Fbxo7 does not rescue climbing (c,e) or flight (d,f) defects in PINK1 male mutants (PINK1B9) or PINK1:parkin double mutants (PINK1B9;park25, daG4). Control genotypes are PINK1B9/+; da-GAL4/+ (c,d) and da-GAL4/+ (e,f). Histograms indicate mean ± s.e.m. One-way ANOVA with Bonferroni correction (***P 0.001). For climbing and flight assays, at least 50 flies were assessed; number of flies is shown for each bar for c-f.


figf6 ( a , b ) Ubiquitination of Mfn1 following treatment with CCCP ( 10 μM ) is reduced in the mitochondrial fraction of both SH - SY5Y cells stably expressing Fbxo7 short hairpin RNA ( Fbxo7 KD ) compared to an empty vector control line ( a ) and in patient fibroblasts with homozygous R378G mutation compared to fibroblasts from healthy controls ( b ) . Arrowheads indicate ubiquitinated Mfn1 . IB , immunoblot . ( c , d ) Fbxo7 expression restores elevated Mfn steady - state levels in parkin ( c ) but not PINK1 ( d ) mutant Drosophila . Histograms show mean ± s . e . m . of densitometry analysis of Mfn immunoblots above , normalized to complex Vα ( CxVα ) . Control genotypes are park25 / + ; da - GAL4 / + ( c ) and PINK1B9 / + ; da - GAL4 / + ( d ) . ( e ) Mitochondria in control Drosophila S2R + cells stained with MitoTracker Red show a heterogeneous morphology , with a mixture of tubules and fragmented mitochondria . RNAi knockdown of parkin or PINK1 causes excessive fusion and elongated mitochondria compared to control double - stranded RNA ( Caenorhabditis elegans gene ZK686 . 3 ) . Expression of Fbxo7 restores parkin but not PINK1 knockdown phenotype to WT appearance . Scale bar shows 5 μm . ( f ) Quantification of mitochondrial morphology in dsRNA treated cells . Scoring system : 1 , fragmented ; 2 , WT ; 3 , tubular ; 4 , hyper - fused ( clumped ) . Histograms indicate mean ± s . e . m . Two - tailed Student t - tests ( * * * P 0 . 001 , * P 0 . 05 ) . All western blots were performed a minimum of three times and images are representative of 100 cells scored per condition . Full - length blots are presented in Supplementary Figure 9 .

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figf6(a,b) Ubiquitination of Mfn1 following treatment with CCCP (10 μM) is reduced in the mitochondrial fraction of both SH-SY5Y cells stably expressing Fbxo7 short hairpin RNA (Fbxo7 KD) compared to an empty vector control line (a) and in patient fibroblasts with homozygous R378G mutation compared to fibroblasts from healthy controls (b). Arrowheads indicate ubiquitinated Mfn1. IB, immunoblot.(c,d) Fbxo7 expression restores elevated Mfn steady-state levels in parkin (c) but not PINK1 (d) mutant Drosophila. Histograms show mean ± s.e.m. of densitometry analysis of Mfn immunoblots above, normalized to complex Vα (CxVα). Control genotypes are park25/+; da-GAL4/+ (c) and PINK1B9/+; da-GAL4/+ (d).(e) Mitochondria in control Drosophila S2R+ cells stained with MitoTracker Red show a heterogeneous morphology, with a mixture of tubules and fragmented mitochondria. RNAi knockdown of parkin or PINK1 causes excessive fusion and elongated mitochondria compared to control double-stranded RNA (Caenorhabditis elegans gene ZK686.3). Expression of Fbxo7 restores parkin but not PINK1 knockdown phenotype to WT appearance. Scale bar shows 5 μm. (f) Quantification of mitochondrial morphology in dsRNA treated cells. Scoring system: 1, fragmented; 2, WT; 3, tubular; 4, hyper-fused (clumped). Histograms indicate mean ± s.e.m. Two-tailed Student t-tests (***P 0.001, *P 0.05). All western blots were performed a minimum of three times and images are representative of 100 cells scored per condition. Full-length blots are presented in Supplementary Figure 9.


figf7 ( a ) Treatment with CCCP ( 10 μM ) results in an increase in LC3 - II in the mitochondrial but not the cytosolic fraction of cells stably expressing the empty shRNA vector ( control ) , and this is delayed in stable Fbxo7 knockdown ( KD ) SH - SY5Y cells . ( b ) As in a , an accumulation of LC3 - II was observed in the mitochondrial fraction of healthy control fibroblasts following 1 and 5 h CCCP treatment , but this was reduced in fibroblasts from a patient carrying the R378G mutation . Western blots were performed a minimum of three times . ( c ) Mitochondrial accumulation of p62 following CCCP treatment is inhibited by Fbxo7 siRNA . Flag - Parkin overexpressing SH - SY5Y cells were transfected with scrambled ( scr ) or Fbxo7 siRNA as indicated and treated with either DMSO or CCCP ( 10 μM ) for 6 h . Colocalization of p62 with HtrA2 , a mitochondrial marker , was assessed by Pearson ' s correlation coefficient ( Rr ) on a cell - by - cell basis . Histogram shows the percentage of cells in which Rr was greater than 0 . 5 . Data are represented as mean ± s . e . m . , * P 0 . 05 . Scale bars , 10 μm . ( d ) Mitophagy was analyzed in untransfected ( UT ) SH - SY5Y cells or in stable Flag - Parkin overexpressing SH - SY5Y cells transfected with either scrambled ( scr ) or Fbxo7 siRNA . Histogram indicates the percentage of cells with no remaining mitochondria following 24 h treatment with CCCP ( 10 μM ) for each condition . Complex Vβ subunit ( CxVβ ) was used as a mitochondrial marker . Data are presented as mean ± s . e . m . , * * P 0 . 01 . Scale bars , 10 μm . ( e ) Mitochondrial mass was measured in Flag - Parkin overexpressing SH - SY5Y cells transfected with scrambled ( scr ) or Fbxo7 siRNA and treated for 24 h with either dimethylsulfoxide ( DMSO ) vehicle or CCCP ( 10 μM ) . For representative images , see Supplementary Figure 8d . Full - length blots are presented in Supplementary Figure 9 .

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figf7(a) Treatment with CCCP (10 μM) results in an increase in LC3-II in the mitochondrial but not the cytosolic fraction of cells stably expressing the empty shRNA vector (control), and this is delayed in stable Fbxo7 knockdown (KD) SH-SY5Y cells.(b) As in a, an accumulation of LC3-II was observed in the mitochondrial fraction of healthy control fibroblasts following 1 and 5 h CCCP treatment, but this was reduced in fibroblasts from a patient carrying the R378G mutation. Western blots were performed a minimum of three times.(c) Mitochondrial accumulation of p62 following CCCP treatment is inhibited by Fbxo7 siRNA. Flag-Parkin overexpressing SH-SY5Y cells were transfected with scrambled (scr) or Fbxo7 siRNA as indicated and treated with either DMSO or CCCP (10 μM) for 6 h. Colocalization of p62 with HtrA2, a mitochondrial marker, was assessed by Pearson's correlation coefficient (Rr) on a cell-by-cell basis. Histogram shows the percentage of cells in which Rr was greater than 0.5. Data are represented as mean ± s.e.m., *P 0.05. Scale bars, 10 μm.(d) Mitophagy was analyzed in untransfected (UT) SH-SY5Y cells or in stable Flag-Parkin overexpressing SH-SY5Y cells transfected with either scrambled (scr) or Fbxo7 siRNA. Histogram indicates the percentage of cells with no remaining mitochondria following 24 h treatment with CCCP (10 μM) for each condition. Complex Vβ subunit (CxVβ) was used as a mitochondrial marker. Data are presented as mean ± s.e.m., **P 0.01. Scale bars, 10 μm.(e) Mitochondrial mass was measured in Flag-Parkin overexpressing SH-SY5Y cells transfected with scrambled (scr) or Fbxo7 siRNA and treated for 24 h with either dimethylsulfoxide (DMSO) vehicle or CCCP (10 μM). For representative images, see Supplementary Figure 8d. Full-length blots are presented in Supplementary Figure 9.


Figure 1 . Non - endosomal ubiquitin - positive structures accumulate in ESCRT depleted cells . HeLa cells were left untransfected ( A ) or transfected with control ( B ) , Hrs ( C ) , Tsg101 ( D ) , Vps22 ( E ) , or Vps24 ( F ) siRNA for 5 d and processed for immunofluorescence microscopy . Cells were labeled with antibodies against EEA1 ( blue ) , ubiquitin ( mono and poly - Ub , red ) , and Lamp2 ( green ) . Colocalization between EEA1 and Ub is indicated in purple and between Ub and Lamp2 in yellow . Bar , 10 μm . Single channel images of the insets are shown in Fig . S1 ( available at http : / / www . jcb . org / cgi / content / full / jcb . 200702115 / DC1 ) . ( G ) Knock - down efficiencies were analyzed by Western blotting . Equal loading was verified by anti - α - tubulin immunoblotting .

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Figure 1.Non-endosomal ubiquitin-positive structures accumulate in ESCRT depleted cells. HeLa cells were left untransfected (A) or transfected with control (B), Hrs (C), Tsg101 (D), Vps22 (E), or Vps24 (F) siRNA for 5 d and processed for immunofluorescence microscopy. Cells were labeled with antibodies against EEA1 (blue), ubiquitin (mono and poly-Ub, red), and Lamp2 (green). Colocalization between EEA1 and Ub is indicated in purple and between Ub and Lamp2 in yellow. Bar, 10 μm. Single channel images of the insets are shown in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200702115/DC1).(G) Knock-down efficiencies were analyzed by Western blotting. Equal loading was verified by anti-α-tubulin immunoblotting.


Figure 2 . p62 - and Alfy - positive structures accumulate in cells depleted of Tsg101 or Vps24 . HeLa cells transfected with siRNA against Tsg101 or Vps24 were fixed , permeabilized , and stained with antibodies against ubiquitin ( red ) and p62 ( green ) ( A ) or against ubiquitin ( green ) and Alfy ( red ) ( B ) . Colocalization is indicated in yellow . Bar , 10 μm . ( C ) The number and size ( area ) of p62 - positive particles were quantified using ImageJ software . Approximately 100 cells from three different experiments ( n = 334 ( control ) , 309 ( Tsg101 ) , and 246 ( Vps24 ) ) were used for the quantification and the data is presented as average number per 100 cells . Error bars = SEM .

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Figure 2.p62- and Alfy-positive structures accumulate in cells depleted of Tsg101 or Vps24. HeLa cells transfected with siRNA against Tsg101 or Vps24 were fixed, permeabilized, and stained with antibodies against ubiquitin (red) and p62 (green) (A) or against ubiquitin (green) and Alfy (red) (B). Colocalization is indicated in yellow. Bar, 10 μm. (C) The number and size (area) of p62-positive particles were quantified using ImageJ software. Approximately 100 cells from three different experiments (n = 334 (control), 309 (Tsg101), and 246 (Vps24)) were used for the quantification and the data is presented as average number per 100 cells. Error bars = SEM.


Figure 3 . Autophagic degradation is impeded in Tsg101 - and Vps24 - depleted cells . HeLa cells stably expressing GFP - LC3 ( green ) were transfected with control ( A ) , Vps24 ( B ) , or Tsg101 ( C ) siRNA for 5 d and processed for immunofluorescence analysis . Cells were labeled with antibodies against p62 ( red ) and analyzed by confocal microscopy . Colocalization is indicated in yellow . Bar , 10 μm . ( D ) The total level of p62 and GFP - LC3 ( normalized to control cells ) and their degree of colocalization ( % of total ) in control and Tsg101 - and Vps24 - depleted cells were quantified using the Zeiss LSM 510 Meta software . 30 cells from three independent experiments were used for quantification . Error bars = SEM . ( E ) Western blot showing accumulation of LC3 - II and p62 in HeLa cells depleted of Vps24 and Tsg101 . p62 also accumulates in the insoluble fraction ( bottom picture ) , indicating a shift to a more aggregated form upon depleted of Vps24 and Tsg101 . ( F ) Western blot showing that LC3 - II levels were similar in control and Vps24 - depleted cells treated with Bafilomycin A .

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Figure 3.Autophagic degradation is impeded in Tsg101- and Vps24-depleted cells. HeLa cells stably expressing GFP-LC3 (green) were transfected with control (A), Vps24 (B), or Tsg101 (C) siRNA for 5 d and processed for immunofluorescence analysis. Cells were labeled with antibodies against p62 (red) and analyzed by confocal microscopy. Colocalization is indicated in yellow. Bar, 10 μm.(D) The total level of p62 and GFP-LC3 (normalized to control cells) and their degree of colocalization (% of total) in control and Tsg101- and Vps24-depleted cells were quantified using the Zeiss LSM 510 Meta software. 30 cells from three independent experiments were used for quantification. Error bars = SEM.(E) Western blot showing accumulation of LC3-II and p62 in HeLa cells depleted of Vps24 and Tsg101. p62 also accumulates in the insoluble fraction (bottom picture), indicating a shift to a more aggregated form upon depleted of Vps24 and Tsg101.(F) Western blot showing that LC3-II levels were similar in control and Vps24-depleted cells treated with Bafilomycin A.


Figure 4 . Formation of autolysosomes is inhibited in cells depleted of Tsg101 or Vps24 . ( A ) The double - tagged LC3 protein ( mCherry - GFP - LC3 ) will emit yellow ( green merged with red ) fluorescence in non - acidic structures and appear as red only in the autolysosomes due to quenching of GFP in these acidic structures . ( B - F ) HeLa cells were transfected with control ( B ) , Tsg101 ( C ) , or Vps24 ( D ) siRNA for 4 d and then with mCherry - GFP - LC3 for another 24 h before confocal microscopy analysis . Bar , 5 μm ( E ) The mCherry - LC3 ( red ) signal was quantified in ∼ 30 cells with similar expression levels from three independent experiments using the Zeiss LSM 510 Meta software and the data are presented as % of total mCherry - GFP - LC3 . Error bar = SEM . ( F ) Degradation of [ 14C ] - valine - labeled proteins in cells incubated in complete medium ( control ) , serum - and amino acid - deficient medium ( starve ) , or starvation media in the presence of 3 - methyl adenine ( 3 - MA ) to inhibit autophagic degradation . Average degradation per hour from four experiments done in duplicates ± 1 SEM are shown .

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Figure 4.Formation of autolysosomes is inhibited in cells depleted of Tsg101 or Vps24. (A) The double-tagged LC3 protein (mCherry-GFP-LC3) will emit yellow (green merged with red) fluorescence in non-acidic structures and appear as red only in the autolysosomes due to quenching of GFP in these acidic structures. (B-F) HeLa cells were transfected with control (B), Tsg101 (C), or Vps24 (D) siRNA for 4 d and then with mCherry-GFP-LC3 for another 24 h before confocal microscopy analysis. Bar, 5 μm(E) The mCherry-LC3 (red) signal was quantified in ∼30 cells with similar expression levels from three independent experiments using the Zeiss LSM 510 Meta software and the data are presented as % of total mCherry-GFP-LC3. Error bar = SEM.(F) Degradation of [14C]-valine- labeled proteins in cells incubated in complete medium (control), serum- and amino acid-deficient medium (starve), or starvation media in the presence of 3-methyl adenine (3-MA) to inhibit autophagic degradation. Average degradation per hour from four experiments done in duplicates ±1 SEM are shown.


Figure 5 . Autophagosomes and amphisomes do form in ESCRT - depleted cells . HeLa cells stably expressing GFP - LC3 were transfected with control ( A ) , Tsg101 ( B , D , and E ) , or Vps24 ( C ) siRNA and processed for immuno - EM analysis . Cryosections were incubated with antibodies against GFP ( 15 - nm gold ) and LBPA ( 10 - nm gold ) . Amphisomes ( Am ) , characterized by their content of electron - dense LC3 - positive areas and intraluminal vesicles , were observed in both control ( A ) and ESCRT - depleted cells ( B and C ) . ( D ) In the Tsg101 - depleted cells we found large clusters of more typical autophagosomes ( Au ) and ( E ) clusters of double - membrane structures , consisting of autophagosomes and tubular structures which might represent phagophores , all labeling strongly for GFP - LC3 . We never observed similar clusters in control cells and very rarely in Vps24 - depleted cells . Bars , 200 nm . ( F ) Quantification of amphisomes in control cells and cells depleted for Tsg101 or Vps24 . Approximately 20 cells were included per siRNA depletion per experiment ( n = 3 ) . Error bars = SEM .

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Figure 5.Autophagosomes and amphisomes do form in ESCRT-depleted cells. HeLa cells stably expressing GFP-LC3 were transfected with control (A), Tsg101 (B, D, and E), or Vps24 (C) siRNA and processed for immuno-EM analysis. Cryosections were incubated with antibodies against GFP (15-nm gold) and LBPA (10-nm gold). Amphisomes (Am), characterized by their content of electron-dense LC3-positive areas and intraluminal vesicles, were observed in both control (A) and ESCRT-depleted cells (B and C). (D) In the Tsg101-depleted cells we found large clusters of more typical autophagosomes (Au) and (E) clusters of double-membrane structures, consisting of autophagosomes and tubular structures which might represent phagophores, all labeling strongly for GFP-LC3. We never observed similar clusters in control cells and very rarely in Vps24-depleted cells. Bars, 200 nm. (F) Quantification of amphisomes in control cells and cells depleted for Tsg101 or Vps24. Approximately 20 cells were included per siRNA depletion per experiment (n = 3). Error bars = SEM.


Figure 6 . Immuno - EM of p62 - positive structures in Vps24 - and Tsg101 - depleted cells . Cryosections of HeLa cells transfected with Vps24 ( A , C , and E ) or Tsg101 ( B , D , and F ) siRNA were incubated with antibodies against p62 ( 15 - nm protein A gold , 10 - nm in D ) . Membrane - free p62 - positive aggregates ( C and D ) or p62 - positive structures contained within dense clusters of vesicular - tubular elements ( A and B ) were detected . Vesicles with early endosomal morphology ( EE ) or multivesicular appearance ( MVB ) associated with these clusters . We also observed p62 labeling in electron - dense structures sequestered within amphisomes in cell depleted of Vps24 ( E ) or Tsg101 ( F ) . The amphisomes typically consist of an electron - dense p62 positive body ( E and F , arrowhead ) , and a multivesicular endosomal structure ( E and F , arrow ) . Bars , 200 nm .

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Figure 6.Immuno-EM of p62-positive structures in Vps24- and Tsg101-depleted cells. Cryosections of HeLa cells transfected with Vps24 (A, C, and E) or Tsg101 (B, D, and F) siRNA were incubated with antibodies against p62 (15-nm protein A gold, 10-nm in D). Membrane-free p62-positive aggregates (C and D) or p62-positive structures contained within dense clusters of vesicular-tubular elements (A and B) were detected. Vesicles with early endosomal morphology (EE) or multivesicular appearance (MVB) associated with these clusters. We also observed p62 labeling in electron-dense structures sequestered within amphisomes in cell depleted of Vps24 (E) or Tsg101 (F). The amphisomes typically consist of an electron-dense p62 positive body (E and F, arrowhead), and a multivesicular endosomal structure (E and F, arrow). Bars, 200 nm.


Figure 7 . Ubiquitin and p62 positive structures accumulate in cells expressing CHMP2 mutants . HeLa cells transfected with myc - tagged wild - type CHMP2B ( A ) , CHMP2BIntron5 ( B ) , or CHMP2BΔ10 ( C ) were labeled with antibodies against c - Myc ( red ) , ubiquitin ( green ) , and p62 ( blue ) and analyzed by confocal microscopy . Single channel images in black and white are shown . Bars , 10 μm . ( D ) Total levels of p62 in these cells were quantified in 20 cells from three independent experiments using the Zeiss LSM 510 Meta software and the average normalized to the average p62 level in control ( mock transfected ) cells . Error bars = SEM . ( E ) Western blot analysis showing accumulation of p62 in cells transfected with myc - CHMP2BIntron5 compared with mock - transfected cells ( control ) and cells transfected with CHMP2Bwt ( F ) Immuno - EM showing p62 - positive aggregates containing dense clusters of vesicular - tubular structures in cells transfected with myc - CHMP2BIntron5 . Bar , 200 nm .

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Figure 7.Ubiquitin and p62 positive structures accumulate in cells expressing CHMP2 mutants. HeLa cells transfected with myc-tagged wild-type CHMP2B (A), CHMP2BIntron5 (B), or CHMP2BΔ10 (C) were labeled with antibodies against c-Myc (red), ubiquitin (green), and p62 (blue) and analyzed by confocal microscopy. Single channel images in black and white are shown. Bars,10 μm.(D) Total levels of p62 in these cells were quantified in 20 cells from three independent experiments using the Zeiss LSM 510 Meta software and the average normalized to the average p62 level in control (mock transfected) cells. Error bars = SEM.(E) Western blot analysis showing accumulation of p62 in cells transfected with myc-CHMP2BIntron5 compared with mock-transfected cells (control) and cells transfected with CHMP2Bwt(F) Immuno-EM showing p62-positive aggregates containing dense clusters of vesicular-tubular structures in cells transfected with myc-CHMP2BIntron5. Bar, 200 nm.


Figure 8 . TDP - 43 accumulates in cytoplasmic ubiquitin - positive structures in Tsg101 - and Vps24 - depleted cells . HeLa cells transfected with control ( A ) , Tsg101 ( B ) , or Vps24 ( C ) siRNA were fixed , permeabilized , and stained with antibodies against TDP - 43 ( green ) , ubiquitin ( red ) , and p62 ( blue ) . Single channel images in black and white are shown . Colocalization of all proteins is indicated in white in the merged picture . Bars , 10 μm .

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Figure 8.TDP-43 accumulates in cytoplasmic ubiquitin-positive structures in Tsg101- and Vps24-depleted cells. HeLa cells transfected with control (A), Tsg101 (B), or Vps24 (C) siRNA were fixed, permeabilized, and stained with antibodies against TDP-43 (green), ubiquitin (red), and p62 (blue). Single channel images in black and white are shown. Colocalization of all proteins is indicated in white in the merged picture. Bars, 10 μm.


Figure 9 . Vps24 is required for efficient clearance of Htt inclusions . HeLa cells expressing HttQ65 - mCFP ( A ) or HttQ103 - mCFP ( B ) and N2a cells expressing HttQ103 - mCFP ( C ) were transfected with control or Vps24 siRNA for 2 d and then incubated in the absence or presence of doxycycline for 3 d to turn off expression of HttQ65 / Q103 - mCFP . Vps24 depletion was determined by Western blot analysis . ( A and C ) Clearance of SDS - insoluble Htt inclusions was analyzed by filter - trap assays . ( B ) Alternatively , aggregate clearance was analyzed by confocal quantification of the number of HeLa HttQ103 cells having visible inclusions after 3 d in the absence or presence of dox . 300 cells from three independent experiments were counted for each condition . Error bars = SD .

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Figure 9.Vps24 is required for efficient clearance of Htt inclusions. HeLa cells expressing HttQ65-mCFP (A) or HttQ103-mCFP (B) and N2a cells expressing HttQ103-mCFP (C) were transfected with control or Vps24 siRNA for 2 d and then incubated in the absence or presence of doxycycline for 3 d to turn off expression of HttQ65/Q103-mCFP. Vps24 depletion was determined by Western blot analysis. (A and C) Clearance of SDS-insoluble Htt inclusions was analyzed by filter-trap assays. (B) Alternatively, aggregate clearance was analyzed by confocal quantification of the number of HeLa HttQ103 cells having visible inclusions after 3 d in the absence or presence of dox. 300 cells from three independent experiments were counted for each condition. Error bars = SD.


Figure 1E . Normalized ThT kinetics of 10μM Biot - Aβ1 - 42 with timepoints of samples that incubated with Aβ1 - 42 membranes . I . ThT kinetics of Biot - Aβ1 - 42 seeding . 10μM of Biot - Aβ1 - 42 incubated with 0 . 5 or 1 μM of Biot - Aβ1 - 42 seeds .

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Figure 1E. Normalized ThT kinetics of 10μM Biot-Aβ1-42 with timepoints of samples that incubated with Aβ1-42 membranes.I. ThT kinetics of Biot-Aβ1-42 seeding. 10μM of Biot-Aβ1-42 incubated with 0.5 or 1 μM of Biot-Aβ1-42 seeds.


Figure 2D . Binding of Biot - Aβ1 - 42 to homologue peptides derived from ~ 520 randomly selected proteins . E . Summary of Biot - Aβ1 - 42 binding throughout 8 membranes , color indicates the mean between membranes and the size of the outline the standard deviation .

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Figure 2D. Binding of Biot-Aβ1-42 to homologue peptides derived from ~520 randomly selected proteins.E. Summary of Biot-Aβ1-42 binding throughout 8 membranes, color indicates the mean between membranes and the size of the outline the standard deviation.


Figure 3A - C . ThT kinetics of 10μM rAβ1 - 42 alone or in presence ( 1 : 1 ) of 3 homologue peptides ( full screen on Appendix Figure S3 , S4 , n = 2 independent experiments with 4 repeats ) . Reused images in Appendix figure S3I . Representative AFM height images of Aβ fibrils alone or in a 1 : 1 mixture with P3 , P5 , P8 and P12 peptides are show in the top row . The boxes indicate the magnified regions shown in the second row . Arrows indicate the locations of representative individual fibrils shown in magnified detail , each shown as a 200 nm digitally straightened segment and a 100 nm segment of the corresponding 3D surface envelope model that was calculated from the image data . The scale bar for each row are shown to the left , with both the 3D model and the straightened image data representing 10 nm . The colour scale of the 3D models from blue to yellow indicate the distance ( from low to high ) between the fibril surface and fibril centre axis to demonstrate their twist patterns . The average fibril height distribution of around 80 manually selected filaments per sample that showed twist patterns characteristic of single , not fragmented , amyloid fibrils are shown in the bottom row .

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Figure 3A-C. ThT kinetics of 10μM rAβ1-42 alone or in presence (1:1) of 3 homologue peptides (full screen on Appendix Figure S3,S4, n= 2 independent experiments with 4 repeats). Reused images in Appendix figure S3I. Representative AFM height images of Aβ fibrils alone or in a 1:1 mixture with P3, P5, P8 and P12 peptides are show in the top row. The boxes indicate the magnified regions shown in the second row. Arrows indicate the locations of representative individual fibrils shown in magnified detail, each shown as a 200 nm digitally straightened segment and a 100 nm segment of the corresponding 3D surface envelope model that was calculated from the image data. The scale bar for each row are shown to the left, with both the 3D model and the straightened image data representing 10 nm. The colour scale of the 3D models from blue to yellow indicate the distance (from low to high) between the fibril surface and fibril centre axis to demonstrate their twist patterns. The average fibril height distribution of around 80 manually selected filaments per sample that showed twist patterns characteristic of single, not fragmented, amyloid fibrils are shown in the bottom row.


Figure 4A . Overrepresentation of Aβ APRs in amyloid plaques of AD brains ( statistics : hypergeometric test with Bonferroni correction ) * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . The original data was taken from Xiong et al and included three biological replicates . G . Overrepresentation of Aβ APRs in amyloid plaques of nonAD brains . ( statistics : hypergeometric test with Bonferroni correction ) . * P ≤ 0 . 05 , * * P ≤ 0 . 01 The original data was taken from Xiong et al and included three biological replicates .

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Figure 4A. Overrepresentation of Aβ APRs in amyloid plaques of AD brains (statistics: hypergeometric test with Bonferroni correction) * P ≤0.05, *** P≤0.001, **** P≤0.0001. The original data was taken from Xiong et al and included three biological replicates.G. Overrepresentation of Aβ APRs in amyloid plaques of nonAD brains.(statistics: hypergeometric test with Bonferroni correction). * P ≤0.05, ** P≤0.01 The original data was taken from Xiong et al and included three biological replicates.


Figure 5A . Overrepresentation of Aβ APRs in amyloid plaques of APP / PS1 mouse brains ( mean ± SD ) from two biological replicates . ( statistics : hypergeometric test with Bonferroni correction ) D . No overrepresentation of Aβ APRs was observed in proteins from Glial cytoplasmic inclusions ( α - synuclein aggregates ) . ( statistics : hypergeometric test with Bonferroni correction )

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Figure 5A. Overrepresentation of Aβ APRs in amyloid plaques of APP/PS1 mouse brains (mean ±SD) from two biological replicates. (statistics: hypergeometric test with Bonferroni correction)D. No overrepresentation of Aβ APRs was observed in proteins from Glial cytoplasmic inclusions (α-synuclein aggregates). (statistics: hypergeometric test with Bonferroni correction)


Figure 6D . Representative images of 3 proteins that can induce aggregation of Aβ1 - 42 in biosensor cell . Increase aggregation is observed in cells expressing the construct but not GFP alone ( left panels ) . Scale bar : 100μm . FRAP of the resulting aggregates shows no recovery ( in detail at Appendix Figure S11 ) . Scale bar : 2 . 5μm Removal of the homologue regions resulting in reduced aggregation ( right panels ) . ( n = 3 independent experiments ) . E . Quantification of number of spots per cell in cells expressing / not expressing the construct ( identified by GFP , transfection reporter ) ( n = 3 independent experiments , statistics : ordinary one - way Anova with Dunnett T3 multiple comparison correction , unpaired t - test for transfected / non - transfected cells ) . Bar plot : mean with 95 % CI . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001F - G . Quantification of Spots per cell for construct6 and construct6 control ( 6Ctrl , removal of homologue region ) ( F ) and construct10 and construct10 control ( 10ctrl , removal of homologue region ) ( G ) . ( n = 4 independent experiments , statistics : ordinary one - way Anova ) . Bar plot : mean with 95 % CI . * * * * P ≤ 0 . 0001H - I ) Confocal images of colocalization of protein of interest with Aβ aggregates . ( Contrast of images were enhanced to 0 . 1 % saturated pixels using Fiji ) . Scale bars :

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Figure 6D. Representative images of 3 proteins that can induce aggregation of Aβ1-42 in biosensor cell. Increase aggregation is observed in cells expressing the construct but not GFP alone (left panels). Scale bar: 100μm. FRAP of the resulting aggregates shows no recovery (in detail at Appendix Figure S11). Scale bar: 2.5μm Removal of the homologue regions resulting in reduced aggregation (right panels). (n=3 independent experiments).E. Quantification of number of spots per cell in cells expressing/not expressing the construct (identified by GFP, transfection reporter) (n=3 independent experiments, statistics: ordinary one-way Anova with Dunnett T3 multiple comparison correction, unpaired t-test for transfected/non-transfected cells). Bar plot: mean with 95%CI. * P ≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001F-G. Quantification of Spots per cell for construct6 and construct6 control (6Ctrl, removal of homologue region) (F) and construct10 and construct10 control (10ctrl, removal of homologue region) (G). (n=4 independent experiments, statistics: ordinary one-way Anova). Bar plot: mean with 95%CI. **** P≤0.0001H-I) Confocal images of colocalization of protein of interest with Aβ aggregates. (Contrast of images were enhanced to 0.1% saturated pixels using Fiji). Scale bars: 22μm