Relative GSEC - APP / Aβn stabilities were assessed in - GSEC thermoactivity assays , which evaluate changes in GSEC processivity ( Aβ ( 38 + 40 ) / 42 ) upon incubation at 42 ° C for 24h , relative to 37 ° C . Aβ peptides in conditioned medium collected from indicated MEF lines ( transiently expressing APPC99 ) were quantified Aβ ( 38 + 40 ) / 42 ratios determined . data are shown as mean ± 95 % CI , as % of WT GSEC incubated at 37 ° C for 24h , N = 6 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc with comparison to WT was used to determine statistical significance ( F ( DFn , DFd ) : F ( 6 , 46 ) = 100 ) ; * * * * p > 0 . 0001 compared to WT at 37 ° C , # p > 0 . 05 , # # p > 0 . 01 , # # # # p > 0 . 0001 compared to WT at 42 ° C . Note : Aβ profiles ( including Aβ43 ) normalized to total Aβ quantified in - thermoactivity assays with different mNCT WT / mutants are shown in Figure EV2 .

O B-SUBCELLULAR O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

Relative GSEC-APP/Aβn stabilities were assessed in cell-based GSEC thermoactivity assays, which evaluate changes in GSEC processivity (Aβ(38+40)/42 ratio) upon incubation at 42°C for 24h, relative to 37°C. Aβ peptides in the conditioned medium collected from the indicated MEF cell lines (transiently expressing APPC99) were quantified and the Aβ(38+40)/42 ratios determined. The data are shown as mean ± 95% CI, as % of the WT GSEC cell line incubated at 37°C for 24h, N = 6 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine the statistical significance (F(DFn, DFd): F(6, 46) = 100); ****p > 0.0001 compared to WT at 37°C, #p > 0.05, ##p>0.01, ####p>0.0001 compared to WT at 42°C. Note: Aβ profiles (including Aβ43 levels) normalized to total Aβ quantified in the cell-based thermoactivity assays with the different mNCT WT/mutants are shown in Figure EV2.


Aβ profiles ( % contribution of Aβ37 , Aβ38 , Aβ40 , Aβ42 peptides to total Aβ ( Aβ37 + 38 + 40 + 42 ) ) in conditioned medium collected from WT or mutant ( NCT ) MEF lines co - expressing WT or mutant APPC99 substrates were assayed by ELISA . Schematic models of tested GSEC - APP complexes are shown .

B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O B-GENEPROD B-SUBCELLULAR O O O

Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from WT or mutant (NCT) MEF cells lines co-expressing WT or mutant APPC99 substrates were assayed by ELISA. Schematic models of the tested GSEC-APP complexes are shown.


To account for increased production of Aβ37 Aβ38 at expense of Aβ40 Aβ42 in tested conditions , of ( ( Aβ 37 / 40 ) + ( Aβ 38 / 42 ) ) / 2 was determined used to estimate GSEC processivity of APPC99 . data are presented as mean ± SD , N ≥ 3 , Unpaired , two - Student ' s t - was used to determine statistical significance . * * p > 0 . 01 , * * * * p > 0 . 0001 . Calculated T - Df values were : T = 11 , 63 ; Df = 16 ( I241 - K28 vs I241E - K28 ) , T = 11 , 37 ; Df = 14 ( I241 - K28 vs I241 - K28E ) , T = 4 , 572 ; Df = 8 ( I241E - K28 vs I241 - K28E ) , T = 4 , 497 ; Df = 5 ( I241 - K28E vs I241K - K28E ) , T = 2 , 561 ; Df = 7 .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

To account for the increased production of Aβ37 and Aβ38 at the expense of Aβ40 and Aβ42 in the tested conditions, the ratio of ((Aβ 37/40) + (Aβ 38/42))/2 was determined and used to estimate GSEC processivity of APPC99. All the data are presented as mean ± SD, N ≥ 3, Unpaired, two-tailed Student's t-test was used to determine the statistical significance. **p > 0.01, ****p > 0.0001. Calculated T- and Df values were: T = 11,63; Df = 16 (I241-K28 vs I241E-K28), T = 11,37; Df = 14 (I241-K28 vs I241-K28E), T = 4,572; Df = 8 (I241E-K28 vs I241-K28E), T = 4,497; Df = 5 (I241-K28E vs I241K-K28E), T = 2,561; Df = 7.


Introduction of Cys at position 241 in NCT allows chemical cross - linking of NCT APPC99 with BMPS . Representative SDS - PAGE / western blot show cross - linked NCT - APPC99 , NCT APPC99 bands in detergent - extracted from BMPS cross - linker - treated membranes prepared from MEF lines expressing indicated WT or mutant NCT GSEC complexes together with APPC99 or in absence of substrate . Orange , black , arrows indicate NCT - APPC99 / Aβ crosslinked , mature NCT , immature NCT bands , respectively . Arrowheads indicate position of molecular markers . of western blot density profiles in panel ( B ) shows that co - migration of NCT ( orange arrow ) with APPC99 shifts cross - linked band towards a higher molecular than observed for mature NCT ( black arrow ) . Immature NCT is indicated with an open ( ) arrow . Full - range molecular density profiles are shown in Figure

O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

Introduction of Cys at position 241 in NCT allows chemical cross-linking of NCT and APPC99 with BMPS. Representative SDS-PAGE/western blot show cross-linked NCT-APPC99, NCT and APPC99 bands in detergent-extracted membrane proteins from BMPS cross-linker-treated membranes prepared from MEF cell lines expressing the indicated WT or mutant NCT GSEC complexes together with APPC99 or in the absence of the substrate. Orange, black, and white arrows indicate the NCT-APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the western blot density profiles in panel (B) shows that co-migration of NCT (orange arrow) with APPC99 shifts the cross-linked band towards a higher molecular weight than the observed for mature NCT (black arrow). Immature NCT is indicated with an open (white) arrow. Full-range molecular weight density profiles are shown in Figure EV3C


Representative SDS - PAGE / western blot presents NCT - APPC99 , NCT APPC99 expression in detergent - extracted membranes prepared from MEF lines co - expressing mutant NCT - I241C GSEC with APPC99 - K28C substrate . As negative control , same sample was supplemented with β - mercaptoethanol ( BME ) to reduce disulfide bridges . A full - range molecular western blot is shown in Figure EV3B . Orange , black , arrows indicate NCT - APPC99 / Aβ crosslinked , mature NCT , immature NCT bands , respectively . Arrowheads indicate position of molecular markers . of integrated density profiles of respective western blot bands supports spontaneous formation of a disulfide bond between NCT - I241C andAPPC99 K28C ( mature immature NCT are indicated with black arrows , respectively . filled orange arrow points at high molecular APPC99 / Aβ density ( crosslinked NCT - APPC99 / Aβ band ) . Note that peak amplitudes for cross - linked NCT - APPC99 mature NCT band were normalized to similar values for clarity purposes . Full - range molecular density profiles are shown in Figure EV3D .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

Representative SDS-PAGE/western blot presents NCT-APPC99, NCT and APPC99 expression in detergent-extracted membranes prepared from MEF cell lines co-expressing the mutant NCT- I241C GSEC complex with the APPC99-K28C substrate. As negative control, the same sample was supplemented with β-mercaptoethanol (BME) to reduce all disulfide bridges. A full-range molecular weight western blot is shown in Figure EV3B. Orange, black, and white arrows indicate the NCT -APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the integrated density profiles of the respective western blot bands supports the spontaneous formation of a disulfide bond between NCT- I241C andAPPC99 K28C (mature and immature NCT are indicated with black and white arrows, respectively. The filled orange arrow points at the high molecular weight APPC99/Aβ density (crosslinked NCT-APPC99/Aβ band). Note that peak amplitudes for the cross-linked NCT-APPC99 and the mature NCT band were normalized to similar values for clarity purposes. Full-range molecular weight density profiles are shown in Figure EV3D.


Representative SDS - PAGE / western blot presents NCT - APPC99 , NCT APPC99 expression in detergent - extracted membranes prepared from MEF lines co - expressing mutant NCT - I241C GSEC with APPC99 - K28C substrate . As negative control , same sample was supplemented with β - mercaptoethanol ( BME ) to reduce disulfide bridges . A full - range molecular western blot is shown in Figure EV3B . Orange , black , arrows indicate

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

Representative SDS-PAGE/western blot presents NCT-APPC99, NCT and APPC99 expression in detergent-extracted membranes prepared from MEF cell lines co-expressing the mutant NCT- I241C GSEC complex with the APPC99-K28C substrate. As negative control, the same sample was supplemented with β-mercaptoethanol (BME) to reduce all disulfide bridges. A full-range molecular weight western blot is shown in Figure EV3B. Orange, black, and white arrows indicate the NCT


Aβ profiles ( % contribution of Aβ37 , Aβ38 , Aβ40 , Aβ42 peptides to total Aβ ( Aβ37 + 38 + 40 + 42 ) ) in conditioned medium collected from vehicle ( DMSO ) or GSM - treated ( 1 µM final ) MEF co - expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA . Data generated using 0 . 3 µM GSM - treated MEF is shown in Figure EV4 . Data are presented as mean ± SD , N ≥ 3 independent experiments .

B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD B-SUBCELLULAR O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O

Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from vehicle (DMSO) or GSM-treated (1 µM final concentration) MEF cells co-expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA. Data generated using 0.3 µM GSM-treated MEF cells is shown in Figure EV4. Data are presented as mean ± SD, N ≥ 3 independent experiments.


Aβ 37 / 40 Aβ 38 / 42 ratios were calculated to evaluate efficiency of GSEC - mediated cleavage of APPC99 ( Figure EV4 ) statistical ( P values ) ( two - way ANOVA with Sidak ' s post hoc ) for effect of GSMs on WT mutant complexes is presented ( response to GSM normalized to corresponding vehicle condition ) .

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Aβ 37/40 and Aβ 38/42 ratios were calculated to evaluate the efficiency of the GSEC-mediated cleavage of APPC99 (Figure EV4) and statistical analysis (P values) (two-way ANOVA with Sidak's post hoc test) for the effect of GSMs on WT and mutant complexes is presented (response to GSM normalized to the corresponding vehicle condition).


A Immunoblot of HeLa using indicated antibodies that were synchronized in G1 / S phase using a double thymidine block or in mitosis using sequential thymidine nocodazole treatment collected at given time points .

O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

A Immunoblot analysis of HeLa cells using the indicated antibodies that were synchronized in G1/S phase using a double thymidine block or in mitosis using sequential thymidine and nocodazole treatment and collected at the given time points.


B Immunoblot of HeLa that were transfected with siRNA oligonucleotides as indicated left untreated or synchronized in mitosis using sequential thymidine nocodazole treatment .

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B Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides as indicated and left untreated or synchronized in mitosis using sequential thymidine and nocodazole treatment.


C Co - immunoprecipitation of XIAP endogenous USP9X in HEK 293T that were transfected with FLAG - tagged XIAP or empty vector ( EV ) synchronized in mitosis using nocodazole or left untreated .

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C Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were transfected with FLAG-tagged XIAP or empty vector (EV) and synchronized in mitosis using nocodazole or left untreated.


D Co - immunoprecipitation of XIAP endogenous USP9X in HEK 293T that were treated as in ( C ) , but with additional transfection of FLAG - tagged XIAP single acid mutants G188R G188E . Only mitotic samples are shown . asterisk denotes ubiquitylated forms of XIAP .

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D Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were treated as in (C), but with additional transfection of the FLAG-tagged XIAP single amino acid mutants G188R and G188E. Only mitotic samples are shown. The asterisk denotes ubiquitylated forms of XIAP.


E In vivo ubiquitylation of XIAP in HeLa that were infected with indicated expression constructs carrying FLAG - tagged XIAP transfected with siRNA oligonucleotides as specified . were synchronized in mitosis using sequential thymidine / nocodazole treatment , as indicated . Subsequent to treatment with MG132 , whole extracts ( WCE ) were prepared ubiquitylated XIAP was isolated by anti - FLAGimmunoprecipitation ( IP ) under denaturing conditions .

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E In vivo ubiquitylation of XIAP in HeLa cells that were infected with the indicated expression constructs carrying FLAG-tagged XIAP and transfected with siRNA oligonucleotides as specified. Cells were synchronized in mitosis using sequential thymidine/nocodazole treatment, as indicated. Subsequent to treatment with MG132, whole cell extracts (WCE) were prepared and ubiquitylated XIAP was isolated by anti-FLAGimmunoprecipitation (IP) under denaturing conditions.


F Immunoblot of NIH 3T3 that were transfected with expression constructs for USP9XWT or catalytically inactive mutant USP9XC1556S . band in EV control lane of anti - V5 panel marks an unspecific band produced by antibody .

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F Immunoblot analysis of NIH 3T3 cells that were transfected with expression constructs for USP9XWT or the catalytically inactive mutant USP9XC1556S. The band in the EV control lane of the anti-V5 panel marks an unspecific band produced by the antibody.


G Immunoblot of HeLa using antibodies to indicated endogenous that were synchronized in mitosis using thymidine / nocodazole treated with DMSO or USP9X inhibitor WP1130 as indicated .

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G Immunoblot analysis of HeLa cells using antibodies to the indicated endogenous proteins that were synchronized in mitosis using thymidine/nocodazole and treated with DMSO or the USP9X inhibitor WP1130 as indicated.


A Immunoblot of WT , Xiap - / - , Mcl1 - / - or cIap2 - / - mouse embryonic fibroblasts that were lentivirally infected with shRNA constructs directed against a non - relevant mRNA ( Ctrl ) or against Usp9X mRNA treated with taxol as specified .

O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O

A Immunoblot analysis of WT, Xiap-/-, Mcl1-/- or cIap2-/- mouse embryonic fibroblasts that were lentivirally infected with shRNA constructs directed against a non-relevant mRNA (Ctrl) or against Usp9X mRNA and treated with taxol as specified.


B Two - dimensional - cycle ( BrdU / PI ) of described in ( A ) . Sub G1 G2 / M fractions of were quantified averaged with two additional , independent experiments ( n = 3 , ± SD ; WT : * * , P = 0 . 0016 ; * * * , P = 0 . 0004 ; Mcl1 - / - : * * * , P = 0 . 0003 ; * * , P = 0 . 0017 ; cIap2 - / - : * * , P = 0 . 0043 , Student ' s t ) . Black bars exemplify shRNA Ctrl , bars shRNA Usp9X samples .

O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O

B Two-dimensional cell-cycle analysis (BrdU/PI) of cells described in (A). Sub G1 and G2/M fractions of cells were quantified and averaged with two additional, independent experiments (n=3, ± SD; WT: **, P = 0.0016; ***, P = 0.0004; Mcl1-/-: ***, P = 0.0003; **, P = 0.0017; cIap2-/-: **, P = 0.0043, Student's t test). Black bars exemplify shRNA Ctrl, white bars shRNA Usp9X samples.


C Immunoblot of HeLa transfected with a FLAG - tagged USP9X expression construct or empty vector ( EV ) treated with taxol for indicated times .

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C Immunoblot analysis of HeLa cells transfected with a FLAG-tagged USP9X expression construct or empty vector (EV) and treated with taxol for the indicated times.


D Immunoblot of HeLa that were transfected with a FLAG - tagged XIAP expression construct or empty vector treated as in ( C ) .

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D Immunoblot analysis of HeLa cells that were transfected with a FLAG-tagged XIAP expression construct or empty vector and treated as in (C).


E Two dimensional - cycle ( BrdU / PI ) of described in ( C ) ( D ) . Apoptotic indices represent ratios of sub G1 to G1 / S are shown for analyses at indicated timepoints . * * , P = 0 . 0020 ; * * * , P = 0 . 0004 ; Student ' s t .

O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Two dimensional cell-cycle analysis (BrdU/PI) of cells described in (C) and (D). Apoptotic indices represent ratios of sub G1 to G1/S cells and are shown for analyses at the indicated timepoints. **, P = 0.0020; ***, P = 0.0004; Student's t test.


F Immunoblot of HeLa that were transfected with siRNA oligonucleotides directed against a non - relevant mRNA ( Ctrl ) or against Xiap mRNA treated with taxol as specified .

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F Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides directed against a non-relevant mRNA (Ctrl) or against Xiap mRNA and treated with taxol as specified.


A Immunoblot of sorted Eμ - Myc lymphoma that were retrovirally transduced with indicated IRES - GFP shRNA constructs .

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A Immunoblot analysis of sorted Eμ-Myc lymphoma cells that were retrovirally transduced with the indicated IRES-GFP shRNA constructs.


B Kaplan Meier survival curves of mice that were injected intravenously with syngeneic Eμ - Myc lymphoma transduced with indicated shRNA constructs as described in ( A ) . Prior to injection , were sorted for viability ( propidium iodide negative ) GFP positivity ( sh _ Ctrl , black , n = 6 ; sh _ USP9X , gray , n = 5 ) . * P = 0 . 0293 ; Mantel - Cox .

O O O B-EXP_ASSAY O O B-ORGANISM O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O 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-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

B Kaplan Meier survival curves of mice that were injected intravenously with syngeneic Eμ-Myc lymphoma cells transduced with the indicated shRNA constructs as described in (A). Prior to injection, cells were sorted for viability (propidium iodide negative) and GFP positivity (sh_Ctrl, black, n = 6; sh_USP9X, gray, n = 5). *P = 0.0293; Mantel-Cox test.


C Kaplan - Meier survival curves of mice that were injected with syngeneic Eμ - Myc lymphoma transduced with indicated shRNA constructs as described in ( A ) treated as in ( B ) ( sh _ Ctrl , black , n = 6 ; sh _ XIAP , gray , n = 6 ) . * * , P = 0 . 0020 ; Mantel - Cox .

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C Kaplan-Meier survival curves of mice that were injected with syngeneic Eμ-Myc lymphoma cells transduced with the indicated shRNA constructs as described in (A) and treated as in (B) (sh_Ctrl, black, n = 6; sh_XIAP, gray, n = 6). **, P = 0.0020; Mantel-Cox test.


D Histopathology of representative lymph nodes derived from mice shown in ( B ) to visualize histomorphology by haematoxylin / eosin ( H & amp ; amp ; E ) , B - origin ( B220 ) , spontaneous apoptosis ( Caspase 3 ) in situ . Scale bars denote 50 μm 20 μm for insets .

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D Histopathology of representative lymph nodes derived from mice shown in (B) to visualize histomorphology by haematoxylin/eosin (H&amp;E), B-cell origin (B220), and spontaneous apoptosis (cleaved Caspase 3) in situ. Scale bars denote 50 μm and 20 μm for insets.


E Histopathology of representative lymph nodes derived from mice shown in ( C ) which were stained as described in ( D ) .

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E Histopathology of representative lymph nodes derived from mice shown in (C) which were stained as described in (D).


F Mean burden in mice injected with Eμ - Myc lymphoma modified as in ( B ) ( C ) , 15 days after injection , assessed by PET imaging ( 4 per group ) . * * , P = 0 . 0025 ; * * * , P = 0 . 0003 , student ' s t -

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F Mean tumor burden in mice injected with Eμ-Myc lymphoma cells modified as in (B) and (C), 15 days after injection, assessed by PET imaging (4 animals per group). **, P = 0.0025; ***, P = 0.0003, student's t-test


G Representative PET of , injected with Eμ - Myc lymphoma modified as in ( B ) ( C ) , 15 days after injection ( pre treatment ) three days following intraperitoneal vincristine administration ( post treatment ) . Bone marrow infiltration , representative for systemic lymphoma manifestation , is indicated by a blue asterisk .

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G Representative PET images of animals, injected with Eμ-Myc lymphoma cells modified as in (B) and (C), 15 days after injection (pre treatment) and three days following intraperitoneal vincristine administration (post treatment). Bone marrow infiltration, representative for systemic lymphoma manifestation, is indicated by a blue asterisk.


H Mean change in standardized 18FDG uptake of ROI between pre post treatment imaging corrected for initial burden ( sh _ Ctrl , black , n = 6 ; sh _ Usp9X , , n = 8 ; sh _ Xiap , grey , n = 8 ) . Within sh _ Usp9X sh _ Xiap groups lived up to control imaging , while two of eight of sh _ ctrl group had to be sacrificed prematurely because they had reached predefined criteria of maximum burden . These had to be excluded from final , suggesting an underestimation of actual effect . Data was obtained from two independent experiments . * * * * , P < 0 . 0001 ; * , P = 0 . 0167 , student ' s t -

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H Mean change in standardized 18FDG uptake of ROI between pre and post treatment imaging corrected for initial tumor burden (sh_Ctrl, black, n=6; sh_Usp9X, white, n=8; sh_Xiap, grey, n=8). Within the sh_Usp9X and sh_Xiap groups all animals lived up to control imaging, while two of eight animals of the sh_ctrl group had to be sacrificed prematurely because they had reached the predefined criteria of maximum tumor burden. These animals had to be excluded from final analysis, suggesting an underestimation of the actual effect. Data was obtained from two independent experiments. ****, P < 0.0001; *, P = 0.0167, student's t-test


A Immunoblot analyses of different human DLBCL lines using antibodies to indicated .

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A Immunoblot analyses of different human DLBCL lines using antibodies to the indicated proteins.


B FACS ( propidium iodide ( PI ) ) of USP9X high low expressing DLBCL shown in ( A ) to determine survival . were treated with taxol collected at specified time points . PI negative are indicated relative to time point 0 ( n = 3 ± SD ) .

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B FACS analysis (propidium iodide (PI)) of USP9X high and low expressing DLBCL cells shown in (A) to determine cell survival. Cells were treated with taxol and collected at the specified time points. PI negative cells are indicated relative to time point 0 (n=3 ± SD).


C Immunoblot analyses of indicated DLBCL lines that were lentivirally transduced with IRES - GFP shRNA constructs against USP9X or a non - relevant mRNA , FACS sorted for GFP + PI - exposed to taxol for indicated periods of time . of caspase 3 ( CC3 ) at 24 hr post taxol treatment timepoint from four independent experiments is quantified in graph on right side normalized to control shRNA . * * , P = 0 . 0099 , student ' s t -

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C Immunoblot analyses of the indicated DLBCL cell lines that were lentivirally transduced with IRES-GFP shRNA constructs against USP9X or a non-relevant mRNA, FACS sorted for GFP+ PI- cells and exposed to taxol for the indicated periods of time. The quantification of cleaved caspase 3 (CC3) at the 24 hr post taxol treatment timepoint from four independent experiments is quantified in the graph on the right side and normalized to control shRNA. **, P = 0.0099, student's t-test


D Immunoblot analyses of indicated DLBCL lines that were exposed to taxol for indicated periods of time . Three hours before collecting , WP1130 at a of 5μM or DMSO was added as specified .

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D Immunoblot analyses of the indicated DLBCL cell lines that were exposed to taxol for the indicated periods of time. Three hours before collecting, WP1130 at a concentration of 5μM or DMSO was added as specified.


E FACS ( propidium iodide ( PI ) uptake ) of DLBCL lines treated with taxol / or SMAC mimetic BV6 as indicated . Results displayed are from three independent experiments ( n = 3 ± SD ) . * * * , P = 0 . 00085 ; * * * , P = 0 . 00529 ; Student ' s t .

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E FACS analysis (propidium iodide (PI) uptake) of DLBCL cell lines treated with taxol and/or the SMAC mimetic BV6 as indicated. Results displayed are from three independent experiments (n=3 ± SD). ***, P = 0.00085; ***, P = 0.00529; Student's t test.


F Collective assessment of USP9X XIAP expression determined by immunohistochemistry in tissue microarrays ( TMA ) derived from a cohort representing 121 evaluable human CD20 + aggressive B - lymphomas . High USP9X expression was identified in 37 cases ( 31 % ) significantly correlated with high XIAP expression ( * * , P = 0 . 004 ; Student ' s t ) .

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F Collective assessment of USP9X and XIAP expression determined by immunohistochemistry in tissue microarrays (TMA) derived from a patient cohort representing 121 evaluable human CD20+ aggressive B-cell lymphomas. High USP9X expression was identified in 37 cases (31 %) and significantly correlated with high XIAP expression levels (**, P = 0.004; Student's t test).


G Expression data derived from ( E ) was correlated with clinical follow - up . Within a cohort of 58 receiving vincristine - containing chemotherapy ( CHOP regimen : cyclophosphamide , doxorubicin , vincristine , prednisone ) without addition of anti - CD20 antibody rituximab ( left panel ) overexpression of USP9X XIAP ( n = 14 , straight ) was associated with a significantly shortened event - free survival ( EFS ) as compared to remaining ( n = 44 , dotted ) ( * , P = 0 . 050 ; log rank ) . Within a cohort of 63 receiving CHOP regimen with addition of anti - CD20 antibody rituximab overexpression of USP9X XIAP ( n = 14 , straight ) was not associated with a shortened event - free survival ( EFS ) as compared to remaining ( n = 49 , dotted ) ( P = 0 . 454 ) .

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G Expression data derived from (E) was correlated with clinical follow-up. Within a cohort of 58 patients receiving vincristine-containing chemotherapy (CHOP regimen: cyclophosphamide, doxorubicin, vincristine, prednisone) without the addition of the anti-CD20 antibody rituximab (left panel) overexpression of USP9X and XIAP (n = 14, straight line) was associated with a significantly shortened event-free survival (EFS) as compared to all remaining patients (n = 44, dotted line) (*, P = 0.050; log rank test). Within a cohort of 63 patients receiving the CHOP regimen with addition of the anti-CD20 antibody rituximab overexpression of USP9X and XIAP (n = 14, straight line) was not associated with a shortened event-free survival (EFS) as compared to all remaining patients (n = 49, dotted line) (P = 0.454).


molecular detection of nCoV - 2019 in seven during two times of sampling . information can be found in Extended Data Table 1 2 . Details on detection method can be found in material methods . BALF , bronchoalveolar lavage fluid ; OS , oral swab ; AS , anal swab .

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molecular detection of nCoV-2019 in seven patients during two times of sampling. Patient information can be found in Extended Data Table 1 and 2. Details on detection method can be found in material and methods. BALF, bronchoalveolar lavage fluid; OS, oral swab; AS, anal swab.


dynamics of nCoV - 2019 antibodies in one who showed sign of disease on 2019 . 12 . 23 ( ICU - 06 ) . For b c , cut - off was set up as 0 . 2 for IgM 0 . 3 for IgG , according to healthy controls .

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dynamics of nCoV-2019 antibodies in one patient who showed sign of disease on 2019.12.23 (ICU-06). For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.


serological of nCoV - 2019 antibodies in five ( more information can be found in Extended Data Table 2 ) . Star indicates data collected from ICU - 06 on 2020 . 01 . 10 . For b c , cut - off was set up as 0 . 2 for IgM 0 . 3 for IgG , according to healthy controls .

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serological test of nCoV-2019 antibodies in five patients (more information can be found in Extended Data Table 2). Star indicates data collected from patient ICU-06 on 2020.01.10. For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.


viral particles in ultrathin sections under electron microscope at 200 kV , sample from viral infected Vero E6

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viral particles in the ultrathin sections under electron microscope at 200 kV, sample from viral infected Vero E6 cells


usage . Determination of infectivity in HeLa with or without expression of ACE2 . h , human ; b , Rhinolophus sinicus bat ; c , civet ; s , swine ( pig ) ; m , mouse . ACE2 ( green ) , viral ( red ) nuclei ( blue ) was shown . Scale bar = 10 um .

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usage.Determination of virus infectivity in HeLa cells with or without the expression of ACE2. h, human; b, Rhinolophus sinicus bat; c, civet; s, swine (pig); m, mouse. ACE2 protein (green), viral protein (red) and nuclei (blue) was shown. Scale bar=10 um.


A . RT - PCR of expression of β - actin , Trpv1 , Trpv2 , Trpv3 Trpv4 using mouse differentiated brown adipocytes after 29 ( upper ) 35 ( lower ) thermal cycles with ( RT ( + ) ) without ( RT ( - ) ) reverse transcription ( RT ) . Control ( Ct . ) lanes indicate results with each plasmid as a template .

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A. RT-PCR analysis of the expression of β-actin, Trpv1, Trpv2, Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 (upper) and 35 (lower) thermal cycles with (RT (+)) and without (RT (-)) reverse transcription (RT). Control (Ct.) lanes indicate the results with each plasmid DNA as a template.


B . Results of real - time RT - PCR of Trpv1 , Trpv2 , Trpv3 Trpv4 expression using mouse differentiated brown adipocytes . Expression of mRNA were normalized to that of ribosomal gene ( 36B4 ) , a housekeeping gene un - affected by adipogenesis . Data are presented as mean ± , n = 6 .

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B. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes. Expression levels of mRNA were normalized to that of the ribosomal protein gene (36B4), a housekeeping gene un-affected by adipogenesis. Data are presented as mean ± SEM, n = 6.


C . Results of real - time PCR of Trpv1 , Trpv2 , Trpv3 Trpv4 expression using mouse interscapular brown adipose tissue ( iBAT ) . mRNA expression were normalized to that of 36B4 . Data are presented as mean ± , n = 5 .

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C. Results of real-time PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue (iBAT). mRNA expression levels were normalized to that of 36B4. Data are presented as mean ± SEM, n = 5.


D . Western blot results of TRPV2 tubulin from WT TRPV2KO brown adipocytes . Upper bands in TRPV2 blots likely indicate glycosylated forms .

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D. Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes. Upper bands in the TRPV2 blots likely indicate glycosylated forms.


E . Averaged traces of [ Ca2 + ] i changes in response to 500 µmol / L 2APB in differentiated brown adipocytes from WT ( black ) TRPV2KO ( red ) mice . One µmol / L NE was used to confirm differentiation . Five µmol / L ionomycin was used to confirm viability . values correspond to real [ Ca2 + ] i of differentiated mousebrown adipocytes . Data are presented as mean ± , n = 149 of WT , n = 112 of TRPV2KO brown adipocytes ; * * P < 0 . 01 . Unpaired Student ' s t - .

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E. Averaged traces of [Ca2+]i changes in response to 500 µmol/L 2APB in differentiated brown adipocytes from WT (black) and TRPV2KO (red) mice. One µmol/L NE was used to confirm differentiation. Five µmol/L ionomycin was used to confirm cell viability. Ratio values correspond to the real [Ca2+]i of differentiated mousebrown adipocytes. Data are presented as mean ± SEM, n = 149 of WT cells, and n = 112 of TRPV2KO brown adipocytes; ** P < 0.01. Unpaired Student's t-test.


F . A representative trace of whole - current activated by 3 mmol / L of 2APB in presence or absence of 10 µmol / L SKF in mouse differentiated brown adipocyte . left inset indicates a voltage ramp - protocol . right inset indicates current - voltage curves of basal current ( a , black ) , current in presence of 2APB + SKF ( b , blue ) current in presence of 2APB alone ( c , red ) at time points of a , b c , respectively . G . Comparison of mean densities of basal currents ( Base , black ) , currents in presence of 2APB alone ( red ) currents in presence of 2APB + SKF ( blue ) at - 60 mV + 100 mV in mouse differentiated brown adipocytes . Data are presented as mean ± , n = 10 ; * * P < 0 . 01 vs . Base ; # # P < 0 . 01 vs . 2APB alone . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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F. A representative trace of whole-cell current activated by 3 mmol/L of 2APB in the presence or absence of 10 µmol/L SKF in mouse differentiated brown adipocyte. The left inset indicates a voltage ramp-pulse protocol. The right inset indicates the current-voltage curves of basal current (a, black), current in the presence of 2APB+SKF (b, blue) and current in the presence of 2APB alone (c, red) at the time points of a, b and c, respectively.G. Comparison of the mean densities of basal currents (Base, black), currents in the presence of 2APB alone (red) and currents in the presence of 2APB+SKF (blue) at -60 mV and +100 mV in mouse differentiated brown adipocytes. Data are presented as mean ± SEM, n = 10; ** P < 0.01 vs. Base; ##P < 0.01 vs. 2APB alone. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Results of real - time RT - PCR of Trpv1 , Trpv2 , Trpv3 Trpv4 expression in mouse pre - adipocytes differentiated brown adipocytes . Expression of mRNA were normalized to those of 36B4 . Data are presented as mean ± , n = 6 . * P < 0 . 05 vs . pre - adipocytes . Unpaired Student ' s t - .

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A. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression in mouse pre-adipocytes and differentiated brown adipocytes. Expression levels of mRNA were normalized to those of 36B4. Data are presented as mean ± SEM, n = 6. * P < 0.05 vs. pre-adipocytes. Unpaired Student's t-test.


B . number of 6 day - differentiated mousebrown adipocytes treated with TRPV2 agonists , 100 µmol / L 2APB or 10 µmol / L LPC . Mean ± , n = 6 , * P < 0 . 05 * * P < 0 . 01 vs . dimethyl sulfoxide ( DMSO ) group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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B. The number of 6 day-differentiated mousebrown adipocytes treated with TRPV2 agonists, 100 µmol/L 2APB or 10 µmol/L LPC. Mean ± SEM, n = 6, * P < 0.05 and ** P < 0.01 vs. dimethyl sulfoxide (DMSO) group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


C D . Comparison of numbers of 6 day - differentiated mouse brown adipocytes ( C ) triglyceride ( D ) in from WT TRPV2KO mice with different differentiation media . 1 / 10 suppl . indicates differentiation medium ten - time - diluted with DMEM . Mean ± , n = 8 , * * P < 0 . 01 vs . control group , # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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C and D. Comparison of the numbers of 6 day-differentiated mouse brown adipocytes (C) and triglyceride levels (D) in the cells from WT and TRPV2KO mice with different differentiation media. 1/10 suppl. indicates differentiation medium ten-time-diluted with DMEM. Mean ± SEM, n = 8, ** P < 0.01 vs. control group, ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Basal mRNA expression of Ucp1 , peroxisome proliferator - activated receptor gamma coactivator 1 - alpha ( Pgc1α ) , peroxisome proliferator - activated receptorγ ( Pparγ ) β3 - adrenergic receptor 3 ( Adrb3 ) in differentiated brown adipocytes from WT TRPV2KO mice . Data are presented as mean ± , n = 5 ; * * P < 0 . 01 vs . WT . Unpaired Student ' s t - .

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A. Basal mRNA expression of Ucp1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α), peroxisome proliferator-activated receptorγ(Pparγ) and β3-adrenergic receptor 3 (Adrb3) in the differentiated brown adipocytes from WT and TRPV2KO mice. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. WT. Unpaired Student's t-test.


B C . Changes in Ucp1 ( B ) Pgc1α ( C ) mRNA expression in differentiated brown adipocytes from WT TRPV2KO mice with without 10 µmol / L isoproterenol ( ISO ) for 4 h . Data are presented as mean ± , n = 5 ; * * P < 0 . 01 vs . DMSO group ; # P < 0 . 05 ; # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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B and C. Changes in Ucp1 (B) and Pgc1α(C) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol/L isoproterenol (ISO) for 4 h. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. DMSO group; # P < 0.05; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


D E . Changes in Ucp1 mRNA expression ( D ) non - esterified fatty acid ( NEFA ) release ( E ) in differentiated brown adipocytes from WT TRPV2KO mice with or without 10 µmol / L forskolin for 4 h . Data are presented as mean ± , n = 6 ; * P < 0 . 05 ; * * P < 0 . 01 vs . DMSO group ; # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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D and E. Changes in Ucp1 mRNA expression (D) and non-esterified fatty acid (NEFA) release (E) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol/L forskolin for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


F G . Changes in Ucp1 mRNA ( F ) Pgc1α mRNA ( G ) in differentiated brown adipocytes treated with 10 µmol / L ISO alone or 10 µmol / L ISO plus 10 µmol / L BAPTA - AM for 4 h . Data are presented as mean ± , n = 6 ; * P < 0 . 05 ; * * P < 0 . 01 vs . DMSO group ; # # P < 0 . 01 vs . ISO group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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F and G. Changes in Ucp1 mRNA (F) and Pgc1α mRNA (G) in the differentiated brown adipocytes treated with 10 µmol/L ISO alone or 10 µmol/L ISO plus 10 µmol/L BAPTA-AM for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. ISO group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . changes from 3 - to 8 - of between two genotypes ( n = 11 ) . Data are presented as mean ± ; * P < 0 . 05 ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - .

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A. Body weight changes from 3-weeks to 8-weeks of age between the two genotypes (n = 11). Data are presented as mean ± SEM; * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


B C . Changes in food intake ( B ) water intake ( C ) from 3 - to 8 - of ages between WT TRPV2KO mice ( n = 6 ) .

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B and C. Changes in food intake (B) and water intake (C) from 3-weeks to 8-weeks of ages between WT and TRPV2KO mice (n = 6).


D . Weights of tissues related to energy metabolism normalized to their weights in WT TRPV2KO mice ( n = 15 ) .

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D. Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice (n = 15).


E . expression of mRNA related to energy metabolism in iBAT from WT TRPV2KO mice . mRNA expression of Ucp1 , Pgc1a , Ppary , Adrb3 , cytochrome c oxidase subunit 4 isoform 1 ( Cox4i1 ) , PR domain containing 16 ( Prdm16 ) , Ucp2 , lipoprotein lipase ( Lpl ) cluster of differentiation 36 ( Cd36 ) were examined normalized to of 36B4 . Data are presented as mean ± , n = 15 ; * P < 0 . 05 vs . WT group ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - .

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E. The expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice. mRNA expression levels of Ucp1, Pgc1a, Ppary, Adrb3, cytochrome c oxidase subunit 4 isoform 1 (Cox4i1), PR domain containing 16 (Prdm16), Ucp2, lipoprotein lipase (Lpl) and cluster of differentiation 36 (Cd36) were examined and normalized to the levels of 36B4. Data are presented as mean ± SEM, n = 15; * P < 0.05 vs. WT group; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


A . Representative morphological results of iBAT by hematoxylin eosin from 8 - week WT TRPV2KO mice . Scale bars indicate 100 μm . B . Comparison of lipid droplet histograms from 8 - week - old WT ( black ) TRPV2KO ( red ) iBAT with hematoxylin eosin . Mean values are 9 . 5 ± 0 . 2 µm in 8 - week - old WT adipocytes , 14 . 7 ± 0 . 2 µm in 8 - week - old TRPV2KO adipocytes ( P < 0 . 01 ) . 1000 to 1400 droplets were counted . Data are presented as mean ± , n = 4 . C . Comparison of adipocyte histograms from 8 - week - old WT ( black ) TRPV2KO ( red ) iBAT with hematoxylin eosin . 800 to 1000 were counted . Data are presented as mean ± , n = 4 . D . Mean diameters of adipocytes in 8 - week - old WT mice ( black ) TRPV2KO mice ( red ) . Mean values are 15 . 8 ± 0 . 4 µm in 8 - week - old WT adipocytes , 19 . 8 ± 0 . 3 µm in 8 - week - old TRPV2KO adipocytes . Data are presented as mean ± , n = 4 ; * P < 0 . 05 vs . WT group . Unpaired Student ' s t - .

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A. Representative morphological results of iBAT by hematoxylin and eosin staining from 8-week WT and TRPV2KO mice. Scale bars indicate 100 μm.B. Comparison of lipid droplet size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. Mean values are 9.5 ± 0.2 µm in 8-week-old WT adipocytes, and 14.7 ± 0.2 µm in 8-week-old TRPV2KO adipocytes (P < 0.01). 1000 to 1400 droplets were counted. Data are presented as mean ± SEM, n = 4.C. Comparison of adipocyte size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. 800 to 1000 cells were counted. Data are presented as mean ± SEM, n = 4.D. Mean diameters of adipocytes in 8-week-old WT mice (black) and TRPV2KO mice (red). Mean values are 15.8 ± 0.4 µm in 8-week-old WT adipocytes, and 19.8 ± 0.3 µm in 8-week-old TRPV2KO adipocytes. Data are presented as mean ± SEM, n = 4; * P < 0.05 vs. WT group. Unpaired Student's t-test.


A , B , C D . Changes in mRNA expression of Ucp1 ( A ) , Pgc1α ( B ) , Pparγ ( C ) Trpv2 ( D ) in iBAT of WT TRPV2KO mice before ( 25oC ) , after 1 day 3 days cold ( 4oC ) exposure . Mean ± , n = 5 - 8 ; * P < 0 . 05 ; * * P < 0 . 01 vs . 25oC group ; # P < 0 . 05 vs . WT group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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A, B, C and D. Changes in mRNA expression of Ucp1 (A), Pgc1α (B), Pparγ (C) and Trpv2 (D) in iBAT of WT and TRPV2KO mice before (25oC), after 1 day and 3 days cold (4oC) exposure. Mean ± SEM, n = 5 - 8; * P < 0.05; ** P < 0.01 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


E F . Western blot results of UCP1 in iBAT from WT TRPV2KO mice before ( 25oC ) after 3 days cold ( 4oC ) exposure ( E ) . Comparison of UCP1 before after cold exposure in iBAT from WT TRPV2KO mice ( F ) . Mean ± , n = 6 ; * P < 0 . 05 vs . 25oC group ; # P < 0 . 05 vs . WT group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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E and F. Western blot results of UCP1 protein in iBAT from WT and TRPV2KO mice before (25oC) and after 3 days cold (4oC) exposure (E). Comparison of UCP1 protein levels before and after cold exposure in iBAT from WT and TRPV2KO mice (F). Mean ± SEM, n = 6; * P < 0.05 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


G H . Averages of temperatures ( G ) activities ( H ) of WT TRPV2KO mice with or without cold ( 4oC ) exposure . Data are presented as mean ± , n = 5 ; * P < 0 . 05 vs . WT group . Unpaired Student ' s t - .

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G and H. Averages of core body temperatures (G) and activities (H) of WT and TRPV2KO mice with or without cold (4oC) exposure. Data are presented as mean ± SEM, n = 5; * P < 0.05 vs. WT group. Unpaired Student's t-test.


A , B C . Changes in mRNA expression of Ucp1 ( A ) , Pgc1α ( B ) Pparγ ( C ) from WT TRPV2KO iBAT 4 h after intraperitoneal administration ( i . p . , arrows ) of saline or a selective β3 - adrenergic receptor agonist , BRL37344 ( 600 µg / kg ) . Mean ± , n = 5 - 7 ; * P < 0 . 05 vs . saline group ; # P < 0 . 01 vs . WT BRL37344 administration group . One - way ANOVA followed by 2 - t - with Bonferroni correction .

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A, B and C. Changes in mRNA expression of Ucp1 (A), Pgc1α (B) and Pparγ(C) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration (i.p., arrows) of saline or a selective β3-adrenergic receptor agonist, BRL37344 (600 µg/kg body weight). Mean ± SEM, n = 5 - 7; * P < 0.05 vs. saline group; # P < 0.01 vs. WT BRL37344 administration group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


D E . Average traces of changes in iBAT ( D ) or rectal ( E ) from WT TRPV2KO mice after BRL37344 ( 600 µg / kg ) administration ( i . p . , arrows ) . Mean ± , n = 5 . * P < 0 . 05 vs . WT group . Unpaired Student ' s t - .

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D and E. Average traces of changes in iBAT temperature (D) or rectal body temperature (E) from WT and TRPV2KO mice after BRL37344 (600 µg/kg body weight) administration (i.p., arrows). Mean ± SEM, n = 5. * P < 0.05 vs. WT group. Unpaired Student's t-test.


F G . iBAT NE turnover rates ( F ) rate constants k ( G ) in WT TRPV2KO mice upon cold exposure ( 4oC ) for 4 h . Data are presented as mean ± , n = 8 .

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F and G. iBAT NE turnover rates (F) and rate constants k (G) in WT and TRPV2KO mice upon cold exposure (4oC) for 4 h. Data are presented as mean ± SEM, n = 8.


A . changes between WT TRPV2KO mice treated with high fat diet ( HFD ) for continuous 8 from 13 - week - old .

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A. Body weight changes between WT and TRPV2KO mice treated with high fat diet (HFD) for continuous 8 weeks from 13-week-old.


B C . Blood glucose level ( B ) plasma insulin level ( C ) of WT TRPV2KO mice after 8 - week HFD treatment .

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B and C. Blood glucose level (B) and plasma insulin level (C) of WT and TRPV2KO mice after 8-week HFD treatment.


D . Weights of tissues related to energy metabolism in WT TRPV2KO mice after 8 - week HFD treatment .

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D. Weights of tissues related to energy metabolism in WT and TRPV2KO mice after 8-week HFD treatment.


E . mRNA expression of Ucp1 Pgc1α in iBAT from WT TRPV2KO mice after 8 - week HFD treatment . data are presented as mean ± , WT mice ( n = 6 ) , TRPV2KO mice ( n = 8 ) ; * P < 0 . 05 ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - .

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E. mRNA expression of Ucp1 and Pgc1α in iBAT from WT and TRPV2KO mice after 8-week HFD treatment. All data are presented as mean ± SEM, WT mice (n = 6), TRPV2KO mice (n = 8); * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


B of anti - Bax anti - Bak antibody specificity using whole lysates from HCT116 wild - type , Bax KO , Bak KO Bax / Bak DKO . After detection of Bax was redecorated with anti - Bak antibodies ( Bax / Bak ) . Actin is used as loading control .

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B Analysis of anti-Bax and anti-Bak antibody specificity using whole cell lysates from HCT116 wild-type, Bax KO, Bak KO and Bax/Bak DKO. After detection of Bax the membrane was redecorated with anti-Bak antibodies (Bax/Bak). Actin is used as loading control.


C Western blot of Bak in heavy ( HM ) fraction of human heart , cerebral cortex ( CNS GM ) , kidney , brain matter ( CNS WM ) , lung , liver , skeletal muscle tissue . cytosol fraction obtained from CNS cortex ( C ) , LDH , Tom20 serve as fractionation loading controls . n = 3 .

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C Western blot analysis of Bak in the heavy membrane (HM) fraction of human heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue. The cytosol fraction obtained from CNS cortex (C), LDH, and Tom20 serve as fractionation and loading controls. n = 3.


D Presence of Bak in cytosol of heart , cerebral cortex ( CNS GM ) , kidney , brain matter ( CNS WM ) , lung , liver , skeletal muscle tissue analyzed by Western blot in parallel to C . heavy heart fraction ( HM ) , LDH Tom20 serve as fractionation loading controls . n = 3 .

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D Presence of Bak in the cytosol of heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue analyzed by Western blot in parallel to C. The heavy membrane heart fraction (HM), LDH and Tom20 serve as fractionation and loading controls. n = 3.


E Carbonate extraction of heavy fraction of human brain matter ( CNS WM ) lung tissue analyzed for presence of Bak by Western blot . - associated supernatant ( S ) - integral pellet ( P ) are displayed . Smac is released from mitochondrial inter space ( IMS ) during carbonate extraction CoxIV remains in pellet . n = 2 .

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E Carbonate extraction of the heavy membrane protein fraction of human brain white matter (CNS WM) and lung tissue analyzed for the presence of Bak by Western blot. Membrane-associated protein supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the mitochondrial inter membrane space (IMS) during carbonate extraction and CoxIV remains in the pellet. n = 2.


A Confocal of HCT116 Bax / Bak DKO transfected with GFP - Bak in absence ( left ) or presence ( right ) of Bcl - xL overexpression . Scale bar 15 µm . n ≥ 10 .

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A Confocal images of HCT116 Bax/Bak DKO cells transfected with GFP-Bak in the absence (left) or the presence (right) of Bcl-xL overexpression. Scale bar 15 µm. n ≥ 10.


B FLIP ( Loss in Photobleaching ) of mitochondrial GFP - Bak in absence ( ● , straight ) or presence ( ○ , broken ) of overexpressed Bcl - xL . of neighboring ( in Fig . 2C ) is shown as control ( , dotted ) . Data represent averages ± from 16 ( - Bcl - xL ) 40 ( + Bcl - xL ) ROI measurements .

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B FLIP (Fluorescence Loss in Photobleaching) of mitochondrial GFP-Bak in the absence (●, straight line) or the presence (○, broken line) of overexpressed Bcl-xL. Fluorescence of the neighboring cell (in Fig. 2C) is shown as control (, dotted line). Data represent averages ± SEM from 16 (-Bcl-xL) and 40 (+Bcl-xL) ROI measurements.


C FLIP of GFP - Bak in absence ( top ) or presence ( bottom ) of overexpressed Bcl - xL diminishes GFP - Bak in cytosol of targeted ( circled ) completely after 90 s GFP is detected only on mitochondria ( arrows ) . During FLIP measurements mitochondrial GFP - Bak is monitored , while cytosol is bleached repeatedly . Time points in seconds are displayed above .

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C FLIP of GFP-Bak in the absence (top) or the presence (bottom) of overexpressed Bcl-xL diminishes GFP-Bak fluorescence in the cytosol of targeted cells (circled) completely after 90 s and GFP fluorescence is detected only on the mitochondria (arrows). During FLIP measurements mitochondrial GFP-Bak fluorescence is monitored, while the cytosol is bleached repeatedly. Time points in seconds are displayed above the images.


A Western blot of GFP - Bak localization in cytosol ( C ) heavy fraction ( HM ) of HCT116 Bax / Bak DKO with or without overexpressed wild - type Bcl - xL or Bcl - xL G138A . GAPDH Tom20 serve as fractionation controls . n = 10 . Bak shuttling by Bcl - xL is independent of GFP fusion ( Fig . E2a ) . B of predominant mitochondrial localization ( grey bars ) mixed mitochondrial localization ( grey bars ) of Bak in HCT116 Bax / Bak DKO expressing GFP - Bak with or without Bcl - xL overexpression . Data represent averages ± SD from 3 independent experiments with n ≥ 100 .

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A Western blot analysis of GFP-Bak localization in cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells with or without overexpressed wild-type Bcl-xL or Bcl-xL G138A. GAPDH and Tom20 serve as fractionation controls. n = 10. Bak shuttling by Bcl-xL is independent of the GFP fusion (Fig. E2a).B Quantification of predominant mitochondrial localization (light grey bars) and mixed cytosolic and mitochondrial localization (dark grey bars) of Bak in HCT116 Bax/Bak DKO cells expressing GFP-Bak with or without Bcl-xL overexpression. Data represent averages ± SD from 3 independent experiments with n ≥ 100.


C Retrotranslocation rates measured for wild - type Bak or Bak D83R in presence or absence of Bcl - 2 , Mcl - 1 , wild - type Bcl - xL or Bcl - xL G138A . Data represent averages ± SD .

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C Retrotranslocation rates measured for wild-type Bak or Bak D83R in the presence or the absence of Bcl-2, Mcl-1, wild-type Bcl-xL or Bcl-xL G138A. Data represent averages ± SD.


D of endogenous Bak localization in cytosol ( C ) heavy fraction ( HM ) of HeLa expressing degradation - prone DD - FLAG - Bcl - xL . In absence of Shield - 1 DD - FLAG - Bcl - xL is readily degraded , resulting in unaltered Bcl - xL . Addition of 0 . 5 µM Shield - 1 instantly stabilizes DD - FLAG - Bcl - xL resulting in elevated Bcl - xL that increase of endogenous Bak ( Fig . E3C , D ) . Akt1 Tom20 serve as fractionation controls . n = 3 .

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D Analysis of endogenous Bak localization in cytosol (C) and heavy membrane fraction (HM) of HeLa cells expressing degradation-prone DD-FLAG-Bcl-xL. In the absence of Shield-1 DD-FLAG-Bcl-xL is readily degraded, resulting in unaltered Bcl-xL levels. Addition of 0.5 µM Shield-1 instantly stabilizes DD-FLAG-Bcl-xL resulting in elevated Bcl-xL levels that increase cytosolic levels of endogenous Bak (Fig. E3C, D). Akt1 and Tom20 serve as fractionation controls. n = 3.


B of HCT116 Bax / Bak DKO expressing Bax , BaxTBak , BakTBax or Bak with expressed being present largely ( bars ) , in a mixed distribution between cytosol mitochondria ( grey bars ) or largely mitochondrial ( black bars ) . Data represent averages ± from 7 independent experiments with n ≥ 100 .

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B Quantification of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak with the expressed protein being present largely cytosolic (white bars), in a mixed distribution between cytosol and mitochondria (grey bars) or largely mitochondrial (black bars). Data represent averages ± SEM from 7 independent experiments with n ≥ 100 cells.


C Confocal of HCT116 Bax / Bak DKO expressing Bax , BaxTBak , BakTBax or Bak . GFP / YFP of expressed variants is depicted in top panels in green in merged image on bottom . mitochondria were stained by MitoTracker far red depicted in red in merged ( bottom row ) . Scale 10 µm . n ≥ 5 .

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C Confocal images of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak. The GFP/YFP fluorescence of the expressed protein variants is depicted in the top panels and in green in the merged image on the bottom. The mitochondria were stained by MitoTracker far red depicted in red in the merged images (bottom row). Scale 10 µm. n ≥ 5.


D Western blot of Bax , BaxTBak , Bak BakTBax localization expressed in HCT116 Bax / Bak DKO . Cytosol ( C ) heavy fraction ( HM ) of HCT116 Bax / Bak DKO are displayed . GAPDH VDAC serve as fractionation controls . n = 3 .

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D Western blot analysis of Bax, BaxTBak, Bak and BakTBax localization expressed in HCT116 Bax/Bak DKO cells. Cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells are displayed. GAPDH and VDAC serve as fractionation controls. n = 3.


A Caspase 3 / 7 activity measured in HCT116 Bax / Bak DKO overexpressing Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression in absence of apoptosis stimuli . Caspase activity is displayed in relative units ( RFU ) . pcDNA3 . 1 - transfected served as control . Data represent averages ± . n ≥ 3 . p - values according to One Way ANOVA are displayed . B Staurosporine ( STS , 1 µM ) - induced caspase 3 / 7 activity of Bax / Bak DKO overexpressing Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression displayed in relative units ( RFU ) . Data represent averages ± . n ≥ 3 . p - values according to One Way ANOVA . BaxTBak activities with without Bcl - xL expression revealed no significant difference ( n . s . ) , while in presence of Bcl - xL overexpression BaxTBak activity is significantly higher than Bax , Bak or BakTBax activities ( p < 0 . 001 ) .

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A Caspase 3/7 activity measured in HCT116 Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression in the absence of apoptosis stimuli. Caspase activity is displayed in relative fluorescence units (RFU). pcDNA3.1-transfected cells served as control. Data represent averages ± SEM. n ≥ 3. p-values according to One Way ANOVA are displayed.B Staurosporine (STS, 1 µM)-induced caspase 3/7 activity of Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression displayed in relative fluorescence units (RFU). Data represent averages ± SEM. n ≥ 3. p-values according to One Way ANOVA. BaxTBak activities with and without Bcl-xL expression revealed no significant difference (n.s.), while in the presence of Bcl-xL overexpression BaxTBak activity is significantly higher than Bax, Bak or BakTBax activities (p < 0.001).


C of active Bax conformation in HCT116 Bax / Bak DKO expressing wild - type Bax or BaxTBak with ( grey bars ) or without Bcl - xL overexpression ( grey bars ) by monoclonal antibody 6A7 ( Sigma ) detecting active Bax fold by imaging . were analyzed prior or after treatment with 1 µM STS in presence of pan - caspase inhibitor qVD . Data are represented as % of expressing population ± . n = 4 .

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C Analysis of the active Bax conformation in HCT116 Bax/Bak DKO cells expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL overexpression (light grey bars) by the monoclonal antibody 6A7 (Sigma) detecting the active Bax protein fold by fluorescence imaging. Cells were analyzed prior or after treatment with 1 µM STS in the presence of the pan-caspase inhibitor qVD. Data are represented as % of the expressing cell population ± SEM. n = 4.


D HCT116 Bax / Bak DKO ectopically expressing wild - type Bax or BaxTBak with ( grey bars ) or without Bcl - xL ( grey bars ) were analyzed in presence or absence of 1 µM STS qVD for retained mitochondrial cyt c . Data are represented as % of expressing population ± . n = 4 .

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D HCT116 Bax/Bak DKO cells ectopically expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL (light grey bars) were analyzed in the presence or the absence of 1 µM STS and qVD for retained mitochondrial cyt c. Data are represented as % of the expressing cell population ± SEM. n = 4.


E Flow cytometry of Annexin V - of Bax / Bak DKO expressing Bax , BaxTBak , Bak or BakTBax in absence ( red ) or presence of Bcl - xL overexpression ( black ) , following STS - treatment . of gated is displayed in color of corresponding graph . Data represent averages ± SD . n = 4 .

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E Flow cytometry analysis of Annexin V-staining of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in the absence (red line) or the presence of Bcl-xL overexpression (black line), following STS-treatment. The percentage of gated cells is displayed in the color of the corresponding graph. Data represent averages ± SD. n = 4.


F Colony formation of Bax / Bak DKO transfected with pcDNA , Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression . STS ( 1 µM ) was added for 24 hrs before were replated colonies were stained with methylene blue typically 14 days after treatment .

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F Colony formation of Bax/Bak DKO cells transfected with pcDNA, Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression. STS (1 µM) was added for 24 hrs before cells were replated and colonies were stained with methylene blue typically 14 days after treatment.


G of colony formation of Bax / Bak DKO expressing Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression after STS treatment ( Fig . 5F ) . Data represent averages ± . n = 4 . p - values according to One Way ANOVA . BaxTBak expressing with or without Bcl - xL expression showed no significant difference ( n . s . ) . H of colony formation of Bax / Bak DKO expressing Bax , BaxTBak , Bak or BakTBax in presence of Bcl - xL overexpression without apoptosis stimulation . Data represent averages ± . n = 5 . p - values according to One Way ANOVA .

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G Quantification of colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression after STS treatment (Fig. 5F). Data represent averages ± SEM. n = 4. p-values according to One Way ANOVA. BaxTBak expressing cells with or without Bcl-xL expression showed no significant difference (n.s.).H Quantification of the colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in presence of Bcl-xL overexpression without apoptosis stimulation. Data represent averages ± SEM. n = 5. p-values according to One Way ANOVA.


A FLIP of BaxTBakSS without ( ○ , broken ) or with ( ● , straight ) overexpressed Bcl - xL . of a neighboring is shown as control ( , dotted ) . Data represent averages ± from 20 ( - Bcl - xL ) 16 ( + Bcl - xL ) ROI measurements .

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A FLIP of BaxTBakSS without (○, broken line) or with (●, straight line) overexpressed Bcl-xL. Fluorescence of a neighboring cell is shown as control (, dotted line). Data represent averages ± SEM from 20 (-Bcl-xL) and 16 (+Bcl-xL) ROI measurements.


B Western blot of PARP cleavage in HCT116 Bax / Bak DKO overexpressing Bax , Bax S184V , BaxTBak or BaxTBak V197 / 198S . experiment was carried out in absence of apoptotic stimuli . transfected with pcDNA3 . 1 vector serve as control for PARP cleavage . Similar sample loading is controlled by actin . n = 4 .

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B Western blot analysis of PARP cleavage in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. The experiment was carried out in the absence of apoptotic stimuli. Cells transfected with pcDNA3.1 vector serve as control for PARP cleavage. Similar sample loading is controlled by actin. n = 4.


C Colony formation of Bax / Bak DKO transfected with pcDNA , BaxTBak or BaxTBakSS with Bcl - xL overexpression in absence of apoptotic stimuli . n = 3 .

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C Colony formation of Bax/Bak DKO cells transfected with pcDNA, BaxTBak or BaxTBakSS with Bcl-xL overexpression in the absence of apoptotic stimuli. n = 3.


D Caspase 3 / 7 activity was measured in HCT116 Bax / Bak DKO overexpressing Bax , Bax S184V , BaxTBak or BaxTBak V197 / 198S . Caspase activity is displayed in relative units ( RFU ) . experiment was carried out in absence of apoptotic stimuli pcDNA3 . 1 - transfected served as control . Data represent averages ± . n = 4 . p - values according to One Way ANOVA using Holm - Sidak method are displayed .

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D Caspase 3/7 activity was measured in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. Caspase activity is displayed in relative fluorescence units (RFU). The experiment was carried out in the absence of apoptotic stimuli and pcDNA3.1-transfected cells served as control. Data represent averages ± SEM. n = 4. p-values according to One Way ANOVA using the Holm-Sidak method are displayed.


F STS - induced caspase 3 / 7 activity of HCT116 Bax / Bak DKO expressing Bax , Bax S184V , BakTBax or BakTBax S184V is displayed normalized to Bax activity . Data represent averages ± . n ≥ 3 .

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F STS-induced caspase 3/7 activity of HCT116 Bax/Bak DKO cells expressing Bax, Bax S184V, BakTBax or BakTBax S184V is displayed normalized to Bax activity. Data represent averages ± SEM. n ≥ 3.


G Western blot of PARP cleavage after STS treatment in HCT116 Bax / Bak DKO overexpressing Bax , Bax S184V , BakTBax or BakTBax S184V . Empty vector - transfected serve as control for PARP cleavage actin is loading control . n = 3 .

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G Western blot analysis of PARP cleavage after STS treatment in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BakTBax or BakTBax S184V. Empty vector-transfected cells serve as control for PARP cleavage and actin is the loading control. n = 3.


H Plot of Bax ( ● ) Bak ( ○ ) retrotranslocation rates vs . hydrophobicity of MA of constructs . R2 = 0 . 95 .

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H Plot of Bax (●) and Bak (○) retrotranslocation rates vs. hydrophobicity of the MA of the constructs. R2 = 0.95.


A Carbonate extraction of HM fraction of Bax / Bak DKO expressing wild - type Bcl - xL in presence or absence of Bak or Bax analyzed by Western blot . Carbonate extractable supernatant ( S ) - integral pellet ( P ) are displayed . Smac is released from IMS during carbonate extraction VDAC remains in pellet . n = 4 . B Western blot of Bcl - xLTBax carbonate extraction from heavy fraction from Bax / Bak DKO expressing Bcl - xL variant with or without Bak or Bax . Smac VDAC serve as controls for supernatant ( S ) pellet ( P ) . n = 4 . C Carbonate extraction of HM fraction of Bax / Bak DKO expressing Bcl - xL G138A with or without Bak or Bax . Smac VDAC serve as controls for supernatant ( S ) pellet ( P ) . n = 4 .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD 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-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O

A Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing wild-type Bcl-xL in the presence or the absence of Bak or Bax analyzed by Western blot. Carbonate extractable supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the IMS during carbonate extraction and VDAC remains in the pellet. n = 4.B Western blot analysis of Bcl-xLTBax carbonate extraction from the heavy membrane fraction from Bax/Bak DKO cells expressing the Bcl-xL variant with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.C Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing Bcl-xL G138A with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.


D HM fraction carbonate extraction of Bax / Bak DKO expressing Bax , BaxTBak or Bax S184V . Smac VDAC serve as controls for supernatant ( S ) pellet ( P ) . n = 4 .

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D HM protein fraction and carbonate extraction of Bax/Bak DKO cells expressing Bax, BaxTBak or Bax S184V. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.


E Smac release from heavy fraction ( HM ) into cytosol ( C ) in HCT116 Bax / Bak DKO overexpressing Bax BaxTBak without apoptotic stimuli analyzed by Western blot . Bax BaxTBak were expressed differentially to produce similar of mitochondrial Bax . GAPDH Tom20 serve as fractionation controls . n = 3 .

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E Smac release from the heavy membrane fraction (HM) into the cytosol (C) in HCT116 Bax/Bak DKO cells overexpressing Bax and BaxTBak without apoptotic stimuli analyzed by Western blot. Bax and BaxTBak were expressed differentially to produce similar levels of mitochondrial Bax. GAPDH and Tom20 serve as fractionation controls. n = 3.


F PARP cleavage in HCT116 Bax / Bak DKO expressing similar of mitochondrial Bax or BaxTBak analyzed by Western blot without apoptotic stimuli . Actin is used as loading control . n = 3 .

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F PARP cleavage in HCT116 Bax/Bak DKO cells expressing similar levels of mitochondrial Bax or BaxTBak analyzed by Western blot without apoptotic stimuli. Actin is used as loading control. n = 3.


A Luciferase complementation imaging ( LCI ) assays showing that BIC1 interacts with BZR1 . nLUC - BZR1 was co - transformed with cLUC - CCA1 , cLUC - TOC1 , cLUC - ELF3 , cLUC - LUX , cLUC - BIC1 , cLUC , respectively , in leaf epidermal of Nicotiana benthamiana as indicated .

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A Luciferase complementation imaging (LCI) assays showing that BIC1 interacts with BZR1. nLUC-BZR1 was co-transformed with cLUC-CCA1, cLUC-TOC1, cLUC-ELF3, cLUC-LUX, cLUC-BIC1, cLUC, respectively, in leaf epidermal cells of Nicotiana benthamiana as indicated.


B BiFC assays showing interaction of BIC1 BZR1 mainly in nucleus . Leaf epidermal of Nicotiana benthamiana were co - transformed with nYFP - BZR1 cYFP - BIC1 . BF , bright field . Scale bars represent 20 μm .

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B BiFC assays showing the interaction of BIC1 and BZR1 mainly in nucleus. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nYFP-BZR1 and cYFP-BIC1. BF, bright field. Scale bars represent 20 μm.


C In vitro pull - down assays showing BIC1 directly interacts with BZR1 . Purified GST - BIC1 were incubated with MBP or MBP - BZR1 for MBP pull - down . Arrowhead indicates specific bands .

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C In vitro pull-down assays showing BIC1 directly interacts with BZR1. Purified GST-BIC1 proteins were incubated with MBP or MBP-BZR1 for the MBP pull-down assay. Arrowhead indicates specific bands.


D Co - immunoprecipitation ( Co - IP ) assays showing interaction between BIC1 BZR1 in vivo . Seedlings were grown for 6 days under long - day conditions . immunoprecipitates were detected using anti - GFP anti - MYC antibodies , respectively . * indicate specific bands . cross - reacting lower band on IP blot is heavy chain after IP .

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D Co-immunoprecipitation (Co-IP) assays showing the interaction between BIC1 and BZR1 in vivo. Seedlings were grown for 6 days under long-day conditions. The immunoprecipitates were detected using anti-GFP and anti-MYC antibodies, respectively. * indicate specific bands. The cross-reacting lower band on IP blot is the heavy chain after IP.


A , B Hypocotyl elongation of BIC1 overexpression mutant lines in response to BR treatment . Col - 0 , 35S : BIC1 - YFP , 35S : BIC1 - Flag bic1bic2 seedlings were grown for 6 days on 1 / 2 MS medium supplemented with 1 μM brassinazole ( BRZ ) plus different concentrations of epibrassinolide ( eBL ) . of representative seedlings are shown in ( A ) , hypocotyl lengths of indicated genotypes were measured are shown in ( B ) . Data are means ± SD ; n > 20 . Scale bars , 2 mm .

O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B Hypocotyl elongation phenotypes of the BIC1 overexpression and mutant lines in response to BR treatment. Col-0, 35S:BIC1-YFP, 35S:BIC1-Flag and bic1bic2 seedlings were grown for 6 days on 1/2 MS medium supplemented with 1 μM brassinazole (BRZ) plus different concentrations of epibrassinolide (eBL). Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD; n>20. Scale bars, 2 mm.


C RT - qPCR of BZR1 - activated gene expression in bic1bic2 double mutants . 6 - d - old Col - 0 bic1bic2 seedlings grown on 1 / 2 MS medium supplemented with 1 μM BRZ were treated with 5 μM eBL ( + eBL ) or not ( - eBL ) for 1 hour . PP2A gene served as internal control . Data are means ± SD ( n = 3 ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

C RT-qPCR analysis of BZR1-activated gene expression in the bic1bic2 double mutants. The 6-d-old Col-0 and bic1bic2 seedlings grown on 1/2 MS medium supplemented with 1 μM BRZ were treated with 5 μM eBL (+eBL) or not (-eBL) for 1 hour. The PP2A gene served as internal control. Data are means ± SD (n=3).


D Heat map of BIC1 - regulated genes . Col - 0 35S : BIC1 - YFP seedlings were grown on 1 / 2 MS medium for 10 days . Three biologic replicates were performed . colored bar beneath map indicates fold change ( log2 value ) .

O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O 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 Heat map of BIC1-regulated genes. The Col-0 and 35S:BIC1-YFP seedlings were grown on 1/2 MS medium for 10 days. Three biologic replicates were performed. The colored bar beneath the map indicates fold change (log2 value).


E BIC1 facilitates expression of elongation genes . RT - qPCR of genes selected in transcriptomic data . Col - 0 , 35S : BIC1 - YFP 35S : BIC1 - Flag seedlings were grown for 5 days under long - day conditions . PP2A gene served as internal control . Data are means ± SD ( n = 3 ) .

O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

E BIC1 facilitates the expression of cell elongation genes. RT-qPCR analysis of genes selected in transcriptomic data. The Col-0, 35S:BIC1-YFP and 35S:BIC1-Flag seedlings were grown for 5 days under long-day conditions. The PP2A gene served as internal control. Data are means ± SD (n=3).


A , B BIC1 BZR1 synergistically promote hypocotyl elongation . Col - 0 , 35S : BZR1 - MYC , 35S : BIC1 - YFP 35S : BZR1 - MYC / 35S : BIC1 - YFP were grown for 5 days under long - day conditions . of representative seedlings are shown in ( A ) , hypocotyl lengths of indicated genotypes were measured are shown in ( B ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - . Scale bar , 2 mm .

O O O B-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B BIC1 and BZR1 synergistically promote hypocotyl elongation. The Col-0, 35S:BZR1-MYC, 35S:BIC1-YFP and 35S:BZR1-MYC/35S:BIC1-YFP plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.