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  • 99
    Thermo Fisher gene exp bax hs00180269 m1
    Gene Exp Bax Hs00180269 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 402 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti bax
    Expression of peiminine-induced proteins related to proliferation in HCC cells. (A) Expressions of p53, <t>Bcl-2,</t> <t>Bax,</t> PARP, and cleaved PARP in HepG2 cells treated with peiminine were determined by Western blot analysis. (B) Expressions of procaspase-3, -8, and -9, caspase-3, -8, and -9 in HepG2 cells treated with peiminine were determined by Western blot analysis. Data are presented as the mean±SD from three independent experiments.* p
    Anti Bax, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 413 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc bax
    Tanshinol inhibits the growth of HepG2 cell in a xenograft model. Notes: ( A ) A xenograft model of HCC was established using HepG2 cell. Mice in the experimental groups were administrated tanshinol intragastrically daily for 5 weeks. Control mice in vehicle-treated group received the same dose of vehicle only. Tumor volumes in each group were calculated. ( B ) Five weeks after inoculation, tumor tissues in each group were dissected from mice. ( C ) Dissected tumors were weighed. The average weight of tumors from each group of mice was calculated. ( D ) The expression of <t>Bcl-2</t> and <t>Bax</t> was detected in xenografts by IHC. Scale bar: 100 µm. ** P
    Bax, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 13316 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology bax
    Dex-induced caspase activation and cytochrome c release in the absence of <t>BAX</t> and <t>BAK1,</t> but this failed to occur if BIM was also deleted. a Bax + /+ Bak1 + /+ (WT), Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Casp9 −/− , Bax −/− Bak1 −/− Apaf1 −/− , Bim −/− Bmf −/− Puma −/− and Bax −/− Bak1 −/− Bim −/− clonal WEHI7 lines were treated with 1 µM Dex for indicated times. Cell lysates were analyzed by western blot using antibodies specific for cleaved Caspase-3, cleaved Caspase-9, and ACTIN. Note, the first 6 lanes of these blots are also shown in left panel of Fig. 2a . b Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bim −/− p53 −/− T lymphoma lines were treated with 1 µM Dex for indicated times. Whole-cell lysates were analyzed by western blot using antibodies specific for cleaved Caspase-3, cleaved Caspase-9, and ACTIN. Note, the first 6 lanes of these blots are also shown in right panel of Fig. 2a . c Bax + /+ Bak1 + /+ , Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Casp9 −/− , Bax −/− Bak1 −/− Apaf1 −/− , Bim −/− Bmf −/− Puma −/− and Bax −/− Bak1 −/− Bim −/− WEHI7 lines were treated with 1 µM Dex for indicated times. Cytoplasmic extracted fractions were subjected to western blot analysis, using antibodies specific for cytochrome c (CYTC), and ACTIN. d Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bim −/− p53 −/− T lymphoma lines were treated with 1 µM Dex for indicated times. Cytoplasmic fractions were subjected to western blot analysis, using antibodies specific for CYTC, and ACTIN.
    Bax, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 10934 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti bax
    Dex-induced caspase activation and cytochrome c release in the absence of <t>BAX</t> and <t>BAK1,</t> but this failed to occur if BIM was also deleted. a Bax + /+ Bak1 + /+ (WT), Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Casp9 −/− , Bax −/− Bak1 −/− Apaf1 −/− , Bim −/− Bmf −/− Puma −/− and Bax −/− Bak1 −/− Bim −/− clonal WEHI7 lines were treated with 1 µM Dex for indicated times. Cell lysates were analyzed by western blot using antibodies specific for cleaved Caspase-3, cleaved Caspase-9, and ACTIN. Note, the first 6 lanes of these blots are also shown in left panel of Fig. 2a . b Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bim −/− p53 −/− T lymphoma lines were treated with 1 µM Dex for indicated times. Whole-cell lysates were analyzed by western blot using antibodies specific for cleaved Caspase-3, cleaved Caspase-9, and ACTIN. Note, the first 6 lanes of these blots are also shown in right panel of Fig. 2a . c Bax + /+ Bak1 + /+ , Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Casp9 −/− , Bax −/− Bak1 −/− Apaf1 −/− , Bim −/− Bmf −/− Puma −/− and Bax −/− Bak1 −/− Bim −/− WEHI7 lines were treated with 1 µM Dex for indicated times. Cytoplasmic extracted fractions were subjected to western blot analysis, using antibodies specific for cytochrome c (CYTC), and ACTIN. d Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bim −/− p53 −/− T lymphoma lines were treated with 1 µM Dex for indicated times. Cytoplasmic fractions were subjected to western blot analysis, using antibodies specific for CYTC, and ACTIN.
    Anti Bax, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3837 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bax  (Abcam)
    99
    Abcam bax
    Dex-induced caspase activation and cytochrome c release in the absence of <t>BAX</t> and <t>BAK1,</t> but this failed to occur if BIM was also deleted. a Bax + /+ Bak1 + /+ (WT), Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Casp9 −/− , Bax −/− Bak1 −/− Apaf1 −/− , Bim −/− Bmf −/− Puma −/− and Bax −/− Bak1 −/− Bim −/− clonal WEHI7 lines were treated with 1 µM Dex for indicated times. Cell lysates were analyzed by western blot using antibodies specific for cleaved Caspase-3, cleaved Caspase-9, and ACTIN. Note, the first 6 lanes of these blots are also shown in left panel of Fig. 2a . b Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bim −/− p53 −/− T lymphoma lines were treated with 1 µM Dex for indicated times. Whole-cell lysates were analyzed by western blot using antibodies specific for cleaved Caspase-3, cleaved Caspase-9, and ACTIN. Note, the first 6 lanes of these blots are also shown in right panel of Fig. 2a . c Bax + /+ Bak1 + /+ , Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Casp9 −/− , Bax −/− Bak1 −/− Apaf1 −/− , Bim −/− Bmf −/− Puma −/− and Bax −/− Bak1 −/− Bim −/− WEHI7 lines were treated with 1 µM Dex for indicated times. Cytoplasmic extracted fractions were subjected to western blot analysis, using antibodies specific for cytochrome c (CYTC), and ACTIN. d Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bim −/− p53 −/− T lymphoma lines were treated with 1 µM Dex for indicated times. Cytoplasmic fractions were subjected to western blot analysis, using antibodies specific for CYTC, and ACTIN.
    Bax, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 4743 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti bax
    <t>BAX</t> and <t>BAK</t> can be autoactivated by DNA damage independently of activators BID, BIM, PUMA and NOXA through downregulation of BCL-2, BCL-X L and MCL-1 (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase, Bad, Bmf or Bik . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (b) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were infected with retrovirus expressing GFP or the indicated BH3-only proteins to induce spontaneous apoptosis. NOXA denotes human NOXA. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide, tunicamycin (TC) or thapsigargin (TG), were subjected to immunoblot analysis using the indicated antibodies. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 18h, and subjected to immunoblot analysis using the indicated antibodies. (e) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 36h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (f) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bcl-2, Bcl-x L and/or Mcl-1 to induce spontaneous apoptosis. After 2 days, cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (g) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide in the presence of the pancaspase inhibitor Q-VD-OPh to preserve cell integrity upon apoptosis induction. After 24 h, cells were permeabilized with digitonin and subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. (h) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs transfected with scrambled siRNA or siRNA against Bcl-x L and Mcl-1 were subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. **, P
    Anti Bax, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 3959 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti bax
    a Quantitative analysis was carried out for expressions of <t>PAX8,</t> <t>Bax,</t> Bcl-2 and Bak in PTC cells that were treated with X-ray and miR-144-3p. b , c Western blot was performed for expression of PAX8, Bax Bcl-2 and Bak. d , e Western blot was used for expressions of PAX8, Bax Bcl-2 and Bak in PTC cells that were treated with paclitaxel and miR-144-3p. f Quantitative analysis was performed for expression of PAX8, Bax Bcl-2 and Bak. *P
    Anti Bax, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1970 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH bax a j biomol nmr
    a Quantitative analysis was carried out for expressions of <t>PAX8,</t> <t>Bax,</t> Bcl-2 and Bak in PTC cells that were treated with X-ray and miR-144-3p. b , c Western blot was performed for expression of PAX8, Bax Bcl-2 and Bak. d , e Western blot was used for expressions of PAX8, Bax Bcl-2 and Bak in PTC cells that were treated with paclitaxel and miR-144-3p. f Quantitative analysis was performed for expression of PAX8, Bax Bcl-2 and Bak. *P
    Bax A J Biomol Nmr, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti bax antibody e63
    a Quantitative analysis was carried out for expressions of <t>PAX8,</t> <t>Bax,</t> Bcl-2 and Bak in PTC cells that were treated with X-ray and miR-144-3p. b , c Western blot was performed for expression of PAX8, Bax Bcl-2 and Bak. d , e Western blot was used for expressions of PAX8, Bax Bcl-2 and Bak in PTC cells that were treated with paclitaxel and miR-144-3p. f Quantitative analysis was performed for expression of PAX8, Bax Bcl-2 and Bak. *P
    Anti Bax Antibody E63, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 570 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti bax
    Western blots for (A) HIF-1α, (B) BNIP3, (C) Beclin1, (D) LC3-II/LC3-I, (E) p62, (F) Bcl2, (G) <t>Bax</t> and <t>GAPDH.</t> The intensity of the protein blots was normalized with GAPDH. The data are expressed as the mean ± SEM. Optical density (%),
    Rabbit Anti Bax, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 995 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteintech rabbit bax polyclonal
    Western blots for (A) HIF-1α, (B) BNIP3, (C) Beclin1, (D) LC3-II/LC3-I, (E) p62, (F) Bcl2, (G) <t>Bax</t> and <t>GAPDH.</t> The intensity of the protein blots was normalized with GAPDH. The data are expressed as the mean ± SEM. Optical density (%),
    Rabbit Bax Polyclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 99/100, based on 541 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson bax
    Critical role for <t>Bax</t> and Bak in zerumbone <t>(ZER)-induced</t> apoptosis. a Fluorescence microscopy for analysis of active Bax and Bak in MDA-MB-231 and MCF-7 cells after 8 hour treatment with DMSO or 20 µM of ZER. Staining for mitochondria (MitoTracker Green) and activated Bak or Bax is indicated by green and red colors, respectively. b Representative flow histograms depicting apoptotic fraction quantitation in SV40 immortalized mouse embryonic fibroblasts (MEF) derived from wild-type mice (WT) and Bak and Bax double knockout (DKO) mice after 24 hour treatment with DMSO or indicated concentration of ZER. c Quantitation of apoptotic cells (Annexin V-FITC/PI method) in SV40 immortalized MEFs derived from wild type mice (WT) and Bak-Bax double knockout (DKO) mice. The MEF were treated for 24 or 48 hours with DMSO (control) or the indicated concentrations of ZER and processed for flow cytometry. Quantitation relative to DMSO-treated WT MEF is shown. Results shown are mean ± SD (n=3). Significantly different (P
    Bax, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 997 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti bax antibody
    Effects of pristimerin (Pris) on Con A-induced apoptosis. (A) Relative mRNA expression of <t>Bax,</t> caspase-3 and <t>Bcl2.</t> (B) Immuno-expression of Bax and Bcl2 in the hepatic tissue (200×) showing negative nuclear immuno-staining of Bax and high cytoplasmic expression of Bcl2 in both control and Pris groups while Con A group expressed high brown nuclear immuno-staining of Bax and low nuclear brown staining of Bcl2. Pris pretreatment attenuated Con-induced expression of Bax as represent by reduced nuclear brown staining and enhanced the cytoplasmic immunoexpression of Bcl2 . (C) Levels of Bax, caspase-3 and Bcl2 in the hepatic tissue. Data are the mean ± SEM ( n = 8). * P
    Anti Bax Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 449 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit anti bax
    Downregulated Mctp1 and <t>Rarb</t> are associated with apoptotic cell death. a <t>Bax</t> proteins were increased by double knockdown of Mctp1 and Rarb. Bcl2 expression was decreased by siMctp1 and Rarb. b Mctp1 transcripts were downregulated. c Knockdown of Mctp1 enhanced Intracellular Ca 2+ levels (siCont (0.063) versus siMctp1 (0.131) in fluorescence intensities from baseline 490/525 ratio). d Rarb transcripts were downregulated. e-f Transfected siRarb reduced neuronal cell differentiation (MAP2). Scale bar, 40 μm. g and h Double knockdown of Mctp1 and Rarb increased Bax transcripts and decreased Bcl2 transcripts. i Transfected siMctp1 and siRarb activated LDH release. j Flow cytometry verified that downregulated Mctp1 and Rarb significantly induce apoptotic cell death in mouse NSCs. Scrambled siRNA served as a negative control (siCont). Significantly different at *, p
    Rabbit Anti Bax, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 605 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti bax antibody
    Analysis of <t>Beclin</t> 1 protein interactions and Bcl2 and <t>Bax</t> protein expression on 793 cells starved with and without anti-EGF antibody Western blot in ( A ), shows that: phospho-EGFR strongly binds Beclin 1 on 793 cells starved for 4 hrs; p-Bcl2 bind Beclin 1 up to 4 hrs of starvation and then detach; p-JNK and p-Bcl2 protein expressions increase at 4 and 8 hrs of starvation, respectively; Bcl2 and Bax were attached until 8 hrs of starvation and, afterwards, they were detached. ( B ) 793 cells starved and treated with anti-EGF antibody showed that: Beclin 1 and p-EGFR were detached; Beclin 1 and p-Bcl2 stayed attached during starvation; p-JNK and p-Bcl-2 expression levels were constantly down-regulated; p-Bcl2 and Bax were constantly attached. Time of exposure of p-Bcl2, p-EGFR, p-JNK, Beclin-1, Bax and tubulin were 7.5, 2, 15, 9.6, 5 and 4.3 s, respectively.
    Anti Bax Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bax
    Analysis of <t>Beclin</t> 1 protein interactions and Bcl2 and <t>Bax</t> protein expression on 793 cells starved with and without anti-EGF antibody Western blot in ( A ), shows that: phospho-EGFR strongly binds Beclin 1 on 793 cells starved for 4 hrs; p-Bcl2 bind Beclin 1 up to 4 hrs of starvation and then detach; p-JNK and p-Bcl2 protein expressions increase at 4 and 8 hrs of starvation, respectively; Bcl2 and Bax were attached until 8 hrs of starvation and, afterwards, they were detached. ( B ) 793 cells starved and treated with anti-EGF antibody showed that: Beclin 1 and p-EGFR were detached; Beclin 1 and p-Bcl2 stayed attached during starvation; p-JNK and p-Bcl-2 expression levels were constantly down-regulated; p-Bcl2 and Bax were constantly attached. Time of exposure of p-Bcl2, p-EGFR, p-JNK, Beclin-1, Bax and tubulin were 7.5, 2, 15, 9.6, 5 and 4.3 s, respectively.
    Bax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1069 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit polyclonal anti bax
    Ku70 mediates <t>Bax</t> deubiquitylation. ( A ) Time-course analysis of ubiquitylated Bax levels by Western blot with <t>polyclonal</t> antibody against Bax before (0) or at various times after the induction of Bax-mediated apoptosis by staurosporine-STS in Ku70 −/− cells. ( B ) In vitro deubiquitylation of Bax in Ku70 −/− cell extracts supplemented with PBS or recombinant Ku70-rKU70. ( C ) In vitro deubiquitylation of Bax in Ku70 −/− cells that were grown in the presence of the proteasomal inhibitor MG-132. The extracts were supplemented with PBS or rKu70. ( D ) Levels of ubiquitylated Bax in wild-type cells that were grown in medium supplemented with DMSO or the proteasomal inhibitor, MG-132. ( E ) In vitro deubiquitylation assay of homogenous tetraubiquitin chains supplemented with BSA and rKu70 for various time periods, demonstrating that Ku70 possess an intrinsic DUB enzymatic activity. Incubation of tetraubiquitin only or tetraubiquitin together with BSA did not induce hydrolysis of the tetraubiquitin chains into monoubiquitin units. In A–D , β-actin levels served as a loading control. U, unmodified Bax; M, modified Bax.
    Rabbit Polyclonal Anti Bax, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti bax
    Ku70 mediates <t>Bax</t> deubiquitylation. ( A ) Time-course analysis of ubiquitylated Bax levels by Western blot with <t>polyclonal</t> antibody against Bax before (0) or at various times after the induction of Bax-mediated apoptosis by staurosporine-STS in Ku70 −/− cells. ( B ) In vitro deubiquitylation of Bax in Ku70 −/− cell extracts supplemented with PBS or recombinant Ku70-rKU70. ( C ) In vitro deubiquitylation of Bax in Ku70 −/− cells that were grown in the presence of the proteasomal inhibitor MG-132. The extracts were supplemented with PBS or rKu70. ( D ) Levels of ubiquitylated Bax in wild-type cells that were grown in medium supplemented with DMSO or the proteasomal inhibitor, MG-132. ( E ) In vitro deubiquitylation assay of homogenous tetraubiquitin chains supplemented with BSA and rKu70 for various time periods, demonstrating that Ku70 possess an intrinsic DUB enzymatic activity. Incubation of tetraubiquitin only or tetraubiquitin together with BSA did not induce hydrolysis of the tetraubiquitin chains into monoubiquitin units. In A–D , β-actin levels served as a loading control. U, unmodified Bax; M, modified Bax.
    Rabbit Anti Bax, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 390 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti bax
    The ER-targeted <t>Bak</t> can induce apoptosis in the absence of endogenous <t>Bax</t> and Bak. Wild-type and bax − / − bak − / − MEFs were infected with retroviruses expressing GFP or GFP together with wild-type murine Bak, Bak-ActA, Bak-cb5, or Bak-ΔC. 48 h after infection, 1 μg/ml DAPI was added to stain the apoptotic cells. (A) bax − / − bak − / − cells were photographed using a FITC or DAPI filter. (B) The percentage of dead cells was determined by the ratio of DAPI-positive cells to GFP-positive cells. In addition to the retroviral infection of the Bak mutants, bax − / − bak − / − cells were also coinfected with retrovirus expressing Bcl-xL, or infected in the presence of 20 μg/ml Z-VAD-fmk. Data shown are the average of three independent experiments. (C) 24 h after infection, bax − / − bak − / − cells were trypsinized. A portion of each sample was subjected to FACS ® to determine the expression efficiency indicated by GFP-positive cells. The rest of the cells were fixed in 0.25% paraformaldehyde and stained with an anti-Bak antibody that recognizes the active form of murine Bak. The number in each dot plot represents the percentage of gated events (R1).
    Anti Bax, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression of peiminine-induced proteins related to proliferation in HCC cells. (A) Expressions of p53, Bcl-2, Bax, PARP, and cleaved PARP in HepG2 cells treated with peiminine were determined by Western blot analysis. (B) Expressions of procaspase-3, -8, and -9, caspase-3, -8, and -9 in HepG2 cells treated with peiminine were determined by Western blot analysis. Data are presented as the mean±SD from three independent experiments.* p

    Journal: PLoS ONE

    Article Title: The effects and mechanism of peiminine-induced apoptosis in human hepatocellular carcinoma HepG2 cells

    doi: 10.1371/journal.pone.0201864

    Figure Lengend Snippet: Expression of peiminine-induced proteins related to proliferation in HCC cells. (A) Expressions of p53, Bcl-2, Bax, PARP, and cleaved PARP in HepG2 cells treated with peiminine were determined by Western blot analysis. (B) Expressions of procaspase-3, -8, and -9, caspase-3, -8, and -9 in HepG2 cells treated with peiminine were determined by Western blot analysis. Data are presented as the mean±SD from three independent experiments.* p

    Article Snippet: RPMI-1640 medium, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), 4,6-diamidino-2-phenylindile(DAPI), dimethyl sulfoxide (DMSO), and anti-Bax, Bcl-2, procaspase-3, -8, -9, caspase-3, -8, -9, PARP1 (Asp214, 89 kD), PARP1 (Asp214, 89 kD) cleaved and β-actin primary antibodies were from Sigma-Aldrich Chemical Co., Ltd (Shanghai, China).

    Techniques: Expressing, Western Blot

    Tanshinol inhibits the growth of HepG2 cell in a xenograft model. Notes: ( A ) A xenograft model of HCC was established using HepG2 cell. Mice in the experimental groups were administrated tanshinol intragastrically daily for 5 weeks. Control mice in vehicle-treated group received the same dose of vehicle only. Tumor volumes in each group were calculated. ( B ) Five weeks after inoculation, tumor tissues in each group were dissected from mice. ( C ) Dissected tumors were weighed. The average weight of tumors from each group of mice was calculated. ( D ) The expression of Bcl-2 and Bax was detected in xenografts by IHC. Scale bar: 100 µm. ** P

    Journal: OncoTargets and therapy

    Article Title: Tanshinol inhibits the growth, migration and invasion of hepatocellular carcinoma cells via regulating the PI3K-AKT signaling pathway

    doi: 10.2147/OTT.S185997

    Figure Lengend Snippet: Tanshinol inhibits the growth of HepG2 cell in a xenograft model. Notes: ( A ) A xenograft model of HCC was established using HepG2 cell. Mice in the experimental groups were administrated tanshinol intragastrically daily for 5 weeks. Control mice in vehicle-treated group received the same dose of vehicle only. Tumor volumes in each group were calculated. ( B ) Five weeks after inoculation, tumor tissues in each group were dissected from mice. ( C ) Dissected tumors were weighed. The average weight of tumors from each group of mice was calculated. ( D ) The expression of Bcl-2 and Bax was detected in xenografts by IHC. Scale bar: 100 µm. ** P

    Article Snippet: Then, the cells were incubated with primary antibody Bcl-2 or Bax (1:100) (Cell Signaling Technology, Danvers, MA, USA) for overnight.

    Techniques: Mouse Assay, Expressing, Immunohistochemistry

    Tanshinol increases the apoptosis of HepG2 cell. Notes: ( A ) Representative images of Hoechst staining of cells treated with tanshinol. Arrows: apoptotic nuclear changes. Scale bar: 500 µm. ( B ) The apoptosis of HepG2 cell was analyzed by flow cytometry with Annexin V-FITC/PI staining. ( C ) The expression of Bax and Bcl-2 were detected using Western blotting assay. GAPDH was used as loading control. ( D ) The expression of Bcl-2 and Bax in HepG2 cell was analyzed using immunohistochemical staining. * P

    Journal: OncoTargets and therapy

    Article Title: Tanshinol inhibits the growth, migration and invasion of hepatocellular carcinoma cells via regulating the PI3K-AKT signaling pathway

    doi: 10.2147/OTT.S185997

    Figure Lengend Snippet: Tanshinol increases the apoptosis of HepG2 cell. Notes: ( A ) Representative images of Hoechst staining of cells treated with tanshinol. Arrows: apoptotic nuclear changes. Scale bar: 500 µm. ( B ) The apoptosis of HepG2 cell was analyzed by flow cytometry with Annexin V-FITC/PI staining. ( C ) The expression of Bax and Bcl-2 were detected using Western blotting assay. GAPDH was used as loading control. ( D ) The expression of Bcl-2 and Bax in HepG2 cell was analyzed using immunohistochemical staining. * P

    Article Snippet: Then, the cells were incubated with primary antibody Bcl-2 or Bax (1:100) (Cell Signaling Technology, Danvers, MA, USA) for overnight.

    Techniques: Staining, Flow Cytometry, Cytometry, Expressing, Western Blot, Immunohistochemistry

    Dex-induced caspase activation and cytochrome c release in the absence of BAX and BAK1, but this failed to occur if BIM was also deleted. a Bax + /+ Bak1 + /+ (WT), Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Casp9 −/− , Bax −/− Bak1 −/− Apaf1 −/− , Bim −/− Bmf −/− Puma −/− and Bax −/− Bak1 −/− Bim −/− clonal WEHI7 lines were treated with 1 µM Dex for indicated times. Cell lysates were analyzed by western blot using antibodies specific for cleaved Caspase-3, cleaved Caspase-9, and ACTIN. Note, the first 6 lanes of these blots are also shown in left panel of Fig. 2a . b Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bim −/− p53 −/− T lymphoma lines were treated with 1 µM Dex for indicated times. Whole-cell lysates were analyzed by western blot using antibodies specific for cleaved Caspase-3, cleaved Caspase-9, and ACTIN. Note, the first 6 lanes of these blots are also shown in right panel of Fig. 2a . c Bax + /+ Bak1 + /+ , Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Casp9 −/− , Bax −/− Bak1 −/− Apaf1 −/− , Bim −/− Bmf −/− Puma −/− and Bax −/− Bak1 −/− Bim −/− WEHI7 lines were treated with 1 µM Dex for indicated times. Cytoplasmic extracted fractions were subjected to western blot analysis, using antibodies specific for cytochrome c (CYTC), and ACTIN. d Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bim −/− p53 −/− T lymphoma lines were treated with 1 µM Dex for indicated times. Cytoplasmic fractions were subjected to western blot analysis, using antibodies specific for CYTC, and ACTIN.

    Journal: Cell Death & Disease

    Article Title: Glucocorticoids can induce BIM to trigger apoptosis in the absence of BAX and BAK1

    doi: 10.1038/s41419-020-2599-5

    Figure Lengend Snippet: Dex-induced caspase activation and cytochrome c release in the absence of BAX and BAK1, but this failed to occur if BIM was also deleted. a Bax + /+ Bak1 + /+ (WT), Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Casp9 −/− , Bax −/− Bak1 −/− Apaf1 −/− , Bim −/− Bmf −/− Puma −/− and Bax −/− Bak1 −/− Bim −/− clonal WEHI7 lines were treated with 1 µM Dex for indicated times. Cell lysates were analyzed by western blot using antibodies specific for cleaved Caspase-3, cleaved Caspase-9, and ACTIN. Note, the first 6 lanes of these blots are also shown in left panel of Fig. 2a . b Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bim −/− p53 −/− T lymphoma lines were treated with 1 µM Dex for indicated times. Whole-cell lysates were analyzed by western blot using antibodies specific for cleaved Caspase-3, cleaved Caspase-9, and ACTIN. Note, the first 6 lanes of these blots are also shown in right panel of Fig. 2a . c Bax + /+ Bak1 + /+ , Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Casp9 −/− , Bax −/− Bak1 −/− Apaf1 −/− , Bim −/− Bmf −/− Puma −/− and Bax −/− Bak1 −/− Bim −/− WEHI7 lines were treated with 1 µM Dex for indicated times. Cytoplasmic extracted fractions were subjected to western blot analysis, using antibodies specific for cytochrome c (CYTC), and ACTIN. d Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bim −/− p53 −/− T lymphoma lines were treated with 1 µM Dex for indicated times. Cytoplasmic fractions were subjected to western blot analysis, using antibodies specific for CYTC, and ACTIN.

    Article Snippet: Antibodies and reagents Antibodies used were to ACTIN (AC-15, Sigma #A1978), MCL-1 (Rockland #600-401-394S), BAX (N-20 Santa Cruz Biotechnology #sc-493), BAK1 (aa23-38, #B5897 Sigma), BCL-2 (#610539, BD Biosciences), Cytochrome c (#556433, BD Biosciences), VDAC2 (M.T.

    Techniques: Activation Assay, Western Blot

    BOK is undetectable in WEHI7 and p53 −/− lymphoma cells. Whole-cell lysis of Bax −/− Bak1 −/− and Bax −/− Bak1 −/− WEH7 cells (upper panel) and p53 −/− T lymphoma cells (lower panel), were treated with 1 µM Dex for the indicated times and analyzed by western blot. Membranes were incubated with a rabbit anti–BOK polyclonal antibody and subsequently with a monoclonal antibody to ACTIN. Lysates from Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bok −/− mouse embryonic fibroblast lines (MEFs) were used as positive and negative controls. The results of one of two independent experiments are shown.

    Journal: Cell Death & Disease

    Article Title: Glucocorticoids can induce BIM to trigger apoptosis in the absence of BAX and BAK1

    doi: 10.1038/s41419-020-2599-5

    Figure Lengend Snippet: BOK is undetectable in WEHI7 and p53 −/− lymphoma cells. Whole-cell lysis of Bax −/− Bak1 −/− and Bax −/− Bak1 −/− WEH7 cells (upper panel) and p53 −/− T lymphoma cells (lower panel), were treated with 1 µM Dex for the indicated times and analyzed by western blot. Membranes were incubated with a rabbit anti–BOK polyclonal antibody and subsequently with a monoclonal antibody to ACTIN. Lysates from Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bok −/− mouse embryonic fibroblast lines (MEFs) were used as positive and negative controls. The results of one of two independent experiments are shown.

    Article Snippet: Antibodies and reagents Antibodies used were to ACTIN (AC-15, Sigma #A1978), MCL-1 (Rockland #600-401-394S), BAX (N-20 Santa Cruz Biotechnology #sc-493), BAK1 (aa23-38, #B5897 Sigma), BCL-2 (#610539, BD Biosciences), Cytochrome c (#556433, BD Biosciences), VDAC2 (M.T.

    Techniques: Lysis, Western Blot, Incubation

    Induced expression of hBIMs was not sufficient to death of untreated cells, but did restore sensitivity of Bax −/− Bak1 −/− Bim −/− WEHI7 cells to treatment with dexamethasone. a Bax −/− Bak1 −/− Bim − /− WEHI7 cells were transduced with a lentiviral construct expressing human BIMs from a doxycycline-inducible promoter. Five independent ihBIM Bax −/− Bak1 −/− Bim − /− WEHI7 clonal cell lines were cultured for 3 days in the presence or absence of 1 µg/ml doxycycline (Dox). Bim expression was monitored by immunoblotting, using ACTIN as loading control. b Bax −/− Bak1 −/− Bim −/− parental cells (gray bars) and five independent Bax −/− Bak1 −/− Bim −/− inducible hBIMs ( ihBIM ) WEHI7 cell clones (white bars) were cultured for 6 days in the presence or absence of 1 µg/ml Dox and/or 1 µM Dex. Cell death was assessed by PI uptake using flow cytometry. For cell death to occur, both treatment with Dex, as well as induction of BIMs, were necessary. c Dot plots show one of the five independent clones as shown in b , numbers represent the percent of PI-negative cells of a total of 10,000 cells analyzed per condition. d Bax −/− Bak1 −/− Bim −/− WEHI7 cells and those from ihBIM Bax −/− Bak1 −/− Bim −/− WEHI7 lines were cultured for 6 days in the presence or absence of 1 µg/ml Dox and/or 1 µM Dex. Cytoplasmic extracts were subjected to western blot analysis, with antibodies specific for CYTC and ACTIN. For CYTC to be released into the cytosol, both treatment with Dex, as well as induction of BIMs, were necessary.

    Journal: Cell Death & Disease

    Article Title: Glucocorticoids can induce BIM to trigger apoptosis in the absence of BAX and BAK1

    doi: 10.1038/s41419-020-2599-5

    Figure Lengend Snippet: Induced expression of hBIMs was not sufficient to death of untreated cells, but did restore sensitivity of Bax −/− Bak1 −/− Bim −/− WEHI7 cells to treatment with dexamethasone. a Bax −/− Bak1 −/− Bim − /− WEHI7 cells were transduced with a lentiviral construct expressing human BIMs from a doxycycline-inducible promoter. Five independent ihBIM Bax −/− Bak1 −/− Bim − /− WEHI7 clonal cell lines were cultured for 3 days in the presence or absence of 1 µg/ml doxycycline (Dox). Bim expression was monitored by immunoblotting, using ACTIN as loading control. b Bax −/− Bak1 −/− Bim −/− parental cells (gray bars) and five independent Bax −/− Bak1 −/− Bim −/− inducible hBIMs ( ihBIM ) WEHI7 cell clones (white bars) were cultured for 6 days in the presence or absence of 1 µg/ml Dox and/or 1 µM Dex. Cell death was assessed by PI uptake using flow cytometry. For cell death to occur, both treatment with Dex, as well as induction of BIMs, were necessary. c Dot plots show one of the five independent clones as shown in b , numbers represent the percent of PI-negative cells of a total of 10,000 cells analyzed per condition. d Bax −/− Bak1 −/− Bim −/− WEHI7 cells and those from ihBIM Bax −/− Bak1 −/− Bim −/− WEHI7 lines were cultured for 6 days in the presence or absence of 1 µg/ml Dox and/or 1 µM Dex. Cytoplasmic extracts were subjected to western blot analysis, with antibodies specific for CYTC and ACTIN. For CYTC to be released into the cytosol, both treatment with Dex, as well as induction of BIMs, were necessary.

    Article Snippet: Antibodies and reagents Antibodies used were to ACTIN (AC-15, Sigma #A1978), MCL-1 (Rockland #600-401-394S), BAX (N-20 Santa Cruz Biotechnology #sc-493), BAK1 (aa23-38, #B5897 Sigma), BCL-2 (#610539, BD Biosciences), Cytochrome c (#556433, BD Biosciences), VDAC2 (M.T.

    Techniques: Expressing, Transduction, Construct, Cell Culture, Clone Assay, Flow Cytometry, Western Blot

    Deletion of BIM increased clonogenic survival of WEHI7 cells in response to Dex. a One representative WEHI7-derived clone of each genotype ( Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Apaf1 −/− and Bax −/− Bak1 −/− Bim −/− ) was cultured for 10 days in the presence or absence of 1 µM Dex. Cells were then washed, and plated in soft agar without Dex at a density of 5000 cells per well. Cells without Dex pre-treatment were plated at a lower density of 250 cells per well to generate countable numbers of colonies. After 14 days, the cloning efficiency was calculated by dividing the number of colonies by the number of cells initially plated in each well, and expressed as percentages. The results are from three independently conducted experiments. Photos of one of the three experiments are shown on the right. b Three independent WEHI7 cell clones from each genotype ( Bax −/− Bak1 −/− , Bim −/− Bmf −/− Puma −/− Bax −/− Bak1 −/− Apaf1 −/− and Bax −/− Bak1 −/− Bim −/− ) were cultured for 10 days in the presence or absence of 1 µM Dex. Cells were then washed free of Dex, and plated in soft-agar medium for 14 days. Photos of one of the three experiments are shown on the right. c Four independent ihBIM Bax −/− Bak1 −/− Bim −/− WEHI7 cell clones were cultured for 10 days in the presence of 1 µM Dex and/or 1 µg/ml Dox. Cells were then washed free of Dex, and plated in soft-agar medium at a density of 4000 cells per well. Cells without Dex pre-treatment were plated at a lower density of 400 cells per well. Colonies were counted 14 days after plating.

    Journal: Cell Death & Disease

    Article Title: Glucocorticoids can induce BIM to trigger apoptosis in the absence of BAX and BAK1

    doi: 10.1038/s41419-020-2599-5

    Figure Lengend Snippet: Deletion of BIM increased clonogenic survival of WEHI7 cells in response to Dex. a One representative WEHI7-derived clone of each genotype ( Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Apaf1 −/− and Bax −/− Bak1 −/− Bim −/− ) was cultured for 10 days in the presence or absence of 1 µM Dex. Cells were then washed, and plated in soft agar without Dex at a density of 5000 cells per well. Cells without Dex pre-treatment were plated at a lower density of 250 cells per well to generate countable numbers of colonies. After 14 days, the cloning efficiency was calculated by dividing the number of colonies by the number of cells initially plated in each well, and expressed as percentages. The results are from three independently conducted experiments. Photos of one of the three experiments are shown on the right. b Three independent WEHI7 cell clones from each genotype ( Bax −/− Bak1 −/− , Bim −/− Bmf −/− Puma −/− Bax −/− Bak1 −/− Apaf1 −/− and Bax −/− Bak1 −/− Bim −/− ) were cultured for 10 days in the presence or absence of 1 µM Dex. Cells were then washed free of Dex, and plated in soft-agar medium for 14 days. Photos of one of the three experiments are shown on the right. c Four independent ihBIM Bax −/− Bak1 −/− Bim −/− WEHI7 cell clones were cultured for 10 days in the presence of 1 µM Dex and/or 1 µg/ml Dox. Cells were then washed free of Dex, and plated in soft-agar medium at a density of 4000 cells per well. Cells without Dex pre-treatment were plated at a lower density of 400 cells per well. Colonies were counted 14 days after plating.

    Article Snippet: Antibodies and reagents Antibodies used were to ACTIN (AC-15, Sigma #A1978), MCL-1 (Rockland #600-401-394S), BAX (N-20 Santa Cruz Biotechnology #sc-493), BAK1 (aa23-38, #B5897 Sigma), BCL-2 (#610539, BD Biosciences), Cytochrome c (#556433, BD Biosciences), VDAC2 (M.T.

    Techniques: Derivative Assay, Cell Culture, Clone Assay

    In the absence of BAX and BAK1, Dex can still cause cell death, but it takes much longer. a Independent Bax +/+ Bak1 +/+ (wild type; open circles) and Bax −/− Bak1 −/− (filled circles) WEHI7 lines (left), and p53 −/− T lymphoma cell lines (right), were treated with 1 µM Dex for 3 days, and viability was determined by propidium iodide (PI) uptake. Almost all of the wild-type cells died by day 3, whereas the lines lacking BAX and BAK1 remained PI negative. b WEHI7 cells (left) and p53 −/− T lymphoma cells (right) from each genotype ( Bax +/+ Bak1 +/+ , Bax −/− Bak1 −/− , Bim −/− Bmf −/− Puma −/− , and Bax −/− Bak1 −/− Bim −/− ) were treated with 1 µM Dex for indicated times, and viability was determined by PI uptake. When cells were exposed to Dex for longer time periods, cells from Bax −/− Bak1 −/− lines also became PI positive. In comparison, fewer cells from independent lines lacking BAX, BAK1, and BIM (filled triangles), or lacking PUMA, BMF, and BIM (open triangles), died when exposed to Dex. Symbols represent independent clonal lines of each genotype. c Caspase inhibitors QVD or IDN, but not the RIPK1 inhibitor Nec-1, prevented PI uptake by Dex-treated Bax −/− Bak1 −/− WEHI7 cells at day 6. Independent Bax −/− Bak1 −/− WEHI7 lines were treated with 1 µM Dex and/or 10 µM QVD-OPh or 5 µM IDN-6556 or 50 µM Nec-1 for 6 days. Cells were harvested, resuspended in PBS containing PI and analyzed by flow cytometry. Data show means of two independent experiments each using three independent lines. d Dot plots of one of the Bax −/− Bak1 −/− WEHI7 independent clonal lines shown in c ; numbers indicate the percent of PI-negative cells of a total of 10,000 cells analyzed per condition. e To confirm gene mutation in each of the clonal lines, whole-cell lysates from Bax + /+ Bak1 + /+ and Bax −/− Bak1 −/− WEHI7 cells (left panel) and p53 −/− T lymphoma cells (right panel) treated with 1 µM Dex for 24 h were subjected to western blot analysis. Note that the absence of protein in the Dex-treated WT p53 −/− T lymphoma cells is due to the death of most of the cells by 24 h.

    Journal: Cell Death & Disease

    Article Title: Glucocorticoids can induce BIM to trigger apoptosis in the absence of BAX and BAK1

    doi: 10.1038/s41419-020-2599-5

    Figure Lengend Snippet: In the absence of BAX and BAK1, Dex can still cause cell death, but it takes much longer. a Independent Bax +/+ Bak1 +/+ (wild type; open circles) and Bax −/− Bak1 −/− (filled circles) WEHI7 lines (left), and p53 −/− T lymphoma cell lines (right), were treated with 1 µM Dex for 3 days, and viability was determined by propidium iodide (PI) uptake. Almost all of the wild-type cells died by day 3, whereas the lines lacking BAX and BAK1 remained PI negative. b WEHI7 cells (left) and p53 −/− T lymphoma cells (right) from each genotype ( Bax +/+ Bak1 +/+ , Bax −/− Bak1 −/− , Bim −/− Bmf −/− Puma −/− , and Bax −/− Bak1 −/− Bim −/− ) were treated with 1 µM Dex for indicated times, and viability was determined by PI uptake. When cells were exposed to Dex for longer time periods, cells from Bax −/− Bak1 −/− lines also became PI positive. In comparison, fewer cells from independent lines lacking BAX, BAK1, and BIM (filled triangles), or lacking PUMA, BMF, and BIM (open triangles), died when exposed to Dex. Symbols represent independent clonal lines of each genotype. c Caspase inhibitors QVD or IDN, but not the RIPK1 inhibitor Nec-1, prevented PI uptake by Dex-treated Bax −/− Bak1 −/− WEHI7 cells at day 6. Independent Bax −/− Bak1 −/− WEHI7 lines were treated with 1 µM Dex and/or 10 µM QVD-OPh or 5 µM IDN-6556 or 50 µM Nec-1 for 6 days. Cells were harvested, resuspended in PBS containing PI and analyzed by flow cytometry. Data show means of two independent experiments each using three independent lines. d Dot plots of one of the Bax −/− Bak1 −/− WEHI7 independent clonal lines shown in c ; numbers indicate the percent of PI-negative cells of a total of 10,000 cells analyzed per condition. e To confirm gene mutation in each of the clonal lines, whole-cell lysates from Bax + /+ Bak1 + /+ and Bax −/− Bak1 −/− WEHI7 cells (left panel) and p53 −/− T lymphoma cells (right panel) treated with 1 µM Dex for 24 h were subjected to western blot analysis. Note that the absence of protein in the Dex-treated WT p53 −/− T lymphoma cells is due to the death of most of the cells by 24 h.

    Article Snippet: Antibodies and reagents Antibodies used were to ACTIN (AC-15, Sigma #A1978), MCL-1 (Rockland #600-401-394S), BAX (N-20 Santa Cruz Biotechnology #sc-493), BAK1 (aa23-38, #B5897 Sigma), BCL-2 (#610539, BD Biosciences), Cytochrome c (#556433, BD Biosciences), VDAC2 (M.T.

    Techniques: Flow Cytometry, Mutagenesis, Western Blot

    Dexamethasone can induce caspase activation in the absence of BAX and BAK1. a WEHI7 cells (left panel) and p53 −/− lymphoma cells (right panel) from each genotype ( Bax + /+ Bak1 + /+ and Bax −/− Bak1 −/− ) were treated with 1 µM Dex for indicated times. Cell lysates were analyzed by western blot with antibodies to cleaved Caspase-3, cleaved Caspase-9, BAK, BAX, and ACTIN. Data show results of one of two independent experiments. Roman numerals to the left of blots (i–ii) indicate the membrane probed. b Mutating Caspase-9 or Apaf1 genes prevented Dex-induced PI uptake in Bax −/− Bak1 −/− WEHI7 clonal lines. In the lines mutant for Caspase-9 or Apaf1 , addition of QVD did not increase the percentage of PI-negative cells. Independent WEHI7 cell clones from each genotype ( Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Casp9 −/− and Bax −/− Bak1 −/− Apaf1 −/− ) were treated with 1 µM Dex and/or 10 µM QVD for up to 6 days. Cells were harvested, resuspended in PBS containing PI, and analyzed by flow cytometry. Data show one of two independent experiments using four or five independent clonal lines. c Dot plots of independent clones from experiments shown in b ; numbers represent the percent of PI-negative cells in a total of 10,000 cells analyzed per condition. d Dexamethasone induces cytochrome c release in a Bax/Bak1 independent manner in WEHI7 cells. Cytoplasmic extracts from WT and Bax −/− Bak1 −/− WEH7 cells, which were treated with 1 µM DEX for 0 to 6 days, were subjected to western blot analysis, with antibody specific for cytochrome c (CYTC) and ACTIN. Results are from one of three independent experiments.

    Journal: Cell Death & Disease

    Article Title: Glucocorticoids can induce BIM to trigger apoptosis in the absence of BAX and BAK1

    doi: 10.1038/s41419-020-2599-5

    Figure Lengend Snippet: Dexamethasone can induce caspase activation in the absence of BAX and BAK1. a WEHI7 cells (left panel) and p53 −/− lymphoma cells (right panel) from each genotype ( Bax + /+ Bak1 + /+ and Bax −/− Bak1 −/− ) were treated with 1 µM Dex for indicated times. Cell lysates were analyzed by western blot with antibodies to cleaved Caspase-3, cleaved Caspase-9, BAK, BAX, and ACTIN. Data show results of one of two independent experiments. Roman numerals to the left of blots (i–ii) indicate the membrane probed. b Mutating Caspase-9 or Apaf1 genes prevented Dex-induced PI uptake in Bax −/− Bak1 −/− WEHI7 clonal lines. In the lines mutant for Caspase-9 or Apaf1 , addition of QVD did not increase the percentage of PI-negative cells. Independent WEHI7 cell clones from each genotype ( Bax −/− Bak1 −/− , Bax −/− Bak1 −/− Casp9 −/− and Bax −/− Bak1 −/− Apaf1 −/− ) were treated with 1 µM Dex and/or 10 µM QVD for up to 6 days. Cells were harvested, resuspended in PBS containing PI, and analyzed by flow cytometry. Data show one of two independent experiments using four or five independent clonal lines. c Dot plots of independent clones from experiments shown in b ; numbers represent the percent of PI-negative cells in a total of 10,000 cells analyzed per condition. d Dexamethasone induces cytochrome c release in a Bax/Bak1 independent manner in WEHI7 cells. Cytoplasmic extracts from WT and Bax −/− Bak1 −/− WEH7 cells, which were treated with 1 µM DEX for 0 to 6 days, were subjected to western blot analysis, with antibody specific for cytochrome c (CYTC) and ACTIN. Results are from one of three independent experiments.

    Article Snippet: Antibodies and reagents Antibodies used were to ACTIN (AC-15, Sigma #A1978), MCL-1 (Rockland #600-401-394S), BAX (N-20 Santa Cruz Biotechnology #sc-493), BAK1 (aa23-38, #B5897 Sigma), BCL-2 (#610539, BD Biosciences), Cytochrome c (#556433, BD Biosciences), VDAC2 (M.T.

    Techniques: Activation Assay, Western Blot, Mutagenesis, Clone Assay, Flow Cytometry

    In the absence of BIM, the ability of Dex to cause death of Bax −/− Bak1 −/− , and Bmf −/− Puma −/− WEHI7 cells was reduced. a Multiple independent clonal Bax −/− Bak1 −/− , Bim −/− Bmf −/− Puma −/− and Bax −/− Bak1 −/− Bim −/− WEHI7 lines were treated with 1 µM Dex and/or 10 µM QVD-OPh for 6 days. Cells were analyzed by flow cytometry for uptake of PI. Each symbol represents an independently derived clonal line. b Dot plots show independent clones from one of the three independent experiments shown in a . Numbers indicate the percent of PI-negative cells in a total of 10,000 cells analyzed per condition. c In two independent experiments, Bax −/− Bak1 −/− parental, and three independent Bax −/− Bak1 −/− Bim −/− mutant p53 −/− T lymphoma cell lines were treated with 1 µM Dex and/or 10 µM QVD-OPh for 6 days. Cells were harvested, resuspended in PBS containing PI and analyzed by flow cytometry. d Dot plots show the parental Bax −/− Bak1 −/− p53 −/− T lymphoma cell line and one of the three Bax −/− Bak1 −/− Bim −/− mutant p53 −/− T lymphoma cell lines shown in c . Numbers indicate the percentage of PI-negative cells from a total of 10,000 cells analyzed per condition.

    Journal: Cell Death & Disease

    Article Title: Glucocorticoids can induce BIM to trigger apoptosis in the absence of BAX and BAK1

    doi: 10.1038/s41419-020-2599-5

    Figure Lengend Snippet: In the absence of BIM, the ability of Dex to cause death of Bax −/− Bak1 −/− , and Bmf −/− Puma −/− WEHI7 cells was reduced. a Multiple independent clonal Bax −/− Bak1 −/− , Bim −/− Bmf −/− Puma −/− and Bax −/− Bak1 −/− Bim −/− WEHI7 lines were treated with 1 µM Dex and/or 10 µM QVD-OPh for 6 days. Cells were analyzed by flow cytometry for uptake of PI. Each symbol represents an independently derived clonal line. b Dot plots show independent clones from one of the three independent experiments shown in a . Numbers indicate the percent of PI-negative cells in a total of 10,000 cells analyzed per condition. c In two independent experiments, Bax −/− Bak1 −/− parental, and three independent Bax −/− Bak1 −/− Bim −/− mutant p53 −/− T lymphoma cell lines were treated with 1 µM Dex and/or 10 µM QVD-OPh for 6 days. Cells were harvested, resuspended in PBS containing PI and analyzed by flow cytometry. d Dot plots show the parental Bax −/− Bak1 −/− p53 −/− T lymphoma cell line and one of the three Bax −/− Bak1 −/− Bim −/− mutant p53 −/− T lymphoma cell lines shown in c . Numbers indicate the percentage of PI-negative cells from a total of 10,000 cells analyzed per condition.

    Article Snippet: Antibodies and reagents Antibodies used were to ACTIN (AC-15, Sigma #A1978), MCL-1 (Rockland #600-401-394S), BAX (N-20 Santa Cruz Biotechnology #sc-493), BAK1 (aa23-38, #B5897 Sigma), BCL-2 (#610539, BD Biosciences), Cytochrome c (#556433, BD Biosciences), VDAC2 (M.T.

    Techniques: Flow Cytometry, Derivative Assay, Clone Assay, Mutagenesis

    Characterization of clonal lymphoid lines mutant for combinations of pro-apoptotic BCL2 family proteins. a Whole-cell lysates from Bax −/− Bak1 −/− and three independent Bax −/− Bak1 −/− Bim −/− cell clones treated with 1 µM Dex treatment for 24 hrs were subjected to western blot analysis to detect BIM protein. Upper panel: WEHI7 mutant lines; lower panel: p53 −/− T lymphoma mutant lines. b Bax +/+ Bak1 +/+ WEHI7 cells expressing Cas9 were transduced with sgRNAs targeting mouse Bim , Bmf and Puma . Following treatment with doxycycline to induce sgRNA expression, clones were isolated and validated for absence of BIM, BMF, and PUMA by western blotting after 24 hrs treatment with 1 µM Dex. c Wild type (WT) and Bax −/− Bak1 −/− WEHI7 cells were treated for 24 hrs with 1 µM Dex and lysates were run on replicate gels and analyzed by western blot using antibodies specific for the indicated BCL2 family proteins. Roman numerals to the left of blots (i–vi) indicate the membranes probed. d Whole-cell lysates from Bax +/+ Bak1 +/+ and Bax −/− Bak1 −/− p53 −/− T lymphoma cells treated with 1 µM Dex for 24 hrs were tested by western for expression of BIM. Note that the absence of protein in the Dex-treated WT p53 −/− T lymphoma cells is due to the death of most of the cells by 24 hours.

    Journal: Cell Death & Disease

    Article Title: Glucocorticoids can induce BIM to trigger apoptosis in the absence of BAX and BAK1

    doi: 10.1038/s41419-020-2599-5

    Figure Lengend Snippet: Characterization of clonal lymphoid lines mutant for combinations of pro-apoptotic BCL2 family proteins. a Whole-cell lysates from Bax −/− Bak1 −/− and three independent Bax −/− Bak1 −/− Bim −/− cell clones treated with 1 µM Dex treatment for 24 hrs were subjected to western blot analysis to detect BIM protein. Upper panel: WEHI7 mutant lines; lower panel: p53 −/− T lymphoma mutant lines. b Bax +/+ Bak1 +/+ WEHI7 cells expressing Cas9 were transduced with sgRNAs targeting mouse Bim , Bmf and Puma . Following treatment with doxycycline to induce sgRNA expression, clones were isolated and validated for absence of BIM, BMF, and PUMA by western blotting after 24 hrs treatment with 1 µM Dex. c Wild type (WT) and Bax −/− Bak1 −/− WEHI7 cells were treated for 24 hrs with 1 µM Dex and lysates were run on replicate gels and analyzed by western blot using antibodies specific for the indicated BCL2 family proteins. Roman numerals to the left of blots (i–vi) indicate the membranes probed. d Whole-cell lysates from Bax +/+ Bak1 +/+ and Bax −/− Bak1 −/− p53 −/− T lymphoma cells treated with 1 µM Dex for 24 hrs were tested by western for expression of BIM. Note that the absence of protein in the Dex-treated WT p53 −/− T lymphoma cells is due to the death of most of the cells by 24 hours.

    Article Snippet: Antibodies and reagents Antibodies used were to ACTIN (AC-15, Sigma #A1978), MCL-1 (Rockland #600-401-394S), BAX (N-20 Santa Cruz Biotechnology #sc-493), BAK1 (aa23-38, #B5897 Sigma), BCL-2 (#610539, BD Biosciences), Cytochrome c (#556433, BD Biosciences), VDAC2 (M.T.

    Techniques: Mutagenesis, Clone Assay, Western Blot, Expressing, Transduction, Isolation

    BAX and BAK can be autoactivated by DNA damage independently of activators BID, BIM, PUMA and NOXA through downregulation of BCL-2, BCL-X L and MCL-1 (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase, Bad, Bmf or Bik . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (b) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were infected with retrovirus expressing GFP or the indicated BH3-only proteins to induce spontaneous apoptosis. NOXA denotes human NOXA. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide, tunicamycin (TC) or thapsigargin (TG), were subjected to immunoblot analysis using the indicated antibodies. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 18h, and subjected to immunoblot analysis using the indicated antibodies. (e) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 36h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (f) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bcl-2, Bcl-x L and/or Mcl-1 to induce spontaneous apoptosis. After 2 days, cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (g) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide in the presence of the pancaspase inhibitor Q-VD-OPh to preserve cell integrity upon apoptosis induction. After 24 h, cells were permeabilized with digitonin and subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. (h) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs transfected with scrambled siRNA or siRNA against Bcl-x L and Mcl-1 were subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. **, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: BAX and BAK can be autoactivated by DNA damage independently of activators BID, BIM, PUMA and NOXA through downregulation of BCL-2, BCL-X L and MCL-1 (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase, Bad, Bmf or Bik . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (b) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were infected with retrovirus expressing GFP or the indicated BH3-only proteins to induce spontaneous apoptosis. NOXA denotes human NOXA. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide, tunicamycin (TC) or thapsigargin (TG), were subjected to immunoblot analysis using the indicated antibodies. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 18h, and subjected to immunoblot analysis using the indicated antibodies. (e) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 36h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (f) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bcl-2, Bcl-x L and/or Mcl-1 to induce spontaneous apoptosis. After 2 days, cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (g) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide in the presence of the pancaspase inhibitor Q-VD-OPh to preserve cell integrity upon apoptosis induction. After 24 h, cells were permeabilized with digitonin and subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. (h) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs transfected with scrambled siRNA or siRNA against Bcl-x L and Mcl-1 were subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. **, P

    Article Snippet: Antibodies used for immunoblot analysis are listed as followed: anti-BAK (NT, Upstate), anti-BAK (G23, Santa Cruz Biotechnology), anti-BAX (N-20, Santa Cruz Biotechnology), anti-HA (12CA5), anti-BCL-XL (#2762, Cell Signaling Technology), anti-BCL-2 (3F11), anti-MCL-1 (#600-401-394, Rockland), anti-cytochrome c (#556433, Pharmingen), anti-LDH (#100-1173, Rockland), anti-VDAC1 (ab16814, Abcam), anti-cleaved Caspase-3 (#9661, Cell Signaling Technology), anti-PARP (#9542, Cell Signaling Technology), anti-BAD (C-20, Santa Cruz), anti-APAF-1 (AB16941, Upstate Biotechnology), anti-Caspase-9 (#9504, Cell Signaling Technology), and anti-actin (Chemicon).

    Techniques: Transformation Assay, Infection, Expressing, shRNA, Luciferase, Staining, Transfection

    Noxa deficiency further protects Bid −/− Bim −/− Puma −/− mouse embryonic fibroblasts or small intestine from apoptosis (a) The mRNA levels of Noxa in the indicated tissues or cells were assessed by qRT-PCR. Data are normalized against 18S rRNA (mean ± s.d., n = 3 independent experiments). (b) Primary MEFs generated from E13.5 wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO mouse embryos were untreated, or cultured in the absence of serum or glucose for 3 days, or in the presence of tunicamycin (TC) or thapsigargin (TG) for 2 days. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (c and d) Apoptosis in the small intestinal crypts of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), Bid −/− Bim −/− Puma −/− Noxa −/− QKO (n = 2), or conditional Bax and Bak DKO (n = 2) mice at 8 to 17 weeks of age at 4 h after 18 Gy whole body irradiation was assessed by TUNEL staining (brown, magnification 400×). 300 small intestinal crypts from each mouse were analyzed. Representative light microscopy images are shown in (c). Scale bars, 50 µm. The number of TUNEL positive cells in the crypts was quantified and summarized in (d) (mean ± s.d.). (e) CD4 + T cells purified from the spleens of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), or Bid −/− Bim −/− Puma −/− Noxa −/− QKO mice (n = 3) at 8 to 10 weeks of age were cultured in the absence of cytokine, in the presence of etoposide, in the presence of dexamethasone, or after exposure to 2.5 Gy γ-irradiation. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d.). **, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: Noxa deficiency further protects Bid −/− Bim −/− Puma −/− mouse embryonic fibroblasts or small intestine from apoptosis (a) The mRNA levels of Noxa in the indicated tissues or cells were assessed by qRT-PCR. Data are normalized against 18S rRNA (mean ± s.d., n = 3 independent experiments). (b) Primary MEFs generated from E13.5 wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO mouse embryos were untreated, or cultured in the absence of serum or glucose for 3 days, or in the presence of tunicamycin (TC) or thapsigargin (TG) for 2 days. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (c and d) Apoptosis in the small intestinal crypts of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), Bid −/− Bim −/− Puma −/− Noxa −/− QKO (n = 2), or conditional Bax and Bak DKO (n = 2) mice at 8 to 17 weeks of age at 4 h after 18 Gy whole body irradiation was assessed by TUNEL staining (brown, magnification 400×). 300 small intestinal crypts from each mouse were analyzed. Representative light microscopy images are shown in (c). Scale bars, 50 µm. The number of TUNEL positive cells in the crypts was quantified and summarized in (d) (mean ± s.d.). (e) CD4 + T cells purified from the spleens of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), or Bid −/− Bim −/− Puma −/− Noxa −/− QKO mice (n = 3) at 8 to 10 weeks of age were cultured in the absence of cytokine, in the presence of etoposide, in the presence of dexamethasone, or after exposure to 2.5 Gy γ-irradiation. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d.). **, P

    Article Snippet: Antibodies used for immunoblot analysis are listed as followed: anti-BAK (NT, Upstate), anti-BAK (G23, Santa Cruz Biotechnology), anti-BAX (N-20, Santa Cruz Biotechnology), anti-HA (12CA5), anti-BCL-XL (#2762, Cell Signaling Technology), anti-BCL-2 (3F11), anti-MCL-1 (#600-401-394, Rockland), anti-cytochrome c (#556433, Pharmingen), anti-LDH (#100-1173, Rockland), anti-VDAC1 (ab16814, Abcam), anti-cleaved Caspase-3 (#9661, Cell Signaling Technology), anti-PARP (#9542, Cell Signaling Technology), anti-BAD (C-20, Santa Cruz), anti-APAF-1 (AB16941, Upstate Biotechnology), anti-Caspase-9 (#9504, Cell Signaling Technology), and anti-actin (Chemicon).

    Techniques: Quantitative RT-PCR, Generated, Cell Culture, Staining, Mouse Assay, Irradiation, TUNEL Assay, Light Microscopy, Purification

    Quadruple deficiency of Bid, Bim, Puma and Noxa abrogates apoptosis in transformed mouse embryonic fibroblasts triggered by growth factor deprivation and ER stress but not genotoxic stress (a–g) E1A/Ras-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in the absence of serum (a), glucose (b) or glutamine (c), or in the presence of tunicamycin (d), thapsigargin (e) or etoposide (f), or irradiated with UV-C (g). Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). (h) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in in the presence of tunicamycin, thapsigargin or etoposide, or irradiated with UV-C. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). *, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: Quadruple deficiency of Bid, Bim, Puma and Noxa abrogates apoptosis in transformed mouse embryonic fibroblasts triggered by growth factor deprivation and ER stress but not genotoxic stress (a–g) E1A/Ras-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in the absence of serum (a), glucose (b) or glutamine (c), or in the presence of tunicamycin (d), thapsigargin (e) or etoposide (f), or irradiated with UV-C (g). Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). (h) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in in the presence of tunicamycin, thapsigargin or etoposide, or irradiated with UV-C. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). *, P

    Article Snippet: Antibodies used for immunoblot analysis are listed as followed: anti-BAK (NT, Upstate), anti-BAK (G23, Santa Cruz Biotechnology), anti-BAX (N-20, Santa Cruz Biotechnology), anti-HA (12CA5), anti-BCL-XL (#2762, Cell Signaling Technology), anti-BCL-2 (3F11), anti-MCL-1 (#600-401-394, Rockland), anti-cytochrome c (#556433, Pharmingen), anti-LDH (#100-1173, Rockland), anti-VDAC1 (ab16814, Abcam), anti-cleaved Caspase-3 (#9661, Cell Signaling Technology), anti-PARP (#9542, Cell Signaling Technology), anti-BAD (C-20, Santa Cruz), anti-APAF-1 (AB16941, Upstate Biotechnology), anti-Caspase-9 (#9504, Cell Signaling Technology), and anti-actin (Chemicon).

    Techniques: Transformation Assay, Cell Culture, Irradiation, Staining

    DNA damage activates BAX and BAK-dependent mitochondrial apoptosis in transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO mouse embryonic fibroblasts (a) SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide for the indicated times, were subjected to subcellular fractionation. Cytosolic and mitochondrial fractions were analyzed by anti-cytochrome c, anti-LDH and anti-VDAC1 immunoblots. LDH and VDAC1 serve as cytosolic and mitochondrial controls, respectively. (b) SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide for the indicated times, were analyzed for Caspase-3/7 activities (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide (etop) or tunicamycin (TC), were analyzed by anti-PARP, anti-cleaved Caspase-3, and anti-actin immunoblots. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase or Apaf-1 , or transfected with scrambled siRNA (siScr) or siRNA against Cytochrome c or Caspase-9 . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (e) Mitochondria isolated from SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs untreated or treated with etoposide for 15 h (WT) or 36 h (QKO) were subjected to BMH crosslinking. The BAX and BAK homo-oligomers were detected by anti-BAX and anti-BAK immunoblots, respectively. (f) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bax and/or Bak . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). *, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: DNA damage activates BAX and BAK-dependent mitochondrial apoptosis in transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO mouse embryonic fibroblasts (a) SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide for the indicated times, were subjected to subcellular fractionation. Cytosolic and mitochondrial fractions were analyzed by anti-cytochrome c, anti-LDH and anti-VDAC1 immunoblots. LDH and VDAC1 serve as cytosolic and mitochondrial controls, respectively. (b) SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide for the indicated times, were analyzed for Caspase-3/7 activities (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide (etop) or tunicamycin (TC), were analyzed by anti-PARP, anti-cleaved Caspase-3, and anti-actin immunoblots. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase or Apaf-1 , or transfected with scrambled siRNA (siScr) or siRNA against Cytochrome c or Caspase-9 . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (e) Mitochondria isolated from SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs untreated or treated with etoposide for 15 h (WT) or 36 h (QKO) were subjected to BMH crosslinking. The BAX and BAK homo-oligomers were detected by anti-BAX and anti-BAK immunoblots, respectively. (f) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bax and/or Bak . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). *, P

    Article Snippet: Antibodies used for immunoblot analysis are listed as followed: anti-BAK (NT, Upstate), anti-BAK (G23, Santa Cruz Biotechnology), anti-BAX (N-20, Santa Cruz Biotechnology), anti-HA (12CA5), anti-BCL-XL (#2762, Cell Signaling Technology), anti-BCL-2 (3F11), anti-MCL-1 (#600-401-394, Rockland), anti-cytochrome c (#556433, Pharmingen), anti-LDH (#100-1173, Rockland), anti-VDAC1 (ab16814, Abcam), anti-cleaved Caspase-3 (#9661, Cell Signaling Technology), anti-PARP (#9542, Cell Signaling Technology), anti-BAD (C-20, Santa Cruz), anti-APAF-1 (AB16941, Upstate Biotechnology), anti-Caspase-9 (#9504, Cell Signaling Technology), and anti-actin (Chemicon).

    Techniques: Transformation Assay, Fractionation, Western Blot, Infection, Expressing, shRNA, Luciferase, Transfection, Staining, Isolation

    BCL-X L is superior to BCL-2 and MCL-1 in preventing DNA damage-induced apoptosis due to its dual inhibition of BAX and BAK as well as higher protein stability (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, HA-BCL-2, HA-BCL-X L or HA-MCL-1 were subjected to anti-HA immunoprecipitation in 0.2% NP-40 or 1% CHAPS lysis buffer. The input (5%) and immunoprecipitates were analyzed by anti-BAX, anti-BAK, and anti-HA immunoblots. (b) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, HA-BCL-2, HA-BCL-X L or HA-MCL-1 were untreated or treated with etoposide. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). The expression of HA-BCL-2, HA-BCL-X L or HA-MCL-1 was detected by an anti-HA immunoblot with or without etoposide treatment for 12 h. (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, BCL-2 or BCL-X L were transfected with scrambled siRNA (siScr) or siRNA against Bax or Bak . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (d) SV40-transformed wild-type MEFs stably expressing HA-tagged BCL-2 or BCL-X L were untreated or treated with etoposide. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). The expression of HA-tagged BCL-2 and BCL-X L was detected by an anti-HA immunoblot. (e) SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, wild-type BCL-X L , BCL-X L mutant 1 (F131V/D133A) or BCL-X L mutant 8 (G138E/R139L/I140N) were untreated or treated with etoposide. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). The expression of BCL-X L was analyzed by an anti-BCL-X L immunoblot. *, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: BCL-X L is superior to BCL-2 and MCL-1 in preventing DNA damage-induced apoptosis due to its dual inhibition of BAX and BAK as well as higher protein stability (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, HA-BCL-2, HA-BCL-X L or HA-MCL-1 were subjected to anti-HA immunoprecipitation in 0.2% NP-40 or 1% CHAPS lysis buffer. The input (5%) and immunoprecipitates were analyzed by anti-BAX, anti-BAK, and anti-HA immunoblots. (b) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, HA-BCL-2, HA-BCL-X L or HA-MCL-1 were untreated or treated with etoposide. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). The expression of HA-BCL-2, HA-BCL-X L or HA-MCL-1 was detected by an anti-HA immunoblot with or without etoposide treatment for 12 h. (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, BCL-2 or BCL-X L were transfected with scrambled siRNA (siScr) or siRNA against Bax or Bak . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (d) SV40-transformed wild-type MEFs stably expressing HA-tagged BCL-2 or BCL-X L were untreated or treated with etoposide. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). The expression of HA-tagged BCL-2 and BCL-X L was detected by an anti-HA immunoblot. (e) SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, wild-type BCL-X L , BCL-X L mutant 1 (F131V/D133A) or BCL-X L mutant 8 (G138E/R139L/I140N) were untreated or treated with etoposide. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). The expression of BCL-X L was analyzed by an anti-BCL-X L immunoblot. *, P

    Article Snippet: Antibodies used for immunoblot analysis are listed as followed: anti-BAK (NT, Upstate), anti-BAK (G23, Santa Cruz Biotechnology), anti-BAX (N-20, Santa Cruz Biotechnology), anti-HA (12CA5), anti-BCL-XL (#2762, Cell Signaling Technology), anti-BCL-2 (3F11), anti-MCL-1 (#600-401-394, Rockland), anti-cytochrome c (#556433, Pharmingen), anti-LDH (#100-1173, Rockland), anti-VDAC1 (ab16814, Abcam), anti-cleaved Caspase-3 (#9661, Cell Signaling Technology), anti-PARP (#9542, Cell Signaling Technology), anti-BAD (C-20, Santa Cruz), anti-APAF-1 (AB16941, Upstate Biotechnology), anti-Caspase-9 (#9504, Cell Signaling Technology), and anti-actin (Chemicon).

    Techniques: Inhibition, Transformation Assay, Stable Transfection, Expressing, Immunoprecipitation, Lysis, Western Blot, Staining, Transfection, Mutagenesis

    NOXA directly activates BAX and BAK independently of BID, BIM and PUMA (a) Mitochondria isolated from wild-type or Bax −/− Bak −/− MEFs were incubated with the indicated IVTT proteins generated using reticulocyte lysates or wheat germ extract (WGE) at 30°C for 30 min, after which the release of cytochrome c was quantified by ELISA assays (mean ± s.d., n = 3 independent experiments). (b) Isolated wild-type mitochondria were incubated with the indicated amounts of IVTT mouse NOXA protein (WGE) and the release of cytochrome c was quantified (mean ± s.d., n = 3 independent experiments). (c) Isolated wild-type mitochondria were incubated with the indicated IVTT proteins (WGE) and the release of cytochrome c was quantified (mean ± s.d., n = 3 independent experiments). (d) Radiolabeled IVTT NOXA or tBID protein was incubated with GST, GST-BAXΔC or GST-BAKΔC protein immobilized on glutathione beads. The precipitates and input were analyzed by Nu-PAGE and autoradiography. (e) Isolated wild-type mitochondria were incubated with the indicated IVTT proteins for 30 min then treated with BMH crosslinker. The BAX and BAK homo-oligomers were detected by anti-BAX and anti-BAK immunoblots, respectively. (f) Mitochondria isolated from wild-type or Bid −/− Bim −/− Puma −/− MEFs were incubated with IVTT NOXA (WGE) at 30°C for the indicated times and release of cytochrome c was quantified (mean ± s.d., n = 3 independent experiments). (g) Bid −/− Bim −/− Puma −/− MEFs were infected with the indicated retrovirus. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). The expression of the indicated proteins was detected by an anti-HA immunoblot. (h and i) ANTS/DPX (fluorophore/quencher)-encapsulated liposomes were incubated with recombinant BAX protein plus the indicated IVTT proteins generated using WGE. The release of entrapped fluorophore monitored with time is shown in (h) (mean ± s.d., n = 3 independent experiments). The release of entrapped fluorophore at 60 min is shown in (i) (mean ± s.d., n = 3 independent experiments). (j and k) ANTS/DPX-encapsulated liposomes were incubated with recombinant BAX protein plus the indicated IVTT proteins (WGE). The release of entrapped fluorophore monitored with time is shown in (j) (mean ± s.d., n = 3 independent experiments). The release of entrapped fluorophore at 60 min is shown in (k) (mean ± s.d., n =3 independent experiments). **, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: NOXA directly activates BAX and BAK independently of BID, BIM and PUMA (a) Mitochondria isolated from wild-type or Bax −/− Bak −/− MEFs were incubated with the indicated IVTT proteins generated using reticulocyte lysates or wheat germ extract (WGE) at 30°C for 30 min, after which the release of cytochrome c was quantified by ELISA assays (mean ± s.d., n = 3 independent experiments). (b) Isolated wild-type mitochondria were incubated with the indicated amounts of IVTT mouse NOXA protein (WGE) and the release of cytochrome c was quantified (mean ± s.d., n = 3 independent experiments). (c) Isolated wild-type mitochondria were incubated with the indicated IVTT proteins (WGE) and the release of cytochrome c was quantified (mean ± s.d., n = 3 independent experiments). (d) Radiolabeled IVTT NOXA or tBID protein was incubated with GST, GST-BAXΔC or GST-BAKΔC protein immobilized on glutathione beads. The precipitates and input were analyzed by Nu-PAGE and autoradiography. (e) Isolated wild-type mitochondria were incubated with the indicated IVTT proteins for 30 min then treated with BMH crosslinker. The BAX and BAK homo-oligomers were detected by anti-BAX and anti-BAK immunoblots, respectively. (f) Mitochondria isolated from wild-type or Bid −/− Bim −/− Puma −/− MEFs were incubated with IVTT NOXA (WGE) at 30°C for the indicated times and release of cytochrome c was quantified (mean ± s.d., n = 3 independent experiments). (g) Bid −/− Bim −/− Puma −/− MEFs were infected with the indicated retrovirus. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). The expression of the indicated proteins was detected by an anti-HA immunoblot. (h and i) ANTS/DPX (fluorophore/quencher)-encapsulated liposomes were incubated with recombinant BAX protein plus the indicated IVTT proteins generated using WGE. The release of entrapped fluorophore monitored with time is shown in (h) (mean ± s.d., n = 3 independent experiments). The release of entrapped fluorophore at 60 min is shown in (i) (mean ± s.d., n = 3 independent experiments). (j and k) ANTS/DPX-encapsulated liposomes were incubated with recombinant BAX protein plus the indicated IVTT proteins (WGE). The release of entrapped fluorophore monitored with time is shown in (j) (mean ± s.d., n = 3 independent experiments). The release of entrapped fluorophore at 60 min is shown in (k) (mean ± s.d., n =3 independent experiments). **, P

    Article Snippet: Antibodies used for immunoblot analysis are listed as followed: anti-BAK (NT, Upstate), anti-BAK (G23, Santa Cruz Biotechnology), anti-BAX (N-20, Santa Cruz Biotechnology), anti-HA (12CA5), anti-BCL-XL (#2762, Cell Signaling Technology), anti-BCL-2 (3F11), anti-MCL-1 (#600-401-394, Rockland), anti-cytochrome c (#556433, Pharmingen), anti-LDH (#100-1173, Rockland), anti-VDAC1 (ab16814, Abcam), anti-cleaved Caspase-3 (#9661, Cell Signaling Technology), anti-PARP (#9542, Cell Signaling Technology), anti-BAD (C-20, Santa Cruz), anti-APAF-1 (AB16941, Upstate Biotechnology), anti-Caspase-9 (#9504, Cell Signaling Technology), and anti-actin (Chemicon).

    Techniques: Isolation, Incubation, Generated, Enzyme-linked Immunosorbent Assay, Polyacrylamide Gel Electrophoresis, Autoradiography, Western Blot, Infection, Staining, Expressing, Recombinant

    TRIM55 silencing inhibits I/R- and miR-378a-3p inhibitor-induced apoptosis of H9C2 cardiomyocytes. H9C2 cardiomyocytes were transfected with three TRIM55-siRNAs (siRNA-1, siRNA-2, siRNA-3) or scramble siRNA (siNC). ( A , B ) TRIM55 expression was measured. H9C2 cardiomyocytes following I/R injury were transfected with the TRIM55-siRNA and/or miR-378a-3p inhibitor. ( C , D ) Cell apoptosis was measured by flow cytometry. ( E – H ) Expression of TRIM55, DUSP1, JNK1/2, cleaved PARP and caspase-3, Bax, and Bcl-2 was measured. *** P

    Journal: Aging (Albany NY)

    Article Title: miR-378a-3p inhibits ischemia/reperfusion-induced apoptosis in H9C2 cardiomyocytes by targeting TRIM55 via the DUSP1-JNK1/2 signaling pathway

    doi: 10.18632/aging.103106

    Figure Lengend Snippet: TRIM55 silencing inhibits I/R- and miR-378a-3p inhibitor-induced apoptosis of H9C2 cardiomyocytes. H9C2 cardiomyocytes were transfected with three TRIM55-siRNAs (siRNA-1, siRNA-2, siRNA-3) or scramble siRNA (siNC). ( A , B ) TRIM55 expression was measured. H9C2 cardiomyocytes following I/R injury were transfected with the TRIM55-siRNA and/or miR-378a-3p inhibitor. ( C , D ) Cell apoptosis was measured by flow cytometry. ( E – H ) Expression of TRIM55, DUSP1, JNK1/2, cleaved PARP and caspase-3, Bax, and Bcl-2 was measured. *** P

    Article Snippet: The anti-Bcl-2 and anti-Bax antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Transfection, Expressing, Flow Cytometry

    DUSP1 overexpression inhibits I/R- and TRIM55 overexpression-induced apoptosis of H9C2 cardiomyocytes. H9C2 cardiomyocytes following I/R injury were transduced with a TRIM55 and/or DUSP1 expression vector or blank vector. ( A , B ) Cell apoptosis was measured by flow cytometry. ( C – H ) The expression of DUSP1, JNK1/2, JNK1/2, cleavage of PARP and caspase-3, Bax, and Bcl-2 was also measured. *** P

    Journal: Aging (Albany NY)

    Article Title: miR-378a-3p inhibits ischemia/reperfusion-induced apoptosis in H9C2 cardiomyocytes by targeting TRIM55 via the DUSP1-JNK1/2 signaling pathway

    doi: 10.18632/aging.103106

    Figure Lengend Snippet: DUSP1 overexpression inhibits I/R- and TRIM55 overexpression-induced apoptosis of H9C2 cardiomyocytes. H9C2 cardiomyocytes following I/R injury were transduced with a TRIM55 and/or DUSP1 expression vector or blank vector. ( A , B ) Cell apoptosis was measured by flow cytometry. ( C – H ) The expression of DUSP1, JNK1/2, JNK1/2, cleavage of PARP and caspase-3, Bax, and Bcl-2 was also measured. *** P

    Article Snippet: The anti-Bcl-2 and anti-Bax antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Over Expression, Transduction, Expressing, Plasmid Preparation, Flow Cytometry

    The miR-378a-3p mimic inhibits I/R-induced apoptosis in rats. Myocardial I/R model rats were injected with 50 mg/kg of the miR-378a-3p mimic or negative control (NC) 24 h before LCA ligation. ( A – C ) Histological assessment of the myocardium was performed by H E staining and TUNEL. Scale bar: 100 μm. ( D – F ) The expression of miR-378a-3p, TRIM55, Bax, and Bcl-2 was measured by real-time PCR. ( G ) The expression of TRIM55, DUSP1, p-JNK1/2, JNK1/2, cleavage of PARP and caspase-3, Bax, and Bcl-2 was measured by western blotting. *** P

    Journal: Aging (Albany NY)

    Article Title: miR-378a-3p inhibits ischemia/reperfusion-induced apoptosis in H9C2 cardiomyocytes by targeting TRIM55 via the DUSP1-JNK1/2 signaling pathway

    doi: 10.18632/aging.103106

    Figure Lengend Snippet: The miR-378a-3p mimic inhibits I/R-induced apoptosis in rats. Myocardial I/R model rats were injected with 50 mg/kg of the miR-378a-3p mimic or negative control (NC) 24 h before LCA ligation. ( A – C ) Histological assessment of the myocardium was performed by H E staining and TUNEL. Scale bar: 100 μm. ( D – F ) The expression of miR-378a-3p, TRIM55, Bax, and Bcl-2 was measured by real-time PCR. ( G ) The expression of TRIM55, DUSP1, p-JNK1/2, JNK1/2, cleavage of PARP and caspase-3, Bax, and Bcl-2 was measured by western blotting. *** P

    Article Snippet: The anti-Bcl-2 and anti-Bax antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Injection, Negative Control, Ligation, Staining, TUNEL Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    miR-378a-3p is upregulated in I/R-induced H9C2 cardiomyocytes and inhibits cell apoptosis. ( A ) miR-378a-3p expression was measured by Real-time PCR in H9C2 cardiomyocytes following 3 h ischemia and 6, 12 or 24 h reperfusion. H9C2 cardiomyocytes following 3 h ischemia and 24 h reperfusion were transfected with a miR-378a-3p mimic, inhibitor or negative control (NC) and those without I/R injury were used as a control. ( B ) miR-378a-3p expression, ( C , D ) cell apoptosis, and ( E – J ) protein expression of DUSP1, p-JNK1/2, JNK1/2, cleaved PARP and caspase-3, Bax, and Bcl-2 were measured. *** P

    Journal: Aging (Albany NY)

    Article Title: miR-378a-3p inhibits ischemia/reperfusion-induced apoptosis in H9C2 cardiomyocytes by targeting TRIM55 via the DUSP1-JNK1/2 signaling pathway

    doi: 10.18632/aging.103106

    Figure Lengend Snippet: miR-378a-3p is upregulated in I/R-induced H9C2 cardiomyocytes and inhibits cell apoptosis. ( A ) miR-378a-3p expression was measured by Real-time PCR in H9C2 cardiomyocytes following 3 h ischemia and 6, 12 or 24 h reperfusion. H9C2 cardiomyocytes following 3 h ischemia and 24 h reperfusion were transfected with a miR-378a-3p mimic, inhibitor or negative control (NC) and those without I/R injury were used as a control. ( B ) miR-378a-3p expression, ( C , D ) cell apoptosis, and ( E – J ) protein expression of DUSP1, p-JNK1/2, JNK1/2, cleaved PARP and caspase-3, Bax, and Bcl-2 were measured. *** P

    Article Snippet: The anti-Bcl-2 and anti-Bax antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control

    The ER-targeted Bak can induce apoptosis in the absence of endogenous Bax and Bak. Wild-type and bax − / − bak − / − MEFs were infected with retroviruses expressing GFP or GFP together with wild-type murine Bak, Bak-ActA, Bak-cb5, or Bak-ΔC. 48 h after infection, 1 μg/ml DAPI was added to stain the apoptotic cells. (A) bax − / − bak − / − cells were photographed using a FITC or DAPI filter. (B) The percentage of dead cells was determined by the ratio of DAPI-positive cells to GFP-positive cells. In addition to the retroviral infection of the Bak mutants, bax − / − bak − / − cells were also coinfected with retrovirus expressing Bcl-xL, or infected in the presence of 20 μg/ml Z-VAD-fmk. Data shown are the average of three independent experiments. (C) 24 h after infection, bax − / − bak − / − cells were trypsinized. A portion of each sample was subjected to FACS ® to determine the expression efficiency indicated by GFP-positive cells. The rest of the cells were fixed in 0.25% paraformaldehyde and stained with an anti-Bak antibody that recognizes the active form of murine Bak. The number in each dot plot represents the percentage of gated events (R1).

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: The ER-targeted Bak can induce apoptosis in the absence of endogenous Bax and Bak. Wild-type and bax − / − bak − / − MEFs were infected with retroviruses expressing GFP or GFP together with wild-type murine Bak, Bak-ActA, Bak-cb5, or Bak-ΔC. 48 h after infection, 1 μg/ml DAPI was added to stain the apoptotic cells. (A) bax − / − bak − / − cells were photographed using a FITC or DAPI filter. (B) The percentage of dead cells was determined by the ratio of DAPI-positive cells to GFP-positive cells. In addition to the retroviral infection of the Bak mutants, bax − / − bak − / − cells were also coinfected with retrovirus expressing Bcl-xL, or infected in the presence of 20 μg/ml Z-VAD-fmk. Data shown are the average of three independent experiments. (C) 24 h after infection, bax − / − bak − / − cells were trypsinized. A portion of each sample was subjected to FACS ® to determine the expression efficiency indicated by GFP-positive cells. The rest of the cells were fixed in 0.25% paraformaldehyde and stained with an anti-Bak antibody that recognizes the active form of murine Bak. The number in each dot plot represents the percentage of gated events (R1).

    Article Snippet: Immunoblotting was performed by use of antibodies against Bax (N-20; Santa Cruz Biotechnology, Inc.), Bak (Upstate Biotechnology), COX IV (Molecular Probes), Tom40 (Santa Cruz Biotechnology, Inc.), calnexin (C-20; Santa Cruz Biotechnology, Inc.), caspase 12, and CHOP (B-3; Santa Cruz Biotechnology, Inc.).

    Techniques: Infection, Expressing, Staining, FACS

    ER-targeted Bak can induce selective cleavage of caspase 12, but not of caspase 7. bax − / − bak − / − cells were infected with GFP vector control, Bak-IRES-GFP, Bak-ActA-IRES-GFP, Bak-cb5-IRES-GFP, and Bak-ΔC-IRES-GFP at a high multiplicity. After infection, cells were lysed. Caspase 12, caspase 7, or PARP were detected by immunoblotting using respective antibodies.

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: ER-targeted Bak can induce selective cleavage of caspase 12, but not of caspase 7. bax − / − bak − / − cells were infected with GFP vector control, Bak-IRES-GFP, Bak-ActA-IRES-GFP, Bak-cb5-IRES-GFP, and Bak-ΔC-IRES-GFP at a high multiplicity. After infection, cells were lysed. Caspase 12, caspase 7, or PARP were detected by immunoblotting using respective antibodies.

    Article Snippet: Immunoblotting was performed by use of antibodies against Bax (N-20; Santa Cruz Biotechnology, Inc.), Bak (Upstate Biotechnology), COX IV (Molecular Probes), Tom40 (Santa Cruz Biotechnology, Inc.), calnexin (C-20; Santa Cruz Biotechnology, Inc.), caspase 12, and CHOP (B-3; Santa Cruz Biotechnology, Inc.).

    Techniques: Infection, Plasmid Preparation

    Caspase 12 functions downstream of the multidomain proapoptotic Bcl-2 family proteins. (A) ER stress–induced caspase 12 cleavage is dependent on Bax and Bak. Immortalized wild-type and bax − / − bak − / − MEFs and NIH3T3 cells were treated with brefeldin A (BFA; 10 μg/ml), thapsigargin (Thap; 2 μM), or tunicamycin (Tuni; 10 μg/ml) for 30 h. Caspase 12 level and processing were examined by immunoblotting of 20 μg of total cellular protein from samples as indicated. An ∼42-kD caspase 12 fragment is indicated by the arrow. Induction of CHOP expression is shown as an indicator of the ER stress response. A nonspecific band (NS) is shown as a loading control. (B) Caspase 12 kills bax − / − bak − / − cells. Wild-type and bax − / − bak − / − MEFs were cotransfected with pEGFP and constructs expressing caspase 3, caspase 9, caspase 12, and t-caspase 12. 24 h after transfection, cells were stained with DAPI, and cell death percentage was determined by the ratio of DAPI-positive to GFP-positive cells.

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: Caspase 12 functions downstream of the multidomain proapoptotic Bcl-2 family proteins. (A) ER stress–induced caspase 12 cleavage is dependent on Bax and Bak. Immortalized wild-type and bax − / − bak − / − MEFs and NIH3T3 cells were treated with brefeldin A (BFA; 10 μg/ml), thapsigargin (Thap; 2 μM), or tunicamycin (Tuni; 10 μg/ml) for 30 h. Caspase 12 level and processing were examined by immunoblotting of 20 μg of total cellular protein from samples as indicated. An ∼42-kD caspase 12 fragment is indicated by the arrow. Induction of CHOP expression is shown as an indicator of the ER stress response. A nonspecific band (NS) is shown as a loading control. (B) Caspase 12 kills bax − / − bak − / − cells. Wild-type and bax − / − bak − / − MEFs were cotransfected with pEGFP and constructs expressing caspase 3, caspase 9, caspase 12, and t-caspase 12. 24 h after transfection, cells were stained with DAPI, and cell death percentage was determined by the ratio of DAPI-positive to GFP-positive cells.

    Article Snippet: Immunoblotting was performed by use of antibodies against Bax (N-20; Santa Cruz Biotechnology, Inc.), Bak (Upstate Biotechnology), COX IV (Molecular Probes), Tom40 (Santa Cruz Biotechnology, Inc.), calnexin (C-20; Santa Cruz Biotechnology, Inc.), caspase 12, and CHOP (B-3; Santa Cruz Biotechnology, Inc.).

    Techniques: Expressing, Construct, Transfection, Staining

    ER stress induces Bax and Bak conformational changes and oligomerization at the ER. (A) ER stresses induce the conformational changes of Bax and Bak. HeLa, MCF7, and 293T cells were treated with thapsigargin (Thap; 2 μM) or tunicamycin (Tuni; 5 μg/ml) for 36 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were incubated with a control antibody (mouse IgG1) and conformation-sensitive antibodies against Bax or Bak, followed by incubation with FITC-conjugated secondary antibody. (B) ER stress induces Bax oligomerization at the ER. Wild-type MEFs were treated with brefeldin A (BFA; 10 μg/ml), Thap (2 μM), or Tuni (10 μg/ml) for 24 h. Cells were resuspended in hypotonic buffer A and disrupted. 5 mM BMH cross-linking reagent was added to cross-link the oligomerized proteins. Cells were subjected to subcellular fractionation to obtain the HM and LM fractions. 20 μg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax. COX IV and calnexin are shown as indicators of the purity of the fractionation and as loading controls.

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: ER stress induces Bax and Bak conformational changes and oligomerization at the ER. (A) ER stresses induce the conformational changes of Bax and Bak. HeLa, MCF7, and 293T cells were treated with thapsigargin (Thap; 2 μM) or tunicamycin (Tuni; 5 μg/ml) for 36 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were incubated with a control antibody (mouse IgG1) and conformation-sensitive antibodies against Bax or Bak, followed by incubation with FITC-conjugated secondary antibody. (B) ER stress induces Bax oligomerization at the ER. Wild-type MEFs were treated with brefeldin A (BFA; 10 μg/ml), Thap (2 μM), or Tuni (10 μg/ml) for 24 h. Cells were resuspended in hypotonic buffer A and disrupted. 5 mM BMH cross-linking reagent was added to cross-link the oligomerized proteins. Cells were subjected to subcellular fractionation to obtain the HM and LM fractions. 20 μg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax. COX IV and calnexin are shown as indicators of the purity of the fractionation and as loading controls.

    Article Snippet: Immunoblotting was performed by use of antibodies against Bax (N-20; Santa Cruz Biotechnology, Inc.), Bak (Upstate Biotechnology), COX IV (Molecular Probes), Tom40 (Santa Cruz Biotechnology, Inc.), calnexin (C-20; Santa Cruz Biotechnology, Inc.), caspase 12, and CHOP (B-3; Santa Cruz Biotechnology, Inc.).

    Techniques: Incubation, Fractionation

    Bax and Bak are localized to the ER in addition to their mitochondrial location. (A) Wild-type and bax − / − bak − / − MEFs as well as HeLa cells were resuspended in hypotonic buffer A and disrupted. Subcellular fractionation was performed to obtain the fractions for cytosol (S-100), HM (mitochondria enriched), and LM (the ER enriched). 20 μg of total protein from each fraction was separated on a 4–12% gradient NuPAGE gel. Antibodies against Bax, Bak, calnexin, and Cox IV were used for immunoblotting. (B) Mouse liver was homogenized in buffer A and fractionated in sucrose gradient as described in the Materials and methods. Fractions from crude mitochondria and the 1.5/1.8 M sucrose interface were probed with indicated antibodies. (C) The LM fraction was not contaminated by mitochondrial outer membrane. Fractions of wild-type MEFs were probed with an anti-Tom40 antibody. (D) Immunoelectron microscopy of Bax (left) and Bak (right) in wild-type MEFs using 10-nm gold particles. The bottom panels are the enlargements of the boxed areas of the top images. The arrowheads point to the ER. mt, mitochondria.

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: Bax and Bak are localized to the ER in addition to their mitochondrial location. (A) Wild-type and bax − / − bak − / − MEFs as well as HeLa cells were resuspended in hypotonic buffer A and disrupted. Subcellular fractionation was performed to obtain the fractions for cytosol (S-100), HM (mitochondria enriched), and LM (the ER enriched). 20 μg of total protein from each fraction was separated on a 4–12% gradient NuPAGE gel. Antibodies against Bax, Bak, calnexin, and Cox IV were used for immunoblotting. (B) Mouse liver was homogenized in buffer A and fractionated in sucrose gradient as described in the Materials and methods. Fractions from crude mitochondria and the 1.5/1.8 M sucrose interface were probed with indicated antibodies. (C) The LM fraction was not contaminated by mitochondrial outer membrane. Fractions of wild-type MEFs were probed with an anti-Tom40 antibody. (D) Immunoelectron microscopy of Bax (left) and Bak (right) in wild-type MEFs using 10-nm gold particles. The bottom panels are the enlargements of the boxed areas of the top images. The arrowheads point to the ER. mt, mitochondria.

    Article Snippet: Immunoblotting was performed by use of antibodies against Bax (N-20; Santa Cruz Biotechnology, Inc.), Bak (Upstate Biotechnology), COX IV (Molecular Probes), Tom40 (Santa Cruz Biotechnology, Inc.), calnexin (C-20; Santa Cruz Biotechnology, Inc.), caspase 12, and CHOP (B-3; Santa Cruz Biotechnology, Inc.).

    Techniques: Fractionation, Immuno-Electron Microscopy

    The ER-targeted Bak-cb5 induces depletion of the ER Ca 2 + pool. (A) Cells with ER-targeted Bak lack thapsigargin-releasable intracellular Ca 2+ . bax − / − bak − / − cells were infected with retroviruses encoding GFP, Bak-IRES-GFP, Bak-ActA-IRES-GFP, or Bak-cb5-IRES-GFP. 48 h after infection, cells were loaded with Indo-1, and the ER Ca 2+ release was measured by flow cytometry. Propidium iodide was added to determine the cell viability. The Ca 2+ flux traces were derived from gated live GFP-positive cells. The [Ca 2+ ] i was calibrated from the measured Indo-1 fluorescence ratio as described in the Materials and methods. The arrowhead indicates the time of thapsigargin addition. (B) Expression of ER-targeted Bak depleted intracellular Ca 2+ storage in the ER. At the start of measurement, extracellular free Ca 2+ concentration was reduced to zero. The arrowhead indicates the time when the extracellular free Ca 2+ concentration was brought back to 2 mM. [Ca 2+ ] i was analyzed as in A. (C and D) bax − / − bak − / − cells were infected with the Bak-cb5-IRES-GFP. (C) The measured Indo-1 fluorescence ratio between λ 405 and λ 475 is presented as an index of change in cytosolic [Ca 2+ ] over time and presented separately for gated live GFP-positive (expressing Bak-cb5) or GFP-negative (not expressing Bak-cb5) cells. The absolute [Ca 2+ ] i in the linear range is indicated on the right. The arrowhead indicates the time of thapsigargin addition. The arrow indicates the time of the addition of 8 mM CaCl 2 to the medium. (D) Mitochondrial potential in GFP-positive (expressing Bak-cb5, green) and in GFP-negative (not expressing Bak-cb5, black) cells was measured by tetramethyl rhodamine ethyl ester (TMRE) staining.

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: The ER-targeted Bak-cb5 induces depletion of the ER Ca 2 + pool. (A) Cells with ER-targeted Bak lack thapsigargin-releasable intracellular Ca 2+ . bax − / − bak − / − cells were infected with retroviruses encoding GFP, Bak-IRES-GFP, Bak-ActA-IRES-GFP, or Bak-cb5-IRES-GFP. 48 h after infection, cells were loaded with Indo-1, and the ER Ca 2+ release was measured by flow cytometry. Propidium iodide was added to determine the cell viability. The Ca 2+ flux traces were derived from gated live GFP-positive cells. The [Ca 2+ ] i was calibrated from the measured Indo-1 fluorescence ratio as described in the Materials and methods. The arrowhead indicates the time of thapsigargin addition. (B) Expression of ER-targeted Bak depleted intracellular Ca 2+ storage in the ER. At the start of measurement, extracellular free Ca 2+ concentration was reduced to zero. The arrowhead indicates the time when the extracellular free Ca 2+ concentration was brought back to 2 mM. [Ca 2+ ] i was analyzed as in A. (C and D) bax − / − bak − / − cells were infected with the Bak-cb5-IRES-GFP. (C) The measured Indo-1 fluorescence ratio between λ 405 and λ 475 is presented as an index of change in cytosolic [Ca 2+ ] over time and presented separately for gated live GFP-positive (expressing Bak-cb5) or GFP-negative (not expressing Bak-cb5) cells. The absolute [Ca 2+ ] i in the linear range is indicated on the right. The arrowhead indicates the time of thapsigargin addition. The arrow indicates the time of the addition of 8 mM CaCl 2 to the medium. (D) Mitochondrial potential in GFP-positive (expressing Bak-cb5, green) and in GFP-negative (not expressing Bak-cb5, black) cells was measured by tetramethyl rhodamine ethyl ester (TMRE) staining.

    Article Snippet: Immunoblotting was performed by use of antibodies against Bax (N-20; Santa Cruz Biotechnology, Inc.), Bak (Upstate Biotechnology), COX IV (Molecular Probes), Tom40 (Santa Cruz Biotechnology, Inc.), calnexin (C-20; Santa Cruz Biotechnology, Inc.), caspase 12, and CHOP (B-3; Santa Cruz Biotechnology, Inc.).

    Techniques: Infection, Flow Cytometry, Cytometry, Derivative Assay, Fluorescence, Expressing, Concentration Assay, Staining

    Bax and Bak localized to the ER can initiate apoptosis. In addition to their mitochondrial localization and activity, Bax and Bak also reside at the ER. Upon ER stress treatment, Bax and Bak can initiate apoptosis from the ER.

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: Bax and Bak localized to the ER can initiate apoptosis. In addition to their mitochondrial localization and activity, Bax and Bak also reside at the ER. Upon ER stress treatment, Bax and Bak can initiate apoptosis from the ER.

    Article Snippet: Immunoblotting was performed by use of antibodies against Bax (N-20; Santa Cruz Biotechnology, Inc.), Bak (Upstate Biotechnology), COX IV (Molecular Probes), Tom40 (Santa Cruz Biotechnology, Inc.), calnexin (C-20; Santa Cruz Biotechnology, Inc.), caspase 12, and CHOP (B-3; Santa Cruz Biotechnology, Inc.).

    Techniques: Activity Assay

    Erdr1 induces apoptosis via regulation of Bcl-2 and Bax in murine melanoma cell lines. ( A ) B16F10 cells were treated with different doses (0, 1, 10, and 100 ng/mL) of recombinant murine Erdr1 for 24 h. Staining with 7-AAD and Annexin V was performed to measure apoptosis induced by recombinant murine Erdr1. After staining, stained cells were analyzed with flow cytometry analysis. The image shows one of the representative experiments of three experiments performed independently; ( B ) Flow cytometry analysis data were converted into a bar graph. The data represents the mean ± SD of one of three independent experiments. *, p

    Journal: International Journal of Molecular Sciences

    Article Title: Erdr1 Suppresses Murine Melanoma Growth via Regulation of Apoptosis

    doi: 10.3390/ijms17010107

    Figure Lengend Snippet: Erdr1 induces apoptosis via regulation of Bcl-2 and Bax in murine melanoma cell lines. ( A ) B16F10 cells were treated with different doses (0, 1, 10, and 100 ng/mL) of recombinant murine Erdr1 for 24 h. Staining with 7-AAD and Annexin V was performed to measure apoptosis induced by recombinant murine Erdr1. After staining, stained cells were analyzed with flow cytometry analysis. The image shows one of the representative experiments of three experiments performed independently; ( B ) Flow cytometry analysis data were converted into a bar graph. The data represents the mean ± SD of one of three independent experiments. *, p

    Article Snippet: Briefly, sections were fixed with cold acetone and then subjected to the blocking step with methanol containing 0.3% H2 O2 for 30 min and 20% normal goat serum for 1 h. After blocking, sections were incubated with rabbit anti-mouse Bcl-2 or Bax antibody (1:100 dilution, Santa Cruz Biotechnology) for 1 h at room temperature.

    Techniques: Recombinant, Staining, Flow Cytometry, Cytometry

    Erdr1 suppresses Bcl-2 and induces Bax in vivo . ( A ) Tumor tissues were lysed, and identical amounts of lysate were loaded onto an acrylamide gel. Western blotting was performed using rabbit anti-mouse Bcl-2 antibody, rabbit anti-mouse Bax antibody, and mouse anti-α-tubulin antibody; ( B ) Bcl-2 and Bax levels in tumor tissues were measured using immunohistochemistry. Sections from tumors were stained with rabbit anti-mouse Bcl-2 antibody and rabbit anti-mouse Bax antibody. Stained sections were examined with the microscope and photographed. Original magnification (100×).

    Journal: International Journal of Molecular Sciences

    Article Title: Erdr1 Suppresses Murine Melanoma Growth via Regulation of Apoptosis

    doi: 10.3390/ijms17010107

    Figure Lengend Snippet: Erdr1 suppresses Bcl-2 and induces Bax in vivo . ( A ) Tumor tissues were lysed, and identical amounts of lysate were loaded onto an acrylamide gel. Western blotting was performed using rabbit anti-mouse Bcl-2 antibody, rabbit anti-mouse Bax antibody, and mouse anti-α-tubulin antibody; ( B ) Bcl-2 and Bax levels in tumor tissues were measured using immunohistochemistry. Sections from tumors were stained with rabbit anti-mouse Bcl-2 antibody and rabbit anti-mouse Bax antibody. Stained sections were examined with the microscope and photographed. Original magnification (100×).

    Article Snippet: Briefly, sections were fixed with cold acetone and then subjected to the blocking step with methanol containing 0.3% H2 O2 for 30 min and 20% normal goat serum for 1 h. After blocking, sections were incubated with rabbit anti-mouse Bcl-2 or Bax antibody (1:100 dilution, Santa Cruz Biotechnology) for 1 h at room temperature.

    Techniques: In Vivo, Acrylamide Gel Assay, Western Blot, Immunohistochemistry, Staining, Microscopy

    a Quantitative analysis was carried out for expressions of PAX8, Bax, Bcl-2 and Bak in PTC cells that were treated with X-ray and miR-144-3p. b , c Western blot was performed for expression of PAX8, Bax Bcl-2 and Bak. d , e Western blot was used for expressions of PAX8, Bax Bcl-2 and Bak in PTC cells that were treated with paclitaxel and miR-144-3p. f Quantitative analysis was performed for expression of PAX8, Bax Bcl-2 and Bak. *P

    Journal: Cancer Cell International

    Article Title: MiR-144-3p promotes the tumor growth and metastasis of papillary thyroid carcinoma by targeting paired box gene 8

    doi: 10.1186/s12935-018-0550-y

    Figure Lengend Snippet: a Quantitative analysis was carried out for expressions of PAX8, Bax, Bcl-2 and Bak in PTC cells that were treated with X-ray and miR-144-3p. b , c Western blot was performed for expression of PAX8, Bax Bcl-2 and Bak. d , e Western blot was used for expressions of PAX8, Bax Bcl-2 and Bak in PTC cells that were treated with paclitaxel and miR-144-3p. f Quantitative analysis was performed for expression of PAX8, Bax Bcl-2 and Bak. *P

    Article Snippet: The primary antibodies used in the experiment were as follows: N-cadherin (ab18203, 1:1000), Vimentin (ab137321, 1:1500), anti-PAX8 (ab53940,1:200), anti-p-Erk1/2 (ab201015, 1:1000), anti-Bcl-2 (ab593480, 1:700), anti-Bax (ab53154, 1:1000), anti-Bak (ab32371, 1:10,000) purchased from Abcam.

    Techniques: Western Blot, Expressing

    Western blots for (A) HIF-1α, (B) BNIP3, (C) Beclin1, (D) LC3-II/LC3-I, (E) p62, (F) Bcl2, (G) Bax and GAPDH. The intensity of the protein blots was normalized with GAPDH. The data are expressed as the mean ± SEM. Optical density (%),

    Journal: Molecular Medicine Reports

    Article Title: Autophagy may play an important role in varicocele

    doi: 10.3892/mmr.2017.7253

    Figure Lengend Snippet: Western blots for (A) HIF-1α, (B) BNIP3, (C) Beclin1, (D) LC3-II/LC3-I, (E) p62, (F) Bcl2, (G) Bax and GAPDH. The intensity of the protein blots was normalized with GAPDH. The data are expressed as the mean ± SEM. Optical density (%),

    Article Snippet: Rabbit anti Bax (CST 14796) and rabbit anti GAPDH, AB-P-R 001, were provided by Hangzhou Goodhere Biotechnology Co. (LTD, Hangzhou, China).

    Techniques: Western Blot

    Western blots for HIF-1α, Beclin1, p62, Bcl2, Bax, BNIP3, LC3-II, LC3-I, and GAPDH. HIF-1α, hypoxia-inducible factor 1α; Beclin1, BCL2 interacting protein.

    Journal: Molecular Medicine Reports

    Article Title: Autophagy may play an important role in varicocele

    doi: 10.3892/mmr.2017.7253

    Figure Lengend Snippet: Western blots for HIF-1α, Beclin1, p62, Bcl2, Bax, BNIP3, LC3-II, LC3-I, and GAPDH. HIF-1α, hypoxia-inducible factor 1α; Beclin1, BCL2 interacting protein.

    Article Snippet: Rabbit anti Bax (CST 14796) and rabbit anti GAPDH, AB-P-R 001, were provided by Hangzhou Goodhere Biotechnology Co. (LTD, Hangzhou, China).

    Techniques: Western Blot

    Critical role for Bax and Bak in zerumbone (ZER)-induced apoptosis. a Fluorescence microscopy for analysis of active Bax and Bak in MDA-MB-231 and MCF-7 cells after 8 hour treatment with DMSO or 20 µM of ZER. Staining for mitochondria (MitoTracker Green) and activated Bak or Bax is indicated by green and red colors, respectively. b Representative flow histograms depicting apoptotic fraction quantitation in SV40 immortalized mouse embryonic fibroblasts (MEF) derived from wild-type mice (WT) and Bak and Bax double knockout (DKO) mice after 24 hour treatment with DMSO or indicated concentration of ZER. c Quantitation of apoptotic cells (Annexin V-FITC/PI method) in SV40 immortalized MEFs derived from wild type mice (WT) and Bak-Bax double knockout (DKO) mice. The MEF were treated for 24 or 48 hours with DMSO (control) or the indicated concentrations of ZER and processed for flow cytometry. Quantitation relative to DMSO-treated WT MEF is shown. Results shown are mean ± SD (n=3). Significantly different (P

    Journal: Breast cancer research and treatment

    Article Title: Zerumbone causes Bax and Bak-mediated apoptosis in human breast cancer cells and inhibits orthotopic xenograft growth in vivo

    doi: 10.1007/s10549-012-2280-5

    Figure Lengend Snippet: Critical role for Bax and Bak in zerumbone (ZER)-induced apoptosis. a Fluorescence microscopy for analysis of active Bax and Bak in MDA-MB-231 and MCF-7 cells after 8 hour treatment with DMSO or 20 µM of ZER. Staining for mitochondria (MitoTracker Green) and activated Bak or Bax is indicated by green and red colors, respectively. b Representative flow histograms depicting apoptotic fraction quantitation in SV40 immortalized mouse embryonic fibroblasts (MEF) derived from wild-type mice (WT) and Bak and Bax double knockout (DKO) mice after 24 hour treatment with DMSO or indicated concentration of ZER. c Quantitation of apoptotic cells (Annexin V-FITC/PI method) in SV40 immortalized MEFs derived from wild type mice (WT) and Bak-Bax double knockout (DKO) mice. The MEF were treated for 24 or 48 hours with DMSO (control) or the indicated concentrations of ZER and processed for flow cytometry. Quantitation relative to DMSO-treated WT MEF is shown. Results shown are mean ± SD (n=3). Significantly different (P

    Article Snippet: We performed immunocytochemistry to determine activation of Bax and Bak in cells treated with 20 µM of ZER (8 hours) using antibodies specific for detection of active Bax (6A7; BD Biosciences) and active Bak (AM03; Calbiochem).

    Techniques: Fluorescence, Microscopy, Multiple Displacement Amplification, Staining, Flow Cytometry, Quantitation Assay, Derivative Assay, Mouse Assay, Double Knockout, Concentration Assay, Cytometry

    Zerumbone (ZER) causes collapse of mitochondrial membrane potential in breast cancer cells. a Immunoblotting for Bak and Bax using lysates from MDA-MB-231 and MCF-7 cells after 24 or 36 hour treatment with DMSO or ZER (20 or 40 µM). Numbers on top of the immunoreactive bands represent changes in protein levels relative to corresponding DMSOtreated control. b Quantitation of monomeric JC-1-associated green fluorescence by flow cytometry in MDA-MB-231 and MCF-7 cells after 16 or 24 hour treatment with DMSO or ZER (20 or 40 µM). Results shown are mean ± SD (n=3). *Significantly different (P

    Journal: Breast cancer research and treatment

    Article Title: Zerumbone causes Bax and Bak-mediated apoptosis in human breast cancer cells and inhibits orthotopic xenograft growth in vivo

    doi: 10.1007/s10549-012-2280-5

    Figure Lengend Snippet: Zerumbone (ZER) causes collapse of mitochondrial membrane potential in breast cancer cells. a Immunoblotting for Bak and Bax using lysates from MDA-MB-231 and MCF-7 cells after 24 or 36 hour treatment with DMSO or ZER (20 or 40 µM). Numbers on top of the immunoreactive bands represent changes in protein levels relative to corresponding DMSOtreated control. b Quantitation of monomeric JC-1-associated green fluorescence by flow cytometry in MDA-MB-231 and MCF-7 cells after 16 or 24 hour treatment with DMSO or ZER (20 or 40 µM). Results shown are mean ± SD (n=3). *Significantly different (P

    Article Snippet: We performed immunocytochemistry to determine activation of Bax and Bak in cells treated with 20 µM of ZER (8 hours) using antibodies specific for detection of active Bax (6A7; BD Biosciences) and active Bak (AM03; Calbiochem).

    Techniques: Multiple Displacement Amplification, Quantitation Assay, Fluorescence, Flow Cytometry, Cytometry

    Effects of pristimerin (Pris) on Con A-induced apoptosis. (A) Relative mRNA expression of Bax, caspase-3 and Bcl2. (B) Immuno-expression of Bax and Bcl2 in the hepatic tissue (200×) showing negative nuclear immuno-staining of Bax and high cytoplasmic expression of Bcl2 in both control and Pris groups while Con A group expressed high brown nuclear immuno-staining of Bax and low nuclear brown staining of Bcl2. Pris pretreatment attenuated Con-induced expression of Bax as represent by reduced nuclear brown staining and enhanced the cytoplasmic immunoexpression of Bcl2 . (C) Levels of Bax, caspase-3 and Bcl2 in the hepatic tissue. Data are the mean ± SEM ( n = 8). * P

    Journal: Frontiers in Pharmacology

    Article Title: Pristimerin as a Novel Hepatoprotective Agent Against Experimental Autoimmune Hepatitis

    doi: 10.3389/fphar.2018.00292

    Figure Lengend Snippet: Effects of pristimerin (Pris) on Con A-induced apoptosis. (A) Relative mRNA expression of Bax, caspase-3 and Bcl2. (B) Immuno-expression of Bax and Bcl2 in the hepatic tissue (200×) showing negative nuclear immuno-staining of Bax and high cytoplasmic expression of Bcl2 in both control and Pris groups while Con A group expressed high brown nuclear immuno-staining of Bax and low nuclear brown staining of Bcl2. Pris pretreatment attenuated Con-induced expression of Bax as represent by reduced nuclear brown staining and enhanced the cytoplasmic immunoexpression of Bcl2 . (C) Levels of Bax, caspase-3 and Bcl2 in the hepatic tissue. Data are the mean ± SEM ( n = 8). * P

    Article Snippet: Primary antibodies against rabbit polyclonal anti NF-κBp65 antibody (Cat. No. RB-9034-P) was purchased from Thermo Fisher Scientific (USA) while that against anti-MPO antibody (Cat. No. ab9535), anti-Bax antibody (Cat. No. ab32503), and anti-Bcl2 antibody (Cat. No. ab59348) was purchased from Abcam, UK.

    Techniques: Expressing, Immunostaining, Staining

    Downregulated Mctp1 and Rarb are associated with apoptotic cell death. a Bax proteins were increased by double knockdown of Mctp1 and Rarb. Bcl2 expression was decreased by siMctp1 and Rarb. b Mctp1 transcripts were downregulated. c Knockdown of Mctp1 enhanced Intracellular Ca 2+ levels (siCont (0.063) versus siMctp1 (0.131) in fluorescence intensities from baseline 490/525 ratio). d Rarb transcripts were downregulated. e-f Transfected siRarb reduced neuronal cell differentiation (MAP2). Scale bar, 40 μm. g and h Double knockdown of Mctp1 and Rarb increased Bax transcripts and decreased Bcl2 transcripts. i Transfected siMctp1 and siRarb activated LDH release. j Flow cytometry verified that downregulated Mctp1 and Rarb significantly induce apoptotic cell death in mouse NSCs. Scrambled siRNA served as a negative control (siCont). Significantly different at *, p

    Journal: Translational Neurodegeneration

    Article Title: Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

    doi: 10.1186/s40035-020-00203-4

    Figure Lengend Snippet: Downregulated Mctp1 and Rarb are associated with apoptotic cell death. a Bax proteins were increased by double knockdown of Mctp1 and Rarb. Bcl2 expression was decreased by siMctp1 and Rarb. b Mctp1 transcripts were downregulated. c Knockdown of Mctp1 enhanced Intracellular Ca 2+ levels (siCont (0.063) versus siMctp1 (0.131) in fluorescence intensities from baseline 490/525 ratio). d Rarb transcripts were downregulated. e-f Transfected siRarb reduced neuronal cell differentiation (MAP2). Scale bar, 40 μm. g and h Double knockdown of Mctp1 and Rarb increased Bax transcripts and decreased Bcl2 transcripts. i Transfected siMctp1 and siRarb activated LDH release. j Flow cytometry verified that downregulated Mctp1 and Rarb significantly induce apoptotic cell death in mouse NSCs. Scrambled siRNA served as a negative control (siCont). Significantly different at *, p

    Article Snippet: Samples were centrifuged at 15000 rpm at 4 °C for 20 min, obtaining a supernatant (insoluble proteins). (Primary antibodies used in this study are mouse anti-Hif1α antibody (NB100–449, NOVUS, Abingdon OX14 3NB UK), rabbit anti-Mef2c antibody (LS-C31031, LSBio, Seattle WA USA), rabbit anti-Mctp1 antibody (NBP1–83604, Novus Biologicals, Oakville ON L6M 2 V5 Canada), rabbit anti-Rarb (ab53161, abcam, Cambridge CB2 0AX UK), rabbit anti-Bax (SC-493, Santa Cruz, Dallas Texas USA), rabbit anti-Bcl2 (SC-492, Santa Cruz, Dallas Texas USA), mouse anti-Flag (F3165, SIGMA, Burlington MA USA), goat anti-GFP antibody (600–101-215, Rockland, Gilbertsville PA USA), rabbit anti-mCherry (M11217, Thermofisher, Waltham MA USA) and mouse anti β-actin (sc-47,778, SantaCruz, Dallas Texas USA), rabbit anti-SOD1 (97,959, abcam, Cambridge CB2 0AX UK).

    Techniques: Expressing, Fluorescence, Transfection, Cell Differentiation, Flow Cytometry, Negative Control

    Downregulated miR-18b (miR-18b-5p) by SOD1 mutations contributes apoptotic cell death in SOD1(G93A) Tg mice and fALS patient spinal cord tissues. a Hif1α, Mef2c and Bax expression were increased in G93A mice. Mctp1, Rarb and Bcl2 proteins were decreased in G93A mice. b mRNAs of Hif1α, Mef2c and Bax was highly expressed in G93A mice. Mctp1, Rarb and Bcl2 transcripts were significantly downregulated in G93A mice. c miR-18b (miR-18b-5p) was deeply reduced and miR-206 was dramatically induced in G93A mice ( n = 5). d The protein levels of Hif1α, Mef2c and Bax was upregulated in fALS (G86S) patient (Cervical and lumber). Mctp1, Rarb and Bcl2 proteins were decreased in fALS (G86S) patient. Normal spinal cord tissues (Cervical (control 1 and 2) served as a negative control (Cont). e The transcripts of Hif1α, Mef2c and Bax was highly upregulated in fALS (G86S) patient (Cervicals). Mctp1, Rarb and Bcl2 transcripts were significantly downregulated in fALS (G86S) patient. f miR-18b (miR-18b-5p) expression was importantly decreased and miR-206 was highly expressed in fALS (G86S) patient (Cervial and Lumber). Normal spinal cord tissues (Cervicals (control 1 and 2) served as a negative control (Cont). hSOD1 immunoreactivity on western blots of the insoluble fraction of the G93A mice and fALS (G86S) patients tissues. Significantly different at *, p

    Journal: Translational Neurodegeneration

    Article Title: Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

    doi: 10.1186/s40035-020-00203-4

    Figure Lengend Snippet: Downregulated miR-18b (miR-18b-5p) by SOD1 mutations contributes apoptotic cell death in SOD1(G93A) Tg mice and fALS patient spinal cord tissues. a Hif1α, Mef2c and Bax expression were increased in G93A mice. Mctp1, Rarb and Bcl2 proteins were decreased in G93A mice. b mRNAs of Hif1α, Mef2c and Bax was highly expressed in G93A mice. Mctp1, Rarb and Bcl2 transcripts were significantly downregulated in G93A mice. c miR-18b (miR-18b-5p) was deeply reduced and miR-206 was dramatically induced in G93A mice ( n = 5). d The protein levels of Hif1α, Mef2c and Bax was upregulated in fALS (G86S) patient (Cervical and lumber). Mctp1, Rarb and Bcl2 proteins were decreased in fALS (G86S) patient. Normal spinal cord tissues (Cervical (control 1 and 2) served as a negative control (Cont). e The transcripts of Hif1α, Mef2c and Bax was highly upregulated in fALS (G86S) patient (Cervicals). Mctp1, Rarb and Bcl2 transcripts were significantly downregulated in fALS (G86S) patient. f miR-18b (miR-18b-5p) expression was importantly decreased and miR-206 was highly expressed in fALS (G86S) patient (Cervial and Lumber). Normal spinal cord tissues (Cervicals (control 1 and 2) served as a negative control (Cont). hSOD1 immunoreactivity on western blots of the insoluble fraction of the G93A mice and fALS (G86S) patients tissues. Significantly different at *, p

    Article Snippet: Samples were centrifuged at 15000 rpm at 4 °C for 20 min, obtaining a supernatant (insoluble proteins). (Primary antibodies used in this study are mouse anti-Hif1α antibody (NB100–449, NOVUS, Abingdon OX14 3NB UK), rabbit anti-Mef2c antibody (LS-C31031, LSBio, Seattle WA USA), rabbit anti-Mctp1 antibody (NBP1–83604, Novus Biologicals, Oakville ON L6M 2 V5 Canada), rabbit anti-Rarb (ab53161, abcam, Cambridge CB2 0AX UK), rabbit anti-Bax (SC-493, Santa Cruz, Dallas Texas USA), rabbit anti-Bcl2 (SC-492, Santa Cruz, Dallas Texas USA), mouse anti-Flag (F3165, SIGMA, Burlington MA USA), goat anti-GFP antibody (600–101-215, Rockland, Gilbertsville PA USA), rabbit anti-mCherry (M11217, Thermofisher, Waltham MA USA) and mouse anti β-actin (sc-47,778, SantaCruz, Dallas Texas USA), rabbit anti-SOD1 (97,959, abcam, Cambridge CB2 0AX UK).

    Techniques: Mouse Assay, Expressing, Negative Control, Western Blot

    SOD1 mutation (G93A) induces apoptosis. a Western blot analysis verified the protein levels of Hif1α, Mef2c, Mctp1, Rarb, Bax, and Bcl2 in three different NSC-34 cell lines. b Quantification analysis of western blot. The data represent the average ± SEM of 3 separate experiments. Significantly different at *, p

    Journal: Translational Neurodegeneration

    Article Title: Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

    doi: 10.1186/s40035-020-00203-4

    Figure Lengend Snippet: SOD1 mutation (G93A) induces apoptosis. a Western blot analysis verified the protein levels of Hif1α, Mef2c, Mctp1, Rarb, Bax, and Bcl2 in three different NSC-34 cell lines. b Quantification analysis of western blot. The data represent the average ± SEM of 3 separate experiments. Significantly different at *, p

    Article Snippet: Samples were centrifuged at 15000 rpm at 4 °C for 20 min, obtaining a supernatant (insoluble proteins). (Primary antibodies used in this study are mouse anti-Hif1α antibody (NB100–449, NOVUS, Abingdon OX14 3NB UK), rabbit anti-Mef2c antibody (LS-C31031, LSBio, Seattle WA USA), rabbit anti-Mctp1 antibody (NBP1–83604, Novus Biologicals, Oakville ON L6M 2 V5 Canada), rabbit anti-Rarb (ab53161, abcam, Cambridge CB2 0AX UK), rabbit anti-Bax (SC-493, Santa Cruz, Dallas Texas USA), rabbit anti-Bcl2 (SC-492, Santa Cruz, Dallas Texas USA), mouse anti-Flag (F3165, SIGMA, Burlington MA USA), goat anti-GFP antibody (600–101-215, Rockland, Gilbertsville PA USA), rabbit anti-mCherry (M11217, Thermofisher, Waltham MA USA) and mouse anti β-actin (sc-47,778, SantaCruz, Dallas Texas USA), rabbit anti-SOD1 (97,959, abcam, Cambridge CB2 0AX UK).

    Techniques: Mutagenesis, Western Blot

    miR-206 post-transcriptionally regulates Mctp1 and Rarb and elevates apoptotic cell death. a and b Luciferases assay with 3′ UTR of Mctp1 showed that Mctp1 is target of miR-206. Mctp1 transcripts was downregulated by increased miR-206. c Intracellular Ca 2+ levels (Cont (0.069) versus miR-206 (0.122) in fluorescence intensities from baseline 490/525 ratio) was enhanced by miR-206. d miR-206 controlled Mctp1 and Rarb protein expression. e Luciferase assay with 3′ UTR of Rarb also verified that Rarb is target of miR-206. Rarb mRNAs was decreased by miR-206. Neuronal cell differentiation (MAP2) was reduced by miR-206. Scale bar, 40 μm. f and g Both Bax protein and mRNAs were increased by miR-206. Bcl2 proteins and mRNAs were decreased by miR-206. h miR-206 was overexpressed in contNSC-34 cells. i ) LDH release assay showed that miR-206 enhances apoptosis. j Flow cytometry analysis explained that overexpressed miR-206 activates apoptotic cell death in mouse NSCs. Empty vector served as a negative control (Cont). Significantly different at *, p

    Journal: Translational Neurodegeneration

    Article Title: Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

    doi: 10.1186/s40035-020-00203-4

    Figure Lengend Snippet: miR-206 post-transcriptionally regulates Mctp1 and Rarb and elevates apoptotic cell death. a and b Luciferases assay with 3′ UTR of Mctp1 showed that Mctp1 is target of miR-206. Mctp1 transcripts was downregulated by increased miR-206. c Intracellular Ca 2+ levels (Cont (0.069) versus miR-206 (0.122) in fluorescence intensities from baseline 490/525 ratio) was enhanced by miR-206. d miR-206 controlled Mctp1 and Rarb protein expression. e Luciferase assay with 3′ UTR of Rarb also verified that Rarb is target of miR-206. Rarb mRNAs was decreased by miR-206. Neuronal cell differentiation (MAP2) was reduced by miR-206. Scale bar, 40 μm. f and g Both Bax protein and mRNAs were increased by miR-206. Bcl2 proteins and mRNAs were decreased by miR-206. h miR-206 was overexpressed in contNSC-34 cells. i ) LDH release assay showed that miR-206 enhances apoptosis. j Flow cytometry analysis explained that overexpressed miR-206 activates apoptotic cell death in mouse NSCs. Empty vector served as a negative control (Cont). Significantly different at *, p

    Article Snippet: Samples were centrifuged at 15000 rpm at 4 °C for 20 min, obtaining a supernatant (insoluble proteins). (Primary antibodies used in this study are mouse anti-Hif1α antibody (NB100–449, NOVUS, Abingdon OX14 3NB UK), rabbit anti-Mef2c antibody (LS-C31031, LSBio, Seattle WA USA), rabbit anti-Mctp1 antibody (NBP1–83604, Novus Biologicals, Oakville ON L6M 2 V5 Canada), rabbit anti-Rarb (ab53161, abcam, Cambridge CB2 0AX UK), rabbit anti-Bax (SC-493, Santa Cruz, Dallas Texas USA), rabbit anti-Bcl2 (SC-492, Santa Cruz, Dallas Texas USA), mouse anti-Flag (F3165, SIGMA, Burlington MA USA), goat anti-GFP antibody (600–101-215, Rockland, Gilbertsville PA USA), rabbit anti-mCherry (M11217, Thermofisher, Waltham MA USA) and mouse anti β-actin (sc-47,778, SantaCruz, Dallas Texas USA), rabbit anti-SOD1 (97,959, abcam, Cambridge CB2 0AX UK).

    Techniques: Fluorescence, Expressing, Luciferase, Cell Differentiation, Lactate Dehydrogenase Assay, Flow Cytometry, Plasmid Preparation, Negative Control

    miR-18b (miR-18b-5p) regulates Hif1α and reduces apoptosis in mtNSC-34 cells. a Overexpressed miR-18b (miR-18b-5p) decreased Hif1α and Mef2c proteins. Both Mctp1 and Rarb expression were increased by miR-18b (miR-18b-5p). Downregulated Bax and upregulated Bcl2 by miR-18b (miR-18b-5p) diminished apoptosis in mtNSC-34 cells. b RT-qPCR analysis showed low expression of Hif1α and Mef2c mRNAs. c Mctp1 and Rarb transcripts were highly expressed by miR-18b (miR-18b-5p). d Bax mRNAs were decreased and Bcl2 mRNAs were increased by overexpressed miR-18b. e miR-18b (miR-18b-5p) was overexpressed in mtNSC-34 cells. f miR-206 was reduced by miR-18b (miR-18b-5p). g LDH release analysis explained that transfected miR-18b (miR-18b-5p) restores apoptosis. h Luciferases assay with 3′ UTR of Hif1α showed that Hif1α is target of miR-18b in contNSC-34 cells. i and j Overexpression of miR-18b (miR-18b-5p) enhanced neuronal differentiation (MAP2) and attenuated intracellular Ca 2+ levels (Cont (0.098) versus miR-18b (miR-18b-5p) (0.051) in fluorescence intensities from baseline 490/525 ratio) in mtNSC-34 cells. Empty vector served as a negative control (Cont). Arrow represents SOD1 aggregation (green). Scale bar, 40 μm. Significantly different at *, p

    Journal: Translational Neurodegeneration

    Article Title: Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

    doi: 10.1186/s40035-020-00203-4

    Figure Lengend Snippet: miR-18b (miR-18b-5p) regulates Hif1α and reduces apoptosis in mtNSC-34 cells. a Overexpressed miR-18b (miR-18b-5p) decreased Hif1α and Mef2c proteins. Both Mctp1 and Rarb expression were increased by miR-18b (miR-18b-5p). Downregulated Bax and upregulated Bcl2 by miR-18b (miR-18b-5p) diminished apoptosis in mtNSC-34 cells. b RT-qPCR analysis showed low expression of Hif1α and Mef2c mRNAs. c Mctp1 and Rarb transcripts were highly expressed by miR-18b (miR-18b-5p). d Bax mRNAs were decreased and Bcl2 mRNAs were increased by overexpressed miR-18b. e miR-18b (miR-18b-5p) was overexpressed in mtNSC-34 cells. f miR-206 was reduced by miR-18b (miR-18b-5p). g LDH release analysis explained that transfected miR-18b (miR-18b-5p) restores apoptosis. h Luciferases assay with 3′ UTR of Hif1α showed that Hif1α is target of miR-18b in contNSC-34 cells. i and j Overexpression of miR-18b (miR-18b-5p) enhanced neuronal differentiation (MAP2) and attenuated intracellular Ca 2+ levels (Cont (0.098) versus miR-18b (miR-18b-5p) (0.051) in fluorescence intensities from baseline 490/525 ratio) in mtNSC-34 cells. Empty vector served as a negative control (Cont). Arrow represents SOD1 aggregation (green). Scale bar, 40 μm. Significantly different at *, p

    Article Snippet: Samples were centrifuged at 15000 rpm at 4 °C for 20 min, obtaining a supernatant (insoluble proteins). (Primary antibodies used in this study are mouse anti-Hif1α antibody (NB100–449, NOVUS, Abingdon OX14 3NB UK), rabbit anti-Mef2c antibody (LS-C31031, LSBio, Seattle WA USA), rabbit anti-Mctp1 antibody (NBP1–83604, Novus Biologicals, Oakville ON L6M 2 V5 Canada), rabbit anti-Rarb (ab53161, abcam, Cambridge CB2 0AX UK), rabbit anti-Bax (SC-493, Santa Cruz, Dallas Texas USA), rabbit anti-Bcl2 (SC-492, Santa Cruz, Dallas Texas USA), mouse anti-Flag (F3165, SIGMA, Burlington MA USA), goat anti-GFP antibody (600–101-215, Rockland, Gilbertsville PA USA), rabbit anti-mCherry (M11217, Thermofisher, Waltham MA USA) and mouse anti β-actin (sc-47,778, SantaCruz, Dallas Texas USA), rabbit anti-SOD1 (97,959, abcam, Cambridge CB2 0AX UK).

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Over Expression, Fluorescence, Plasmid Preparation, Negative Control

    Analysis of Beclin 1 protein interactions and Bcl2 and Bax protein expression on 793 cells starved with and without anti-EGF antibody Western blot in ( A ), shows that: phospho-EGFR strongly binds Beclin 1 on 793 cells starved for 4 hrs; p-Bcl2 bind Beclin 1 up to 4 hrs of starvation and then detach; p-JNK and p-Bcl2 protein expressions increase at 4 and 8 hrs of starvation, respectively; Bcl2 and Bax were attached until 8 hrs of starvation and, afterwards, they were detached. ( B ) 793 cells starved and treated with anti-EGF antibody showed that: Beclin 1 and p-EGFR were detached; Beclin 1 and p-Bcl2 stayed attached during starvation; p-JNK and p-Bcl-2 expression levels were constantly down-regulated; p-Bcl2 and Bax were constantly attached. Time of exposure of p-Bcl2, p-EGFR, p-JNK, Beclin-1, Bax and tubulin were 7.5, 2, 15, 9.6, 5 and 4.3 s, respectively.

    Journal: Oncotarget

    Article Title: Autophagy processes are dependent on EGF receptor signaling

    doi: 10.18632/oncotarget.25708

    Figure Lengend Snippet: Analysis of Beclin 1 protein interactions and Bcl2 and Bax protein expression on 793 cells starved with and without anti-EGF antibody Western blot in ( A ), shows that: phospho-EGFR strongly binds Beclin 1 on 793 cells starved for 4 hrs; p-Bcl2 bind Beclin 1 up to 4 hrs of starvation and then detach; p-JNK and p-Bcl2 protein expressions increase at 4 and 8 hrs of starvation, respectively; Bcl2 and Bax were attached until 8 hrs of starvation and, afterwards, they were detached. ( B ) 793 cells starved and treated with anti-EGF antibody showed that: Beclin 1 and p-EGFR were detached; Beclin 1 and p-Bcl2 stayed attached during starvation; p-JNK and p-Bcl-2 expression levels were constantly down-regulated; p-Bcl2 and Bax were constantly attached. Time of exposure of p-Bcl2, p-EGFR, p-JNK, Beclin-1, Bax and tubulin were 7.5, 2, 15, 9.6, 5 and 4.3 s, respectively.

    Article Snippet: For each sample, total cell lysate containing 120 μg protein was incubated with 5 μl of anti-Beclin or anti-Bax antibody (Cell Signaling Technologies, Danvers, MA, USA) in rotation overnight at 4° C. Then 30 μl of Protein G Agarose beads were added to the mix of protein lysate and anti-Beclin or anti-Bax antibody, and incubation was performed overnight at 4° C. The IP complex was washed 6 times with NP40 lysis buffer.

    Techniques: Expressing, Western Blot

    Ku70 mediates Bax deubiquitylation. ( A ) Time-course analysis of ubiquitylated Bax levels by Western blot with polyclonal antibody against Bax before (0) or at various times after the induction of Bax-mediated apoptosis by staurosporine-STS in Ku70 −/− cells. ( B ) In vitro deubiquitylation of Bax in Ku70 −/− cell extracts supplemented with PBS or recombinant Ku70-rKU70. ( C ) In vitro deubiquitylation of Bax in Ku70 −/− cells that were grown in the presence of the proteasomal inhibitor MG-132. The extracts were supplemented with PBS or rKu70. ( D ) Levels of ubiquitylated Bax in wild-type cells that were grown in medium supplemented with DMSO or the proteasomal inhibitor, MG-132. ( E ) In vitro deubiquitylation assay of homogenous tetraubiquitin chains supplemented with BSA and rKu70 for various time periods, demonstrating that Ku70 possess an intrinsic DUB enzymatic activity. Incubation of tetraubiquitin only or tetraubiquitin together with BSA did not induce hydrolysis of the tetraubiquitin chains into monoubiquitin units. In A–D , β-actin levels served as a loading control. U, unmodified Bax; M, modified Bax.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of the proapoptotic factor Bax by Ku70-dependent deubiquitylation

    doi: 10.1073/pnas.0706700105

    Figure Lengend Snippet: Ku70 mediates Bax deubiquitylation. ( A ) Time-course analysis of ubiquitylated Bax levels by Western blot with polyclonal antibody against Bax before (0) or at various times after the induction of Bax-mediated apoptosis by staurosporine-STS in Ku70 −/− cells. ( B ) In vitro deubiquitylation of Bax in Ku70 −/− cell extracts supplemented with PBS or recombinant Ku70-rKU70. ( C ) In vitro deubiquitylation of Bax in Ku70 −/− cells that were grown in the presence of the proteasomal inhibitor MG-132. The extracts were supplemented with PBS or rKu70. ( D ) Levels of ubiquitylated Bax in wild-type cells that were grown in medium supplemented with DMSO or the proteasomal inhibitor, MG-132. ( E ) In vitro deubiquitylation assay of homogenous tetraubiquitin chains supplemented with BSA and rKu70 for various time periods, demonstrating that Ku70 possess an intrinsic DUB enzymatic activity. Incubation of tetraubiquitin only or tetraubiquitin together with BSA did not induce hydrolysis of the tetraubiquitin chains into monoubiquitin units. In A–D , β-actin levels served as a loading control. U, unmodified Bax; M, modified Bax.

    Article Snippet: The following antibodies were used to detect protein expression by Western blot: rabbit polyclonal anti Bax (N-20; Santa Cruz Biotechnology), mouse monoclonal anti actin (A4700; Sigma), mouse monoclonal anti ubiquitin (P4D1; Santa Cruz Biotechnology), and mouse monoclonal anti HA (HA.11; Covance).

    Techniques: Western Blot, In Vitro, Recombinant, Activity Assay, Incubation, Modification

    Bax ubiquitylation promotes its degradation and decreases upon apoptotic stimuli. ( A ) Treatment of Ku70 −/− cells with the proteasomal inhibitor, MG-132, increased the levels of ubiquitylated Bax. ( B ) Treatment of HEK293 cells overexpressing siRNA against Ku70 with the proteasomal inhibitor Bertozomib (Velcade or PS-341) increased the levels of ubiquitylated Bax. The decrease in Ku70 levels was validated against HEK293 cells overexpressing control siRNA (data not shown) ( C ) Time-course analysis of ubiquitylated Bax levels by Western blot using polyclonal antibody against Bax before (0) or at various times after the induction of Bax-mediated apoptosis by staurosporine-STS in wild-type cells. In all experiments, β-actin levels served as a loading control. U, unmodified Bax; M, modified Bax.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of the proapoptotic factor Bax by Ku70-dependent deubiquitylation

    doi: 10.1073/pnas.0706700105

    Figure Lengend Snippet: Bax ubiquitylation promotes its degradation and decreases upon apoptotic stimuli. ( A ) Treatment of Ku70 −/− cells with the proteasomal inhibitor, MG-132, increased the levels of ubiquitylated Bax. ( B ) Treatment of HEK293 cells overexpressing siRNA against Ku70 with the proteasomal inhibitor Bertozomib (Velcade or PS-341) increased the levels of ubiquitylated Bax. The decrease in Ku70 levels was validated against HEK293 cells overexpressing control siRNA (data not shown) ( C ) Time-course analysis of ubiquitylated Bax levels by Western blot using polyclonal antibody against Bax before (0) or at various times after the induction of Bax-mediated apoptosis by staurosporine-STS in wild-type cells. In all experiments, β-actin levels served as a loading control. U, unmodified Bax; M, modified Bax.

    Article Snippet: The following antibodies were used to detect protein expression by Western blot: rabbit polyclonal anti Bax (N-20; Santa Cruz Biotechnology), mouse monoclonal anti actin (A4700; Sigma), mouse monoclonal anti ubiquitin (P4D1; Santa Cruz Biotechnology), and mouse monoclonal anti HA (HA.11; Covance).

    Techniques: Western Blot, Modification

    The ER-targeted Bak can induce apoptosis in the absence of endogenous Bax and Bak. Wild-type and bax − / − bak − / − MEFs were infected with retroviruses expressing GFP or GFP together with wild-type murine Bak, Bak-ActA, Bak-cb5, or Bak-ΔC. 48 h after infection, 1 μg/ml DAPI was added to stain the apoptotic cells. (A) bax − / − bak − / − cells were photographed using a FITC or DAPI filter. (B) The percentage of dead cells was determined by the ratio of DAPI-positive cells to GFP-positive cells. In addition to the retroviral infection of the Bak mutants, bax − / − bak − / − cells were also coinfected with retrovirus expressing Bcl-xL, or infected in the presence of 20 μg/ml Z-VAD-fmk. Data shown are the average of three independent experiments. (C) 24 h after infection, bax − / − bak − / − cells were trypsinized. A portion of each sample was subjected to FACS ® to determine the expression efficiency indicated by GFP-positive cells. The rest of the cells were fixed in 0.25% paraformaldehyde and stained with an anti-Bak antibody that recognizes the active form of murine Bak. The number in each dot plot represents the percentage of gated events (R1).

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: The ER-targeted Bak can induce apoptosis in the absence of endogenous Bax and Bak. Wild-type and bax − / − bak − / − MEFs were infected with retroviruses expressing GFP or GFP together with wild-type murine Bak, Bak-ActA, Bak-cb5, or Bak-ΔC. 48 h after infection, 1 μg/ml DAPI was added to stain the apoptotic cells. (A) bax − / − bak − / − cells were photographed using a FITC or DAPI filter. (B) The percentage of dead cells was determined by the ratio of DAPI-positive cells to GFP-positive cells. In addition to the retroviral infection of the Bak mutants, bax − / − bak − / − cells were also coinfected with retrovirus expressing Bcl-xL, or infected in the presence of 20 μg/ml Z-VAD-fmk. Data shown are the average of three independent experiments. (C) 24 h after infection, bax − / − bak − / − cells were trypsinized. A portion of each sample was subjected to FACS ® to determine the expression efficiency indicated by GFP-positive cells. The rest of the cells were fixed in 0.25% paraformaldehyde and stained with an anti-Bak antibody that recognizes the active form of murine Bak. The number in each dot plot represents the percentage of gated events (R1).

    Article Snippet: Cells were washed three times with PBS and incubated with 1:50 anti–mouse IgG1 (BD Biosciences), anti-Bax (clone 6A7; BD Biosciences), anti-Bak (AM03; Oncogene Research Products), or anti-Bak (NT; Upstate Biotechnology) in 100 μg/ml digitonin in PBS for 30 min. After being washed with PBS three times, cells were incubated with 1:100 FITC-conjugated anti–mouse Ig and 1 μg/ml DAPI for 30 min.

    Techniques: Infection, Expressing, Staining, FACS

    ER-targeted Bak can induce selective cleavage of caspase 12, but not of caspase 7. bax − / − bak − / − cells were infected with GFP vector control, Bak-IRES-GFP, Bak-ActA-IRES-GFP, Bak-cb5-IRES-GFP, and Bak-ΔC-IRES-GFP at a high multiplicity. After infection, cells were lysed. Caspase 12, caspase 7, or PARP were detected by immunoblotting using respective antibodies.

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: ER-targeted Bak can induce selective cleavage of caspase 12, but not of caspase 7. bax − / − bak − / − cells were infected with GFP vector control, Bak-IRES-GFP, Bak-ActA-IRES-GFP, Bak-cb5-IRES-GFP, and Bak-ΔC-IRES-GFP at a high multiplicity. After infection, cells were lysed. Caspase 12, caspase 7, or PARP were detected by immunoblotting using respective antibodies.

    Article Snippet: Cells were washed three times with PBS and incubated with 1:50 anti–mouse IgG1 (BD Biosciences), anti-Bax (clone 6A7; BD Biosciences), anti-Bak (AM03; Oncogene Research Products), or anti-Bak (NT; Upstate Biotechnology) in 100 μg/ml digitonin in PBS for 30 min. After being washed with PBS three times, cells were incubated with 1:100 FITC-conjugated anti–mouse Ig and 1 μg/ml DAPI for 30 min.

    Techniques: Infection, Plasmid Preparation

    Caspase 12 functions downstream of the multidomain proapoptotic Bcl-2 family proteins. (A) ER stress–induced caspase 12 cleavage is dependent on Bax and Bak. Immortalized wild-type and bax − / − bak − / − MEFs and NIH3T3 cells were treated with brefeldin A (BFA; 10 μg/ml), thapsigargin (Thap; 2 μM), or tunicamycin (Tuni; 10 μg/ml) for 30 h. Caspase 12 level and processing were examined by immunoblotting of 20 μg of total cellular protein from samples as indicated. An ∼42-kD caspase 12 fragment is indicated by the arrow. Induction of CHOP expression is shown as an indicator of the ER stress response. A nonspecific band (NS) is shown as a loading control. (B) Caspase 12 kills bax − / − bak − / − cells. Wild-type and bax − / − bak − / − MEFs were cotransfected with pEGFP and constructs expressing caspase 3, caspase 9, caspase 12, and t-caspase 12. 24 h after transfection, cells were stained with DAPI, and cell death percentage was determined by the ratio of DAPI-positive to GFP-positive cells.

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: Caspase 12 functions downstream of the multidomain proapoptotic Bcl-2 family proteins. (A) ER stress–induced caspase 12 cleavage is dependent on Bax and Bak. Immortalized wild-type and bax − / − bak − / − MEFs and NIH3T3 cells were treated with brefeldin A (BFA; 10 μg/ml), thapsigargin (Thap; 2 μM), or tunicamycin (Tuni; 10 μg/ml) for 30 h. Caspase 12 level and processing were examined by immunoblotting of 20 μg of total cellular protein from samples as indicated. An ∼42-kD caspase 12 fragment is indicated by the arrow. Induction of CHOP expression is shown as an indicator of the ER stress response. A nonspecific band (NS) is shown as a loading control. (B) Caspase 12 kills bax − / − bak − / − cells. Wild-type and bax − / − bak − / − MEFs were cotransfected with pEGFP and constructs expressing caspase 3, caspase 9, caspase 12, and t-caspase 12. 24 h after transfection, cells were stained with DAPI, and cell death percentage was determined by the ratio of DAPI-positive to GFP-positive cells.

    Article Snippet: Cells were washed three times with PBS and incubated with 1:50 anti–mouse IgG1 (BD Biosciences), anti-Bax (clone 6A7; BD Biosciences), anti-Bak (AM03; Oncogene Research Products), or anti-Bak (NT; Upstate Biotechnology) in 100 μg/ml digitonin in PBS for 30 min. After being washed with PBS three times, cells were incubated with 1:100 FITC-conjugated anti–mouse Ig and 1 μg/ml DAPI for 30 min.

    Techniques: Expressing, Construct, Transfection, Staining

    ER stress induces Bax and Bak conformational changes and oligomerization at the ER. (A) ER stresses induce the conformational changes of Bax and Bak. HeLa, MCF7, and 293T cells were treated with thapsigargin (Thap; 2 μM) or tunicamycin (Tuni; 5 μg/ml) for 36 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were incubated with a control antibody (mouse IgG1) and conformation-sensitive antibodies against Bax or Bak, followed by incubation with FITC-conjugated secondary antibody. (B) ER stress induces Bax oligomerization at the ER. Wild-type MEFs were treated with brefeldin A (BFA; 10 μg/ml), Thap (2 μM), or Tuni (10 μg/ml) for 24 h. Cells were resuspended in hypotonic buffer A and disrupted. 5 mM BMH cross-linking reagent was added to cross-link the oligomerized proteins. Cells were subjected to subcellular fractionation to obtain the HM and LM fractions. 20 μg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax. COX IV and calnexin are shown as indicators of the purity of the fractionation and as loading controls.

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: ER stress induces Bax and Bak conformational changes and oligomerization at the ER. (A) ER stresses induce the conformational changes of Bax and Bak. HeLa, MCF7, and 293T cells were treated with thapsigargin (Thap; 2 μM) or tunicamycin (Tuni; 5 μg/ml) for 36 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were incubated with a control antibody (mouse IgG1) and conformation-sensitive antibodies against Bax or Bak, followed by incubation with FITC-conjugated secondary antibody. (B) ER stress induces Bax oligomerization at the ER. Wild-type MEFs were treated with brefeldin A (BFA; 10 μg/ml), Thap (2 μM), or Tuni (10 μg/ml) for 24 h. Cells were resuspended in hypotonic buffer A and disrupted. 5 mM BMH cross-linking reagent was added to cross-link the oligomerized proteins. Cells were subjected to subcellular fractionation to obtain the HM and LM fractions. 20 μg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax. COX IV and calnexin are shown as indicators of the purity of the fractionation and as loading controls.

    Article Snippet: Cells were washed three times with PBS and incubated with 1:50 anti–mouse IgG1 (BD Biosciences), anti-Bax (clone 6A7; BD Biosciences), anti-Bak (AM03; Oncogene Research Products), or anti-Bak (NT; Upstate Biotechnology) in 100 μg/ml digitonin in PBS for 30 min. After being washed with PBS three times, cells were incubated with 1:100 FITC-conjugated anti–mouse Ig and 1 μg/ml DAPI for 30 min.

    Techniques: Incubation, Fractionation

    Bax and Bak are localized to the ER in addition to their mitochondrial location. (A) Wild-type and bax − / − bak − / − MEFs as well as HeLa cells were resuspended in hypotonic buffer A and disrupted. Subcellular fractionation was performed to obtain the fractions for cytosol (S-100), HM (mitochondria enriched), and LM (the ER enriched). 20 μg of total protein from each fraction was separated on a 4–12% gradient NuPAGE gel. Antibodies against Bax, Bak, calnexin, and Cox IV were used for immunoblotting. (B) Mouse liver was homogenized in buffer A and fractionated in sucrose gradient as described in the Materials and methods. Fractions from crude mitochondria and the 1.5/1.8 M sucrose interface were probed with indicated antibodies. (C) The LM fraction was not contaminated by mitochondrial outer membrane. Fractions of wild-type MEFs were probed with an anti-Tom40 antibody. (D) Immunoelectron microscopy of Bax (left) and Bak (right) in wild-type MEFs using 10-nm gold particles. The bottom panels are the enlargements of the boxed areas of the top images. The arrowheads point to the ER. mt, mitochondria.

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: Bax and Bak are localized to the ER in addition to their mitochondrial location. (A) Wild-type and bax − / − bak − / − MEFs as well as HeLa cells were resuspended in hypotonic buffer A and disrupted. Subcellular fractionation was performed to obtain the fractions for cytosol (S-100), HM (mitochondria enriched), and LM (the ER enriched). 20 μg of total protein from each fraction was separated on a 4–12% gradient NuPAGE gel. Antibodies against Bax, Bak, calnexin, and Cox IV were used for immunoblotting. (B) Mouse liver was homogenized in buffer A and fractionated in sucrose gradient as described in the Materials and methods. Fractions from crude mitochondria and the 1.5/1.8 M sucrose interface were probed with indicated antibodies. (C) The LM fraction was not contaminated by mitochondrial outer membrane. Fractions of wild-type MEFs were probed with an anti-Tom40 antibody. (D) Immunoelectron microscopy of Bax (left) and Bak (right) in wild-type MEFs using 10-nm gold particles. The bottom panels are the enlargements of the boxed areas of the top images. The arrowheads point to the ER. mt, mitochondria.

    Article Snippet: Cells were washed three times with PBS and incubated with 1:50 anti–mouse IgG1 (BD Biosciences), anti-Bax (clone 6A7; BD Biosciences), anti-Bak (AM03; Oncogene Research Products), or anti-Bak (NT; Upstate Biotechnology) in 100 μg/ml digitonin in PBS for 30 min. After being washed with PBS three times, cells were incubated with 1:100 FITC-conjugated anti–mouse Ig and 1 μg/ml DAPI for 30 min.

    Techniques: Fractionation, Immuno-Electron Microscopy

    The ER-targeted Bak-cb5 induces depletion of the ER Ca 2 + pool. (A) Cells with ER-targeted Bak lack thapsigargin-releasable intracellular Ca 2+ . bax − / − bak − / − cells were infected with retroviruses encoding GFP, Bak-IRES-GFP, Bak-ActA-IRES-GFP, or Bak-cb5-IRES-GFP. 48 h after infection, cells were loaded with Indo-1, and the ER Ca 2+ release was measured by flow cytometry. Propidium iodide was added to determine the cell viability. The Ca 2+ flux traces were derived from gated live GFP-positive cells. The [Ca 2+ ] i was calibrated from the measured Indo-1 fluorescence ratio as described in the Materials and methods. The arrowhead indicates the time of thapsigargin addition. (B) Expression of ER-targeted Bak depleted intracellular Ca 2+ storage in the ER. At the start of measurement, extracellular free Ca 2+ concentration was reduced to zero. The arrowhead indicates the time when the extracellular free Ca 2+ concentration was brought back to 2 mM. [Ca 2+ ] i was analyzed as in A. (C and D) bax − / − bak − / − cells were infected with the Bak-cb5-IRES-GFP. (C) The measured Indo-1 fluorescence ratio between λ 405 and λ 475 is presented as an index of change in cytosolic [Ca 2+ ] over time and presented separately for gated live GFP-positive (expressing Bak-cb5) or GFP-negative (not expressing Bak-cb5) cells. The absolute [Ca 2+ ] i in the linear range is indicated on the right. The arrowhead indicates the time of thapsigargin addition. The arrow indicates the time of the addition of 8 mM CaCl 2 to the medium. (D) Mitochondrial potential in GFP-positive (expressing Bak-cb5, green) and in GFP-negative (not expressing Bak-cb5, black) cells was measured by tetramethyl rhodamine ethyl ester (TMRE) staining.

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: The ER-targeted Bak-cb5 induces depletion of the ER Ca 2 + pool. (A) Cells with ER-targeted Bak lack thapsigargin-releasable intracellular Ca 2+ . bax − / − bak − / − cells were infected with retroviruses encoding GFP, Bak-IRES-GFP, Bak-ActA-IRES-GFP, or Bak-cb5-IRES-GFP. 48 h after infection, cells were loaded with Indo-1, and the ER Ca 2+ release was measured by flow cytometry. Propidium iodide was added to determine the cell viability. The Ca 2+ flux traces were derived from gated live GFP-positive cells. The [Ca 2+ ] i was calibrated from the measured Indo-1 fluorescence ratio as described in the Materials and methods. The arrowhead indicates the time of thapsigargin addition. (B) Expression of ER-targeted Bak depleted intracellular Ca 2+ storage in the ER. At the start of measurement, extracellular free Ca 2+ concentration was reduced to zero. The arrowhead indicates the time when the extracellular free Ca 2+ concentration was brought back to 2 mM. [Ca 2+ ] i was analyzed as in A. (C and D) bax − / − bak − / − cells were infected with the Bak-cb5-IRES-GFP. (C) The measured Indo-1 fluorescence ratio between λ 405 and λ 475 is presented as an index of change in cytosolic [Ca 2+ ] over time and presented separately for gated live GFP-positive (expressing Bak-cb5) or GFP-negative (not expressing Bak-cb5) cells. The absolute [Ca 2+ ] i in the linear range is indicated on the right. The arrowhead indicates the time of thapsigargin addition. The arrow indicates the time of the addition of 8 mM CaCl 2 to the medium. (D) Mitochondrial potential in GFP-positive (expressing Bak-cb5, green) and in GFP-negative (not expressing Bak-cb5, black) cells was measured by tetramethyl rhodamine ethyl ester (TMRE) staining.

    Article Snippet: Cells were washed three times with PBS and incubated with 1:50 anti–mouse IgG1 (BD Biosciences), anti-Bax (clone 6A7; BD Biosciences), anti-Bak (AM03; Oncogene Research Products), or anti-Bak (NT; Upstate Biotechnology) in 100 μg/ml digitonin in PBS for 30 min. After being washed with PBS three times, cells were incubated with 1:100 FITC-conjugated anti–mouse Ig and 1 μg/ml DAPI for 30 min.

    Techniques: Infection, Flow Cytometry, Cytometry, Derivative Assay, Fluorescence, Expressing, Concentration Assay, Staining

    Bax and Bak localized to the ER can initiate apoptosis. In addition to their mitochondrial localization and activity, Bax and Bak also reside at the ER. Upon ER stress treatment, Bax and Bak can initiate apoptosis from the ER.

    Journal: The Journal of Cell Biology

    Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

    doi: 10.1083/jcb.200302084

    Figure Lengend Snippet: Bax and Bak localized to the ER can initiate apoptosis. In addition to their mitochondrial localization and activity, Bax and Bak also reside at the ER. Upon ER stress treatment, Bax and Bak can initiate apoptosis from the ER.

    Article Snippet: Cells were washed three times with PBS and incubated with 1:50 anti–mouse IgG1 (BD Biosciences), anti-Bax (clone 6A7; BD Biosciences), anti-Bak (AM03; Oncogene Research Products), or anti-Bak (NT; Upstate Biotechnology) in 100 μg/ml digitonin in PBS for 30 min. After being washed with PBS three times, cells were incubated with 1:100 FITC-conjugated anti–mouse Ig and 1 μg/ml DAPI for 30 min.

    Techniques: Activity Assay