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  • 88
    Thermo Fisher gene exp bak1 hs00832876 g1
    Gene Exp Bak1 Hs00832876 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore anti bak
    Western blot analysis of the expression of Bid, <t>Bax,</t> <t>Bak,</t> and actin (as loading control) in Bid +/+ and Bid −/− cells. Equal amounts of protein from whole cell lysates were resolved on SDS-PAGE and processed for western blot analysis
    Anti Bak, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 316 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc bak
    Effects of TIIA and CT on expressions of Bcl-2 family proteins on hypoxia induced H9c2 cells. (A) Anti-apoptotic proteins expression: Bcl-2 protein (50 µg) and Bcl-xl protein (20 µg) expression. (B) Pro-apoptotic proteins expression: <t>Bak</t> and <t>Bax</t> proteins expression (80 µg). (C) Bax/Bcl-2 ratio. Data shown are representative mean ± SEM of 4–5 independent experiments. * P
    Bak, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1375 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti bak
    Proposed model illustrating the role of <t>ANP32B</t> and its relationship with other molecules in apoptosis. ANP32B inhibits Bax and phosphorylation of Bad, and promotes <t>Bak</t> expression in the mitochondrial apoptosis pathway.
    Anti Bak, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bak
    Autophagy induced after <t>Bax/Bak-activation</t> inhibits type I IFN secretion during apoptosis a Bax/Bak-activation activates the cGAS/STING pathway to induce IFN-β production, but the cytokine is degraded by apoptotic caspases. b Both autophagy genes and caspases inhibit IFN-β secretion following Bax/Bak-activation. <t>Mcl-1</t> −/− MEFs, those with CRISPR/Cas-9 deleted ATG5 or ATG7 (gRNA ATG5.299, ATG.405, ATG7.113 or ATG7.160), and Bax −/− Bak −/− MEFs were treated with 1 μM ABT-737 and 10 μM QVD-OPh as indicated. After 16 h, supernatants were harvested and IFN-β was measured by ELISA ( n = 10–11 independent experiments). c IFN-β secretion after autophagy inhibition requires mitochondrial (mt) DNA. MEFs lacking Mcl-1 and either ATG5 or ATG7 were depleted of mtDNA (ρ 0 ) and subsequently treated as in b ( n = 4). d IL-6 secretion measured by ELISA in supernatants from b . e Autophagy inhibition does not increase Infb1 mRNA expression following ABT-737 treatment. Mcl-1 −/− MEFs, and those with deleted ATG5 or ATG7, were treated for 4 h with 1 μM ABT-737 and/or 10 μM QVD-OPh prior to RNA purification. After cDNA synthesis, the fold change of ABT-737 treatment to untreated of Ifnb1/Gapdh was calculated ( n = 3–13, using four different gRNAs independently: ATG5.299, ATG5.405, ATG7.113, and ATG7.160). f IFN-β is produced to similar extents irrespective of a functional canonical autophagy system. Cells were treated with 1 μM ABT-737 and/or 10 μM QVD-OPh for 4 h prior to lysis and immunoblotting. Representative of 3 experiments. g Apaf-1 −/− ]). Samples were normalised to contain equal amount of total protein (30 μg), except for fractions 1–10 where the entire sample was loaded. Representative of three independent experiments. All graphs represent the mean and SEM
    Bak, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 794 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti bak
    Western blot analysis of <t>BCL-XL</t> and GAPDH in J/Neo and J/BCL-XL cells (A), and flow cytometric analysis of the effect of CMEP-NQ on NOC-, 2-MeO-E 2 -, and CPT-induced alterations in cell cycle distribution (B and C) and Δψm loss (D and E) in Jurkat T cell clone transfected with a BCL-XL-expression vector (J/BCL-XL cells). After pretreatment with 7.5 μM CMEP-NQ for 1 h, J/BCL-XL cells (5 × 10 5 /ml) were treated with 0.3 μM NOC, 1.0 μM 2-MeO-E 2 , or 0.02 μM CPT for 17 h and then subjected to flow cytometric analysis of cell cycle distribution, Δψm loss, and <t>BAK</t> activation as described in the Materials and Methods. Western blot and flow cytometric analyses were performed as described in the Materials and Methods. A representative result is shown; two additional experiments yielded similar results. Error bars represent standard deviations.
    Anti Bak, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology bak
    Bcl-X L protein levels are selectively regulated by hemopoietins. ( A ) Levels of Bcl-2 family members were assessed by immunoblot analyses with antibodies that detect murine Bcl-2, Bcl-X L , Mcl-1, <t>Bax,</t> Bad, and <t>Bak</t> proteins. Protein extracts (50 μg each) were prepared from 32D.3 ( left ) and FDC-P1.2 ( right ) myeloid cells growing in IL-3 or deprived of IL-3 for the indicated intervals and analyzed by immunoblots. Results shown are representative of six independent experiments. ( B ) Fetal liver-derived myeloid cells were grown in IL-3 (20 U/ml), IL-6 (10 ng/ml), and SCF (10 ng/ml) and then were deprived of all hemopoietins. Protein extracts (40 μg each) were prepared from cells after the indicated times after removal of hemopoietins and analyzed by immunoblot. Results shown are representative of three independent experiments. ( C ) The rates of death of 32D.3, FDC-P1.2, and primary myeloid cells were assessed following withdrawal of cytokines by trypan blue dye exclusion. Cell death was always apoptotic based on morphological criteria, and AnnexinV and TUNEL assays (data not shown). Results shown are representative of 10 independent experiments for 32D.3 and FDC-P1 cells and three for primary myeloid cells.
    Bak, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 768 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc bak
    Increased immunogenicity of <t>Bax</t> −/− <t>Bak</t> −/− DC
    Bak, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti bak
    Effect of ES, <t>ES-BAX,</t> <t>ES-BAK,</t> and ES-BAX-ES in the ex vivo rat aortic ring assay. Ring sections were cut out from freshly isolated aorta of euthanized rats. The rings were cultivated in Matrigel, in the absence or presence of 10 μ g/ml of each protein for 7 days. The aortic rings and the angiogenic sprouting were stained with MTT and photographed. ( a ) Photographs from representative rings from assays performed in triplicate. Scale bar: 1000 μ m. ( b ) Quantification of microvessel outgrowth in each condition. The mean of the pixels number of the outgrowth of the control group was set as 100%. Error bars: mean±S.E.M. The statistical significance of differences between the groups was assessed by one-way ANOVA with Bonferroni post-test, ** P
    Anti Bak, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc anti bak
    BID and <t>BAK</t> physically interact, and a BAK-blocking Ab prevents cytochrome c release. ( A ) In vitro binding between GST–BAK and IVT <t>BCL-X</t> L , p15 BID, and BIDα345/TM, but not p15 BID mIII.4 or BIDα345/TM mIII.4 (lanes 1–5 ). None of the IVT proteins bound GST itself (data not shown). Preincubation with an anti-BAK Ab inhibits GST–-BAK in vitro binding to p15 BID (lanes 6,7 ). ( B ) Coimmunoprecipitation of BAK and wild-type p15 BID. 35 S-labeled p15 BID wild-type (lane 1 ) and mutant p15 BID mIII.4 (lane 2 ) were targeted to mitochondria in vitro. The mitochondria were then solubilized and immunoprecipitated with an anti-BAK Ab. Coimmunoprecipitated p15 was detected by autoradiography. The anti-BAK Ab did not directly immunoprecipitate p15 BID wild type from solution (lane 3 ). BAK (24 kD) comigrates with light chain (25 kD), precluding detection of BAK by IP Western. Consequently, 35 S-labeled IVT BAK was mixed with IVT p15 wild type or p15 mIII.4 in buffer B. BID was immunoprecipitated with an anti-BID Ab, and coimmunoprecipitated BAK was detected by autoradiography (lanes 4,5 ). ( C ) Inhibition of the p15 BID/BAK interaction prevents cytochrome c release. Wild-type mitochondria were incubated with the indicated amounts of anti-BAK Ab (G-23) or anti-BCL-X L Ab (SC-18) for 20 min at room temperature. A total of 25 ng of recombinant p15 BID was targeted to the mitochondria. The cytochrome c released into the supernatant and the p15 BID targeted to the mitochondrial pellet are shown.
    Anti Bak, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc bak d4e4 rabbit mab
    BID and <t>BAK</t> physically interact, and a BAK-blocking Ab prevents cytochrome c release. ( A ) In vitro binding between GST–BAK and IVT <t>BCL-X</t> L , p15 BID, and BIDα345/TM, but not p15 BID mIII.4 or BIDα345/TM mIII.4 (lanes 1–5 ). None of the IVT proteins bound GST itself (data not shown). Preincubation with an anti-BAK Ab inhibits GST–-BAK in vitro binding to p15 BID (lanes 6,7 ). ( B ) Coimmunoprecipitation of BAK and wild-type p15 BID. 35 S-labeled p15 BID wild-type (lane 1 ) and mutant p15 BID mIII.4 (lane 2 ) were targeted to mitochondria in vitro. The mitochondria were then solubilized and immunoprecipitated with an anti-BAK Ab. Coimmunoprecipitated p15 was detected by autoradiography. The anti-BAK Ab did not directly immunoprecipitate p15 BID wild type from solution (lane 3 ). BAK (24 kD) comigrates with light chain (25 kD), precluding detection of BAK by IP Western. Consequently, 35 S-labeled IVT BAK was mixed with IVT p15 wild type or p15 mIII.4 in buffer B. BID was immunoprecipitated with an anti-BID Ab, and coimmunoprecipitated BAK was detected by autoradiography (lanes 4,5 ). ( C ) Inhibition of the p15 BID/BAK interaction prevents cytochrome c release. Wild-type mitochondria were incubated with the indicated amounts of anti-BAK Ab (G-23) or anti-BCL-X L Ab (SC-18) for 20 min at room temperature. A total of 25 ng of recombinant p15 BID was targeted to the mitochondria. The cytochrome c released into the supernatant and the p15 BID targeted to the mitochondrial pellet are shown.
    Bak D4e4 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    alcon travoprost bak free
    BID and <t>BAK</t> physically interact, and a BAK-blocking Ab prevents cytochrome c release. ( A ) In vitro binding between GST–BAK and IVT <t>BCL-X</t> L , p15 BID, and BIDα345/TM, but not p15 BID mIII.4 or BIDα345/TM mIII.4 (lanes 1–5 ). None of the IVT proteins bound GST itself (data not shown). Preincubation with an anti-BAK Ab inhibits GST–-BAK in vitro binding to p15 BID (lanes 6,7 ). ( B ) Coimmunoprecipitation of BAK and wild-type p15 BID. 35 S-labeled p15 BID wild-type (lane 1 ) and mutant p15 BID mIII.4 (lane 2 ) were targeted to mitochondria in vitro. The mitochondria were then solubilized and immunoprecipitated with an anti-BAK Ab. Coimmunoprecipitated p15 was detected by autoradiography. The anti-BAK Ab did not directly immunoprecipitate p15 BID wild type from solution (lane 3 ). BAK (24 kD) comigrates with light chain (25 kD), precluding detection of BAK by IP Western. Consequently, 35 S-labeled IVT BAK was mixed with IVT p15 wild type or p15 mIII.4 in buffer B. BID was immunoprecipitated with an anti-BID Ab, and coimmunoprecipitated BAK was detected by autoradiography (lanes 4,5 ). ( C ) Inhibition of the p15 BID/BAK interaction prevents cytochrome c release. Wild-type mitochondria were incubated with the indicated amounts of anti-BAK Ab (G-23) or anti-BCL-X L Ab (SC-18) for 20 min at room temperature. A total of 25 ng of recombinant p15 BID was targeted to the mitochondria. The cytochrome c released into the supernatant and the p15 BID targeted to the mitochondrial pellet are shown.
    Travoprost Bak Free, supplied by alcon, used in various techniques. Bioz Stars score: 88/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti bak
    mt DNA is released from mitochondria following MOMP in a <t>BAX</t> / <t>BAK</t> ‐dependent manner Fixed super‐resolution Airyscan images of U2OS cells immunostained with anti‐TOM20 (green) and anti‐DNA (red) antibodies. Scale bar = 10 μm. Representative images from three independent experiments. Airyscan images of U2OS cells treated with 10 μM ABT‐737, 1 μM ActD and 20 μM qVD‐OPh for 3 h, immunostained with anti‐TOM20 and anti‐DNA antibodies. Scale bar = 10 μm. Representative images from three independent experiments. Quantification of cells exhibiting > 10% mtDNA release following treatment with 10 μM ABT‐737, 1 μM ActD and 20 μM qVD‐OPh. Data are expressed as mean ± SD from three independent experiments and analysed by Student's t ‐test. Quantification of the extent of mitochondrial DNA (mtDNA) nucleoid release per cell following treatment with 10 μM ABT‐737, 1 μM ActD and 20 μM qVD‐OPh. Data are expressed as mean ± SD from two independent experiments and analysed by Student's t ‐test. Airyscan images of MEF cells untreated or treated with 10 μM ABT‐737 and 20 μM qVD‐OPh for 3 h, immunostained with anti‐TOM20 and anti‐DNA antibodies. Scale bar = 10 μm. Representative images from three independent experiments. BAX and BAK expression levels in U2OS cells with CRISPR‐Cas9‐mediated deletion of BAX, BAK or BAX/BAK. U2OS cells with BAX, BAK or BAX/BAK deletion by CRISPR‐Cas9 treated with 10 μM ABT‐737 and 2 μM S63845 and analysed for cell viability using an IncuCyte live‐cell imager and SYTOX Green exclusion. Data are expressed as mean ± SEM, representative of three independent experiments, and have been normalised to starting confluency. Airyscan images of U2OS (i) control cells or with CRISPR‐Cas9‐mediated deletion of either (ii) BAX, (iii) BAK or (iv) BAX and BAK treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh for 3 h. Scale bar = 10 μm. Representative images from three independent experiments. Quantification of mtDNA nucleoid release per cell in U2OS EMPTY CRISPR , BAX CRISPR , BAK CRISPR and BAX/BAK CRISPR cells. Data are expressed as mean ± SD from three independent experiments and analysed using Student's t ‐test. Source data are available online for this figure.
    Rabbit Anti Bak, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti bak
    a Quantitative analysis was carried out for expressions of <t>PAX8,</t> Bax, Bcl-2 and <t>Bak</t> 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 Bak, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson bak
    Acetylated <t>Sp1</t> does not bind at the <t>bak</t> or p21 promoters . Panel A shows the organisation and sequence of the bak (Ai) and p21 (Aii) probes, with hypothetical and proven Sp1/3 binding sites underlined. Sequence numbers refer to distance from the transcriptional start site of each gene. Panel B shows that binding of Sp1 to both target sequences is decreased following butyrate treatment. Panel B: Western of mobility shift assay (WeMSA) analysis of binding to the Bak and p21 probes shows that Sp1 binding decreases following treatment with 10 mM sodium butyrate for 24 h compared to an untreated control (upper panel). Binding of acetyl-Sp1 could not be detected by WeMSA (lower panel). These data are representative of three independent repeats. Panel C: The binding of Sp1 to the bak (panel Ci) and p21 (panel Cii) promoter sequences was determined following treatment of HCT116 cells with a range of butyrate concentrations (0-20 mM). The upper panels show WeMSA gels immunoprobed for Sp1. As a loading control, the same extracts were also separated by SDS page, and immunoprobed with the same antibody (lower panels). Data shown in Ci and Cii are representative of at least two independent repeat experiments. Panel Ciii shows mean (+/- SD) of response at the Bak promoter. Whilst the levels of Sp1 are broadly constant, levels of Sp1 binding for both probes are reduced following treatment with butyrate.
    Bak, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    OriGene bak1
    Reciprocal expression of miR‐29c and Birc2 and <t>Bak1.</t> ( A ) The expression of miR‐29c in three groups. ( B ) and ( C ) The expression of Birc2 mRNA and Bak1 mRNA in rat brains. ( D ) and ( E ) The expression of Birc2 protein and Bak1 protein in three groups. * P
    Bak1, supplied by OriGene, used in various techniques. Bioz Stars score: 89/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam anti bak antibody y164
    Anti-apoptotic prohibitin shown by knockdown analysis. (A) ARPE-19 cells were transfected by siRNA and random sequence. Anti-apoptotic BCL-xL increased when prohibitin was down-regulated. Pro-apoptotic proteins, including <t>AIF,</t> caspase-9, and <t>BAK,</t> were
    Anti Bak Antibody Y164, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Western blot analysis of the expression of Bid, Bax, Bak, and actin (as loading control) in Bid +/+ and Bid −/− cells. Equal amounts of protein from whole cell lysates were resolved on SDS-PAGE and processed for western blot analysis

    Journal: Photochemistry and photobiology

    Article Title: A Requirement for Bid for Induction of Apoptosis by Photodynamic Therapy with a Lysosome- but not a Mitochondrion-Targeted Photosensitizer

    doi: 10.1111/j.1751-1097.2010.00766.x

    Figure Lengend Snippet: Western blot analysis of the expression of Bid, Bax, Bak, and actin (as loading control) in Bid +/+ and Bid −/− cells. Equal amounts of protein from whole cell lysates were resolved on SDS-PAGE and processed for western blot analysis

    Article Snippet: After transfer to PVDF membranes, the proteins were probed with one or more of the following antibodies: anti-Bax (1:500 dilution, cat no. N-20, Santa Cruz, Santa Cruz, CA), anti-Bak (1:1000 dilution, cat no.06536, Millipore, Billerica, MA), anti-Bid (1:500 dilution, cat no. 550365, BD Biosciences, San Diego, CA), anti-active caspase-3 (1:1000 dilution, cat. No. 559565, BD Pharmingen, San Diego, CA) or anti-actin (1:2000 dilution, cat.no.

    Techniques: Western Blot, Expressing, SDS Page

    Effects of TIIA and CT on expressions of Bcl-2 family proteins on hypoxia induced H9c2 cells. (A) Anti-apoptotic proteins expression: Bcl-2 protein (50 µg) and Bcl-xl protein (20 µg) expression. (B) Pro-apoptotic proteins expression: Bak and Bax proteins expression (80 µg). (C) Bax/Bcl-2 ratio. Data shown are representative mean ± SEM of 4–5 independent experiments. * P

    Journal: PLoS ONE

    Article Title: TanshinoneIIA and Cryptotanshinone Protect against Hypoxia-Induced Mitochondrial Apoptosis in H9c2 Cells

    doi: 10.1371/journal.pone.0051720

    Figure Lengend Snippet: Effects of TIIA and CT on expressions of Bcl-2 family proteins on hypoxia induced H9c2 cells. (A) Anti-apoptotic proteins expression: Bcl-2 protein (50 µg) and Bcl-xl protein (20 µg) expression. (B) Pro-apoptotic proteins expression: Bak and Bax proteins expression (80 µg). (C) Bax/Bcl-2 ratio. Data shown are representative mean ± SEM of 4–5 independent experiments. * P

    Article Snippet: Bax and Bak were purchased from Cell signaling Technology (MA, USA).

    Techniques: Expressing

    Proposed model illustrating the role of ANP32B and its relationship with other molecules in apoptosis. ANP32B inhibits Bax and phosphorylation of Bad, and promotes Bak expression in the mitochondrial apoptosis pathway.

    Journal: PLoS ONE

    Article Title: Downregulation of ANP32B exerts anti-apoptotic effects in hepatocellular carcinoma

    doi: 10.1371/journal.pone.0177343

    Figure Lengend Snippet: Proposed model illustrating the role of ANP32B and its relationship with other molecules in apoptosis. ANP32B inhibits Bax and phosphorylation of Bad, and promotes Bak expression in the mitochondrial apoptosis pathway.

    Article Snippet: Western blotting For Western blotting, 20 μg of protein was applied to the lanes of 4% to 12% Bis-Tris gels (Life Technologies) for electrophoresis, blotted onto Immobilon-P membranes (Millipore, Bedford, MA, USA) after electrophoresis, and incubated with the relevant primary antibody: anti-beta-actin (Chemicon, Temecula, Ca, USA); anti-PHAPI1/APRIL (ANP32B) (Abcam); anti-GFP (MBL, Nagoya, Japan); anti-Bad (9292) anti-phospho-Bad (5284), anti-Bcl-2 (2870), anti-Bak (12105), anti-Bcl-xL (2764), anti-Bcl-2 (2870), anti-phospho-Akt (4060), anti-Akt (9272), anti-caspase-3 (9662), anti-caspase-8 (9746), and anti-caspase-9 (9502) antibodies (Cell Signaling).

    Techniques: Expressing

    Silencing of ANP32B regulated Bak expression and the phosphorylation of Bad. (A) Expression of genes involved in the mitochondrial pathway and Akt; phosphorylated Akt was analyzed by Western blotting after ANP32B knockdown (left panels; Huh7 cells, right panels; HLE cells). (B) The altered levels of BAD and BAK mRNA expression were analyzed by real time RT-PCR using ANP32B siRNA (left panels; Huh7 cells, right panels; HLE cells). Mean ± SEM of six replicates. **p

    Journal: PLoS ONE

    Article Title: Downregulation of ANP32B exerts anti-apoptotic effects in hepatocellular carcinoma

    doi: 10.1371/journal.pone.0177343

    Figure Lengend Snippet: Silencing of ANP32B regulated Bak expression and the phosphorylation of Bad. (A) Expression of genes involved in the mitochondrial pathway and Akt; phosphorylated Akt was analyzed by Western blotting after ANP32B knockdown (left panels; Huh7 cells, right panels; HLE cells). (B) The altered levels of BAD and BAK mRNA expression were analyzed by real time RT-PCR using ANP32B siRNA (left panels; Huh7 cells, right panels; HLE cells). Mean ± SEM of six replicates. **p

    Article Snippet: Western blotting For Western blotting, 20 μg of protein was applied to the lanes of 4% to 12% Bis-Tris gels (Life Technologies) for electrophoresis, blotted onto Immobilon-P membranes (Millipore, Bedford, MA, USA) after electrophoresis, and incubated with the relevant primary antibody: anti-beta-actin (Chemicon, Temecula, Ca, USA); anti-PHAPI1/APRIL (ANP32B) (Abcam); anti-GFP (MBL, Nagoya, Japan); anti-Bad (9292) anti-phospho-Bad (5284), anti-Bcl-2 (2870), anti-Bak (12105), anti-Bcl-xL (2764), anti-Bcl-2 (2870), anti-phospho-Akt (4060), anti-Akt (9272), anti-caspase-3 (9662), anti-caspase-8 (9746), and anti-caspase-9 (9502) antibodies (Cell Signaling).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR

    Depletion of DAP1 inhibits SubAB-induced Bak/Bax conformational changes. (a) NC and DAP1 siRNA-transfected cells were incubated with mt or wt SubAB (0.2 μg/ml) for 3 h at 37°C. Then cells were lysed, and proteins were immunoprecipitated

    Journal: Infection and Immunity

    Article Title: DAP1, a Negative Regulator of Autophagy, Controls SubAB-Mediated Apoptosis and Autophagy

    doi: 10.1128/IAI.02213-14

    Figure Lengend Snippet: Depletion of DAP1 inhibits SubAB-induced Bak/Bax conformational changes. (a) NC and DAP1 siRNA-transfected cells were incubated with mt or wt SubAB (0.2 μg/ml) for 3 h at 37°C. Then cells were lysed, and proteins were immunoprecipitated

    Article Snippet: After immunocomplexes were washed three times with lysis buffer, proteins were dissolved in SDS sample buffer, subjected to SDS-PAGE in 15% gels, and transferred to polyvinylidene difluoride (PVDF) membranes, which were then analyzed by Western blotting using anti-Bax or anti-Bak antibodies (Cell Signaling).

    Techniques: Transfection, Incubation, Immunoprecipitation

    Effects of METTL3 depletion on SHH-GLI pathway and apoptosis. ( A )The protein level of SHH-GLI pathway was analyzed by Western blotting after METTL3 depletion. ( B, C ) The mRNA level of c-Myc and cycling D1 was analyzed by qRT-PCR. * P ˂ 0.05, ** P ˂ 0.01. ( D, E ) The protein level of Bak, Bax, Bcl-2, Bcl-xL, and cleaved PARP was analyzed by Western blotting after METTL3 depletion. ( F ) The relative caspase-3/7 activity was measured using Apo-One™ homogenous caspase-3/7 assay. ** P ˂ 0.01. ( G, H ) LNCaP and PC3 cells were transduced with Scr or METTL3 shRNA for 48 hrs followed by overexpression with wild type or mutant METTL3 plasmid for 24 hrs. GLI1 protein level was detected by Western blotting ( G ) and the methylated GLI1 mRNA level was analyzed by Me-RIP assay ( H ). * P ˂ 0.05.

    Journal: OncoTargets and therapy

    Article Title: RNA m6A Methyltransferase METTL3 Promotes The Growth Of Prostate Cancer By Regulating Hedgehog Pathway

    doi: 10.2147/OTT.S226796

    Figure Lengend Snippet: Effects of METTL3 depletion on SHH-GLI pathway and apoptosis. ( A )The protein level of SHH-GLI pathway was analyzed by Western blotting after METTL3 depletion. ( B, C ) The mRNA level of c-Myc and cycling D1 was analyzed by qRT-PCR. * P ˂ 0.05, ** P ˂ 0.01. ( D, E ) The protein level of Bak, Bax, Bcl-2, Bcl-xL, and cleaved PARP was analyzed by Western blotting after METTL3 depletion. ( F ) The relative caspase-3/7 activity was measured using Apo-One™ homogenous caspase-3/7 assay. ** P ˂ 0.01. ( G, H ) LNCaP and PC3 cells were transduced with Scr or METTL3 shRNA for 48 hrs followed by overexpression with wild type or mutant METTL3 plasmid for 24 hrs. GLI1 protein level was detected by Western blotting ( G ) and the methylated GLI1 mRNA level was analyzed by Me-RIP assay ( H ). * P ˂ 0.05.

    Article Snippet: Antibodies against Bak, Bax, Bcl-2, Bcl-xL, GLI1, GLI2, and PTCH1 were purchased from Cell Signaling Technology (Danvers, MA USA).

    Techniques: Western Blot, Quantitative RT-PCR, Activity Assay, Transduction, shRNA, Over Expression, Mutagenesis, Plasmid Preparation, Methylation

    Autophagy induced after Bax/Bak-activation inhibits type I IFN secretion during apoptosis a Bax/Bak-activation activates the cGAS/STING pathway to induce IFN-β production, but the cytokine is degraded by apoptotic caspases. b Both autophagy genes and caspases inhibit IFN-β secretion following Bax/Bak-activation. Mcl-1 −/− MEFs, those with CRISPR/Cas-9 deleted ATG5 or ATG7 (gRNA ATG5.299, ATG.405, ATG7.113 or ATG7.160), and Bax −/− Bak −/− MEFs were treated with 1 μM ABT-737 and 10 μM QVD-OPh as indicated. After 16 h, supernatants were harvested and IFN-β was measured by ELISA ( n = 10–11 independent experiments). c IFN-β secretion after autophagy inhibition requires mitochondrial (mt) DNA. MEFs lacking Mcl-1 and either ATG5 or ATG7 were depleted of mtDNA (ρ 0 ) and subsequently treated as in b ( n = 4). d IL-6 secretion measured by ELISA in supernatants from b . e Autophagy inhibition does not increase Infb1 mRNA expression following ABT-737 treatment. Mcl-1 −/− MEFs, and those with deleted ATG5 or ATG7, were treated for 4 h with 1 μM ABT-737 and/or 10 μM QVD-OPh prior to RNA purification. After cDNA synthesis, the fold change of ABT-737 treatment to untreated of Ifnb1/Gapdh was calculated ( n = 3–13, using four different gRNAs independently: ATG5.299, ATG5.405, ATG7.113, and ATG7.160). f IFN-β is produced to similar extents irrespective of a functional canonical autophagy system. Cells were treated with 1 μM ABT-737 and/or 10 μM QVD-OPh for 4 h prior to lysis and immunoblotting. Representative of 3 experiments. g Apaf-1 −/− ]). Samples were normalised to contain equal amount of total protein (30 μg), except for fractions 1–10 where the entire sample was loaded. Representative of three independent experiments. All graphs represent the mean and SEM

    Journal: Cell Death and Differentiation

    Article Title: Autophagy induced during apoptosis degrades mitochondria and inhibits type I interferon secretion

    doi: 10.1038/s41418-017-0017-z

    Figure Lengend Snippet: Autophagy induced after Bax/Bak-activation inhibits type I IFN secretion during apoptosis a Bax/Bak-activation activates the cGAS/STING pathway to induce IFN-β production, but the cytokine is degraded by apoptotic caspases. b Both autophagy genes and caspases inhibit IFN-β secretion following Bax/Bak-activation. Mcl-1 −/− MEFs, those with CRISPR/Cas-9 deleted ATG5 or ATG7 (gRNA ATG5.299, ATG.405, ATG7.113 or ATG7.160), and Bax −/− Bak −/− MEFs were treated with 1 μM ABT-737 and 10 μM QVD-OPh as indicated. After 16 h, supernatants were harvested and IFN-β was measured by ELISA ( n = 10–11 independent experiments). c IFN-β secretion after autophagy inhibition requires mitochondrial (mt) DNA. MEFs lacking Mcl-1 and either ATG5 or ATG7 were depleted of mtDNA (ρ 0 ) and subsequently treated as in b ( n = 4). d IL-6 secretion measured by ELISA in supernatants from b . e Autophagy inhibition does not increase Infb1 mRNA expression following ABT-737 treatment. Mcl-1 −/− MEFs, and those with deleted ATG5 or ATG7, were treated for 4 h with 1 μM ABT-737 and/or 10 μM QVD-OPh prior to RNA purification. After cDNA synthesis, the fold change of ABT-737 treatment to untreated of Ifnb1/Gapdh was calculated ( n = 3–13, using four different gRNAs independently: ATG5.299, ATG5.405, ATG7.113, and ATG7.160). f IFN-β is produced to similar extents irrespective of a functional canonical autophagy system. Cells were treated with 1 μM ABT-737 and/or 10 μM QVD-OPh for 4 h prior to lysis and immunoblotting. Representative of 3 experiments. g Apaf-1 −/− ]). Samples were normalised to contain equal amount of total protein (30 μg), except for fractions 1–10 where the entire sample was loaded. Representative of three independent experiments. All graphs represent the mean and SEM

    Article Snippet: Equal amounts of protein were separated on 10% NUPAGE® Bis Tris gels (Invitrogen), transferred to nitrocellulose, and blotted with the antibodies raised against Mcl-1 (19C4; WEHI), Bim (3C5; WEHI), Bax (49F9-13-13; WEHI), Bak (Sigma), Parkin (sc-32282; Santa Cruz), LAMP1 (1D4B; Santa Cruz), TNFR1 (ab19139; Abcam), Calnexin (ab22595; Abcam), Apaf-1 (18H2; WEHI), TIMM44 (HPA043052; Sigma) and β-actin (AC-15; Sigma).

    Techniques: Activation Assay, CRISPR, Enzyme-linked Immunosorbent Assay, Inhibition, Expressing, Purification, Produced, Functional Assay, Lysis

    Model of Bax/Bak-stimulated autophagy Bax/Bak activation induces 3 separate pathways: (1) apoptotic caspase cascade, (2) IFN-β production via the cGAS/STING pathway and (3) autophagy. Both apoptotic caspases and autophagy restrain IFN-β secretion to render apoptotic cells immunologically silent

    Journal: Cell Death and Differentiation

    Article Title: Autophagy induced during apoptosis degrades mitochondria and inhibits type I interferon secretion

    doi: 10.1038/s41418-017-0017-z

    Figure Lengend Snippet: Model of Bax/Bak-stimulated autophagy Bax/Bak activation induces 3 separate pathways: (1) apoptotic caspase cascade, (2) IFN-β production via the cGAS/STING pathway and (3) autophagy. Both apoptotic caspases and autophagy restrain IFN-β secretion to render apoptotic cells immunologically silent

    Article Snippet: Equal amounts of protein were separated on 10% NUPAGE® Bis Tris gels (Invitrogen), transferred to nitrocellulose, and blotted with the antibodies raised against Mcl-1 (19C4; WEHI), Bim (3C5; WEHI), Bax (49F9-13-13; WEHI), Bak (Sigma), Parkin (sc-32282; Santa Cruz), LAMP1 (1D4B; Santa Cruz), TNFR1 (ab19139; Abcam), Calnexin (ab22595; Abcam), Apaf-1 (18H2; WEHI), TIMM44 (HPA043052; Sigma) and β-actin (AC-15; Sigma).

    Techniques: Activation Assay

    Bax/Bak-mediated LC3B lipidation is independent of apoptosome formation a Schematic of mitochondrial-mediated apoptosis. BH3 only proteins (e.g., Bim) and BH3 mimetic compounds sequester the pro-survival Bcl-2 family from Bax and Bak. Bax and Bak become activated, inducing mitochondrial outer membrane permiabilization, thus releasing cytochrome c. This activates the apoptosome and caspase 3 to induce rapid cell death. The pan-caspase inhibitor QVD-OPh (QVD) inhibits apoptotic caspase activity. b The pan caspase inhibitor QVD does not inhibit Bax/Bak-dependent LC3B lipidation. Mouse embryonic fibroblasts (MEFs) were treated with 1 μM ABT-737 and/or 10 μM QVD for 4 h prior to lysis and western blotting. c Wild type MEFs or those lacking Bax and Bak were infected with a doxycycline (Dox)-inducible Bim s lentiviral construct. Cells were treated with 1 μg/ml Dox and/or 10 μM QVD for 4 h prior to lysis. d Apaf-1 is not required for LC3B lipidation following Bax/Bak activation. Wild-type and Apaf-1 −/− MEFs previously bearing a Dox-inducible Bim s lentiviral construct were treated with 1 μg/ml Dox and/or 10 μM QVD for 4 h. e MEFs lacking Apaf-1 were infected with construct expressing Cas9 and a guide RNA against Mcl-1, cloned to isolate knockouts, and subsequently treated for 4 h with 1 μM ABT-737 to induce Bax/Bak activation

    Journal: Cell Death and Differentiation

    Article Title: Autophagy induced during apoptosis degrades mitochondria and inhibits type I interferon secretion

    doi: 10.1038/s41418-017-0017-z

    Figure Lengend Snippet: Bax/Bak-mediated LC3B lipidation is independent of apoptosome formation a Schematic of mitochondrial-mediated apoptosis. BH3 only proteins (e.g., Bim) and BH3 mimetic compounds sequester the pro-survival Bcl-2 family from Bax and Bak. Bax and Bak become activated, inducing mitochondrial outer membrane permiabilization, thus releasing cytochrome c. This activates the apoptosome and caspase 3 to induce rapid cell death. The pan-caspase inhibitor QVD-OPh (QVD) inhibits apoptotic caspase activity. b The pan caspase inhibitor QVD does not inhibit Bax/Bak-dependent LC3B lipidation. Mouse embryonic fibroblasts (MEFs) were treated with 1 μM ABT-737 and/or 10 μM QVD for 4 h prior to lysis and western blotting. c Wild type MEFs or those lacking Bax and Bak were infected with a doxycycline (Dox)-inducible Bim s lentiviral construct. Cells were treated with 1 μg/ml Dox and/or 10 μM QVD for 4 h prior to lysis. d Apaf-1 is not required for LC3B lipidation following Bax/Bak activation. Wild-type and Apaf-1 −/− MEFs previously bearing a Dox-inducible Bim s lentiviral construct were treated with 1 μg/ml Dox and/or 10 μM QVD for 4 h. e MEFs lacking Apaf-1 were infected with construct expressing Cas9 and a guide RNA against Mcl-1, cloned to isolate knockouts, and subsequently treated for 4 h with 1 μM ABT-737 to induce Bax/Bak activation

    Article Snippet: Equal amounts of protein were separated on 10% NUPAGE® Bis Tris gels (Invitrogen), transferred to nitrocellulose, and blotted with the antibodies raised against Mcl-1 (19C4; WEHI), Bim (3C5; WEHI), Bax (49F9-13-13; WEHI), Bak (Sigma), Parkin (sc-32282; Santa Cruz), LAMP1 (1D4B; Santa Cruz), TNFR1 (ab19139; Abcam), Calnexin (ab22595; Abcam), Apaf-1 (18H2; WEHI), TIMM44 (HPA043052; Sigma) and β-actin (AC-15; Sigma).

    Techniques: Activity Assay, Lysis, Western Blot, Infection, Construct, Activation Assay, Expressing, Clone Assay

    Autophagic flux following Bax/Bak activation is stimulated upstream of Apaf-1 a Schematic of the mCherry-EGFP-LC3B autophagic flux reporter. As LC3B is transported to the autolysosome via the autophagosome, EGFP fluorescence is inhibited due to a drop in pH. b Representative dot plots of Apaf-1 −/− MEFs expressing a guide RNA against Mcl-1 and the mCherry-EGFP-LC3B autophagic flux reporter. Cells were treated overnight with 1 μM ABT-737, 0.1 μM bafilomycin A 1 (BafA 1 ), and/or starved by culturing them in HBSS prior to flow cytometry analysis. c Quantitation of (b) using two different clones of Apaf-1 −/− ΔMcl-1 MEFs expressing the mCherry-EGFP-LC3B reporter ( n = 7 experiments). d Representative dot plots of Apaf-1 −/− MEFs expressing a Dox-inducible Bim s and the mCherry-EGFP-LC3B autophagic flux reporter were treated overnight with 1 μg/mL Dox to express Bim s , 0.1 μM bafilomycin A 1 (BafA 1 ), and/or starved by culturing them in HBSS. e Quantitation of (d) ( n = 3 independent experiments). The mean is shown in all graphs; error bars represent the SEM

    Journal: Cell Death and Differentiation

    Article Title: Autophagy induced during apoptosis degrades mitochondria and inhibits type I interferon secretion

    doi: 10.1038/s41418-017-0017-z

    Figure Lengend Snippet: Autophagic flux following Bax/Bak activation is stimulated upstream of Apaf-1 a Schematic of the mCherry-EGFP-LC3B autophagic flux reporter. As LC3B is transported to the autolysosome via the autophagosome, EGFP fluorescence is inhibited due to a drop in pH. b Representative dot plots of Apaf-1 −/− MEFs expressing a guide RNA against Mcl-1 and the mCherry-EGFP-LC3B autophagic flux reporter. Cells were treated overnight with 1 μM ABT-737, 0.1 μM bafilomycin A 1 (BafA 1 ), and/or starved by culturing them in HBSS prior to flow cytometry analysis. c Quantitation of (b) using two different clones of Apaf-1 −/− ΔMcl-1 MEFs expressing the mCherry-EGFP-LC3B reporter ( n = 7 experiments). d Representative dot plots of Apaf-1 −/− MEFs expressing a Dox-inducible Bim s and the mCherry-EGFP-LC3B autophagic flux reporter were treated overnight with 1 μg/mL Dox to express Bim s , 0.1 μM bafilomycin A 1 (BafA 1 ), and/or starved by culturing them in HBSS. e Quantitation of (d) ( n = 3 independent experiments). The mean is shown in all graphs; error bars represent the SEM

    Article Snippet: Equal amounts of protein were separated on 10% NUPAGE® Bis Tris gels (Invitrogen), transferred to nitrocellulose, and blotted with the antibodies raised against Mcl-1 (19C4; WEHI), Bim (3C5; WEHI), Bax (49F9-13-13; WEHI), Bak (Sigma), Parkin (sc-32282; Santa Cruz), LAMP1 (1D4B; Santa Cruz), TNFR1 (ab19139; Abcam), Calnexin (ab22595; Abcam), Apaf-1 (18H2; WEHI), TIMM44 (HPA043052; Sigma) and β-actin (AC-15; Sigma).

    Techniques: Activation Assay, Fluorescence, Expressing, Flow Cytometry, Cytometry, Quantitation Assay, Clone Assay

    Bax/Bak-activation activates ULK1 via AMPK a Schematic of the regulation of autophagy protein ULK1. AMPK activates ULK1 by phosphorylating both S317 and S555 on ULK1, while mTORC1 inhibits ULK1 activity by phosphorylating S757. b Apaf-1 −/− MEFs with Mcl-1 deleted by CRISPR-Cas9 technology were treated with 1 μM ABT-737, 50 μM CCCP, or starved in HBSS for 4 h prior to lysis and western blotting. Identical aliquots of samples were run on four gels as follows: #1 (AMPK, p-ULK1 S757 ), #2 (p-AMPK T172 , p-ULK1 S317 , S6, β-actin), #3 (p-ULK1 S555 , 4E-BP1), #4 (p-S6 S240/244 , ULK1, p-4E-BP1 S65 , LC3B). c Apaf-1 −/− MEFs were treated with 1 μg/mL Dox to express Bim s , 50 μM CCCP, or starved in HBSS for 4 h prior to lysis and western blotting. Identical aliquots of samples were run on four gels as follows: #1 (LC3B, p-ULK1 S757 ), #2 (p-AMPK T172 , p-ULK1 S317 , 4E-BP, S6), #3 (p-4E-BP1 S65 , ULK1, AMPK), #4 (p-S6 S240/244 , p-ULK1 S555 . d AMPK activation is upstream of LC3B lipidation. Mcl-1 −/− MEFs were infected with two different guide RNAs against ATG5 and selected for puromycin resistance. Pools were then treated with 1 μM ABT-737 for 4 h prior to western blotting. Identical amounts of sample were loaded on four different gels. e The intracellular ratio of AMP:ATP increases upon Bax/Bak-activation. MEFs were treated with either 1 μM ABT-737 or 1 μg/mL Dox to express Bim s for 4 h and analysed by mass spectrometry. The mean of five to six experiments is shown; error bars represent the SEM

    Journal: Cell Death and Differentiation

    Article Title: Autophagy induced during apoptosis degrades mitochondria and inhibits type I interferon secretion

    doi: 10.1038/s41418-017-0017-z

    Figure Lengend Snippet: Bax/Bak-activation activates ULK1 via AMPK a Schematic of the regulation of autophagy protein ULK1. AMPK activates ULK1 by phosphorylating both S317 and S555 on ULK1, while mTORC1 inhibits ULK1 activity by phosphorylating S757. b Apaf-1 −/− MEFs with Mcl-1 deleted by CRISPR-Cas9 technology were treated with 1 μM ABT-737, 50 μM CCCP, or starved in HBSS for 4 h prior to lysis and western blotting. Identical aliquots of samples were run on four gels as follows: #1 (AMPK, p-ULK1 S757 ), #2 (p-AMPK T172 , p-ULK1 S317 , S6, β-actin), #3 (p-ULK1 S555 , 4E-BP1), #4 (p-S6 S240/244 , ULK1, p-4E-BP1 S65 , LC3B). c Apaf-1 −/− MEFs were treated with 1 μg/mL Dox to express Bim s , 50 μM CCCP, or starved in HBSS for 4 h prior to lysis and western blotting. Identical aliquots of samples were run on four gels as follows: #1 (LC3B, p-ULK1 S757 ), #2 (p-AMPK T172 , p-ULK1 S317 , 4E-BP, S6), #3 (p-4E-BP1 S65 , ULK1, AMPK), #4 (p-S6 S240/244 , p-ULK1 S555 . d AMPK activation is upstream of LC3B lipidation. Mcl-1 −/− MEFs were infected with two different guide RNAs against ATG5 and selected for puromycin resistance. Pools were then treated with 1 μM ABT-737 for 4 h prior to western blotting. Identical amounts of sample were loaded on four different gels. e The intracellular ratio of AMP:ATP increases upon Bax/Bak-activation. MEFs were treated with either 1 μM ABT-737 or 1 μg/mL Dox to express Bim s for 4 h and analysed by mass spectrometry. The mean of five to six experiments is shown; error bars represent the SEM

    Article Snippet: Equal amounts of protein were separated on 10% NUPAGE® Bis Tris gels (Invitrogen), transferred to nitrocellulose, and blotted with the antibodies raised against Mcl-1 (19C4; WEHI), Bim (3C5; WEHI), Bax (49F9-13-13; WEHI), Bak (Sigma), Parkin (sc-32282; Santa Cruz), LAMP1 (1D4B; Santa Cruz), TNFR1 (ab19139; Abcam), Calnexin (ab22595; Abcam), Apaf-1 (18H2; WEHI), TIMM44 (HPA043052; Sigma) and β-actin (AC-15; Sigma).

    Techniques: Activation Assay, Activity Assay, CRISPR, Lysis, Western Blot, Infection, Mass Spectrometry

    Model of Bim-dependent and Bmf-dependent apoptosis during Ngo-induced apoptosis. Ngo infection leads to a Rac-dependent activation of JNK-1 and a concurrent alteration of the cytoskeletal morphology. Upon JNK-1–mediated phosphorylation, the cytoskeleton-attached proapoptotic proteins Bim and Bmf are released. Subsequently, the antiapoptotic effects of Mcl-1 and Bcl-X L are abrogated by Bim and Bmf, respectively, leading to the activation of Bak and Bax and cell death.

    Journal: PLoS Pathogens

    Article Title: Bim and Bmf Synergize To Induce Apoptosis in Neisseria Gonorrhoeae Infection

    doi: 10.1371/journal.ppat.1000348

    Figure Lengend Snippet: Model of Bim-dependent and Bmf-dependent apoptosis during Ngo-induced apoptosis. Ngo infection leads to a Rac-dependent activation of JNK-1 and a concurrent alteration of the cytoskeletal morphology. Upon JNK-1–mediated phosphorylation, the cytoskeleton-attached proapoptotic proteins Bim and Bmf are released. Subsequently, the antiapoptotic effects of Mcl-1 and Bcl-X L are abrogated by Bim and Bmf, respectively, leading to the activation of Bak and Bax and cell death.

    Article Snippet: The following antibodies and sera were used in this study: anti-β-Actin (Sigma); anti-Bad (Cell Signaling); anti-Bak NT (Upstate); anti-Bak (Ab-1) (Millipore);anti-Bax NT (Upstate); anti-Bax (6A7) (BD Pharmingen); anti-Bid (Cell Signaling); anti-Bim (Sigma); anti-Bmf (Cell Signaling); anti-cleaved Caspase-3 (Cell Signalling); anti-JNK-1 (Santa Cruz); anti-pJNK-1 (Cell Signalling) and anti-Mcl-1 (BD Pharmingen).

    Techniques: Infection, Activation Assay

    Bim-specific and Bmf-specific targeting of Mcl-1 and Bcl-X L . (A,B) The activation of Bak and Bax upon siRNA-mediated knockdown was visualized 15 h post-infection by immunoprecipitation with conformation-specific antibodies followed by SDS-PAGE and immunodetection with the indicated antibodies. (C) The network of Bcl-2 family proteins was analyzed by caspase activation assays after single or double knockdowns, 15 h post-infection. Shown are the means±SD of three independent experiments. (D,E) Knockdowns were validated by Western blot using the indicated antibodies (D) and qRT-PCR (E).

    Journal: PLoS Pathogens

    Article Title: Bim and Bmf Synergize To Induce Apoptosis in Neisseria Gonorrhoeae Infection

    doi: 10.1371/journal.ppat.1000348

    Figure Lengend Snippet: Bim-specific and Bmf-specific targeting of Mcl-1 and Bcl-X L . (A,B) The activation of Bak and Bax upon siRNA-mediated knockdown was visualized 15 h post-infection by immunoprecipitation with conformation-specific antibodies followed by SDS-PAGE and immunodetection with the indicated antibodies. (C) The network of Bcl-2 family proteins was analyzed by caspase activation assays after single or double knockdowns, 15 h post-infection. Shown are the means±SD of three independent experiments. (D,E) Knockdowns were validated by Western blot using the indicated antibodies (D) and qRT-PCR (E).

    Article Snippet: The following antibodies and sera were used in this study: anti-β-Actin (Sigma); anti-Bad (Cell Signaling); anti-Bak NT (Upstate); anti-Bak (Ab-1) (Millipore);anti-Bax NT (Upstate); anti-Bax (6A7) (BD Pharmingen); anti-Bid (Cell Signaling); anti-Bim (Sigma); anti-Bmf (Cell Signaling); anti-cleaved Caspase-3 (Cell Signalling); anti-JNK-1 (Santa Cruz); anti-pJNK-1 (Cell Signalling) and anti-Mcl-1 (BD Pharmingen).

    Techniques: Activation Assay, Infection, Immunoprecipitation, SDS Page, Immunodetection, Western Blot, Quantitative RT-PCR

    Expression of pro- and anti-apoptotic molecules in monocytes and mDCs during primary SIV infection. ( A ) Expression of FLIP and Mcl-1 in purified CD14 + (MØ) from healthy RM (SIV − ) and SIV-infected RMs (SIV + ). After isolation, the cells were lysed and the proteins were immunoblotted with specific antibodies against the anti-apoptotic molecules FLIP and Mcl-1. Actin was used as a control for equal protein loading. Values represent the ratio of the FLIP and Mcl-1 bands and normalized with respect to the loading control. ( B ) Flow cytometric analysis of the active form of the pro-apoptotic molecules Bax and Bak in CD4 + T cells, and monocytes (MØ) at days 0 and 14. ( C ) Percentage of active form of Bax and Bak among monocyte and DC populations at days 0 and 14. Values are means ± sem (n = 6); Significantly different from day 0 (*, p

    Journal: PLoS Pathogens

    Article Title: HIV/SIV Infection Primes Monocytes and Dendritic Cells for Apoptosis

    doi: 10.1371/journal.ppat.1002087

    Figure Lengend Snippet: Expression of pro- and anti-apoptotic molecules in monocytes and mDCs during primary SIV infection. ( A ) Expression of FLIP and Mcl-1 in purified CD14 + (MØ) from healthy RM (SIV − ) and SIV-infected RMs (SIV + ). After isolation, the cells were lysed and the proteins were immunoblotted with specific antibodies against the anti-apoptotic molecules FLIP and Mcl-1. Actin was used as a control for equal protein loading. Values represent the ratio of the FLIP and Mcl-1 bands and normalized with respect to the loading control. ( B ) Flow cytometric analysis of the active form of the pro-apoptotic molecules Bax and Bak in CD4 + T cells, and monocytes (MØ) at days 0 and 14. ( C ) Percentage of active form of Bax and Bak among monocyte and DC populations at days 0 and 14. Values are means ± sem (n = 6); Significantly different from day 0 (*, p

    Article Snippet: After blocking nonspecific sites for 1 hour at room temperature with 5% nonfat milk and 0.2% Tween 20 in phosphate-buffered saline (pH 7.4), the membrane was incubated with rabbit anti-Bak (Calbiochem, clone 2–14), rabbit polyclonal anti-Bax (Santa-Cruz, N-20), rabbit anti-Mcl-1 (S19, Santa-Cruz), or rat anti-FLIP (DAVE-2, Alexis Corporation).

    Techniques: Expressing, Infection, Purification, Isolation, Flow Cytometry

    The effect of MAPO2 knockdown on the activation of BAK proteins. Soluble protein fractions were extracted from HeLa MR and RF101 cells harvested 2, 3, 4 and 5 days after MNU treatment. The extracts were treated with disulphide bonding inducer to form intermolecularly linked active BAK dimers. The samples were boiled with (reduced) or without (CuPhe) 2-mercaptoethanol and subjected onto SDS-PAGE followed by immunoblotting analysis to detect the total amounts of BAK monomers and active BAK dimers, respectively, using anti-BAK monoclonal antibody ab-1. β-Actin was used as a loading control. The molecular weights are shown on the left.

    Journal: PLoS ONE

    Article Title: The Identification of a Novel Gene, MAPO2, That Is Involved in the Induction of Apoptosis Triggered by O6-Methylguanine

    doi: 10.1371/journal.pone.0044817

    Figure Lengend Snippet: The effect of MAPO2 knockdown on the activation of BAK proteins. Soluble protein fractions were extracted from HeLa MR and RF101 cells harvested 2, 3, 4 and 5 days after MNU treatment. The extracts were treated with disulphide bonding inducer to form intermolecularly linked active BAK dimers. The samples were boiled with (reduced) or without (CuPhe) 2-mercaptoethanol and subjected onto SDS-PAGE followed by immunoblotting analysis to detect the total amounts of BAK monomers and active BAK dimers, respectively, using anti-BAK monoclonal antibody ab-1. β-Actin was used as a loading control. The molecular weights are shown on the left.

    Article Snippet: Anti-phospho-CHK1 (S317) (Bethyl), anti-CHK1 (Santa Cruz), anti-BAK ab-1 (Millipore), anti-MSH2 (Invitrogen, Life Technologies Corp.), anti-MSH6 (BD Biosciences), anti-PMS2 (BD Biosciences), anti-MLH1 (BD Biosciences), anti-caspase-3 (Cell Signaling) and anti-β-actin (Sigma) were used as primary antibodies.

    Techniques: Activation Assay, SDS Page

    Western blot analysis of BCL-XL and GAPDH in J/Neo and J/BCL-XL cells (A), and flow cytometric analysis of the effect of CMEP-NQ on NOC-, 2-MeO-E 2 -, and CPT-induced alterations in cell cycle distribution (B and C) and Δψm loss (D and E) in Jurkat T cell clone transfected with a BCL-XL-expression vector (J/BCL-XL cells). After pretreatment with 7.5 μM CMEP-NQ for 1 h, J/BCL-XL cells (5 × 10 5 /ml) were treated with 0.3 μM NOC, 1.0 μM 2-MeO-E 2 , or 0.02 μM CPT for 17 h and then subjected to flow cytometric analysis of cell cycle distribution, Δψm loss, and BAK activation as described in the Materials and Methods. Western blot and flow cytometric analyses were performed as described in the Materials and Methods. A representative result is shown; two additional experiments yielded similar results. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Cytoprotective effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) is mediated by the inhibition of BAK-dependent mitochondrial apoptosis pathway

    doi: 10.1371/journal.pone.0204585

    Figure Lengend Snippet: Western blot analysis of BCL-XL and GAPDH in J/Neo and J/BCL-XL cells (A), and flow cytometric analysis of the effect of CMEP-NQ on NOC-, 2-MeO-E 2 -, and CPT-induced alterations in cell cycle distribution (B and C) and Δψm loss (D and E) in Jurkat T cell clone transfected with a BCL-XL-expression vector (J/BCL-XL cells). After pretreatment with 7.5 μM CMEP-NQ for 1 h, J/BCL-XL cells (5 × 10 5 /ml) were treated with 0.3 μM NOC, 1.0 μM 2-MeO-E 2 , or 0.02 μM CPT for 17 h and then subjected to flow cytometric analysis of cell cycle distribution, Δψm loss, and BAK activation as described in the Materials and Methods. Western blot and flow cytometric analyses were performed as described in the Materials and Methods. A representative result is shown; two additional experiments yielded similar results. Error bars represent standard deviations.

    Article Snippet: Anti-poly (ADP-ribose) polymerase (PARP), anti-BAK, anti-BIM, anti-BAG3, anti-BCL-2, anti-MCL-1, anti-CDK1, anti-cyclin B1, and anti-p53 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Western Blot, Flow Cytometry, Cycling Probe Technology, Transfection, Expressing, Plasmid Preparation, Activation Assay

    Western blot analysis of phosphorylated CDK1 (Thr-161), phosphorylated CDK1 (Tyr-15), CDK1, cyclin B1, phosphorylated-CDC25C (Thr-48), CDC25C, phosphorylated-Histone H3 (Ser-10), Histone H3, phosphorylated-CHK2 (Ser-19), CHK2, phosphorylated p53 (Ser-15), p53, and GAPDH (A), and phosphorylated BCL-2 (Ser-70), BCL-2, BCL-XL, BAG3, phosphorylated MCL-1 (Ser-159/Thr-169), MCL-1, BAK, reduction in the electrophoretic mobility of the BIM isoforms (BIM EL and BIM L ), caspase-9, PARP, and GAPDH (B) in J/Neo cells treated with NOC, 2-MeO-E 2 , or CPT in the absence and presence of CMEP-NQ for 17 h. After pretreatment with 7.5 μM CMEP-NQ for 1 h, J/Neo cells (5 × 10 5 /mL) were treated with 0.3 μM NOC, 1.0 μM 2-MeO-E2, or 0.02 μM CPT for 17 h and then collected for the preparation of total cell lysates. The levels of individual proteins in the cell lysates were examined by western blotting as described in the Materials and Methods. Symbol: ←*, the phosphorylated form of CDC25C and BIM. A representative result is shown; two additional experiments yielded similar results.

    Journal: PLoS ONE

    Article Title: Cytoprotective effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) is mediated by the inhibition of BAK-dependent mitochondrial apoptosis pathway

    doi: 10.1371/journal.pone.0204585

    Figure Lengend Snippet: Western blot analysis of phosphorylated CDK1 (Thr-161), phosphorylated CDK1 (Tyr-15), CDK1, cyclin B1, phosphorylated-CDC25C (Thr-48), CDC25C, phosphorylated-Histone H3 (Ser-10), Histone H3, phosphorylated-CHK2 (Ser-19), CHK2, phosphorylated p53 (Ser-15), p53, and GAPDH (A), and phosphorylated BCL-2 (Ser-70), BCL-2, BCL-XL, BAG3, phosphorylated MCL-1 (Ser-159/Thr-169), MCL-1, BAK, reduction in the electrophoretic mobility of the BIM isoforms (BIM EL and BIM L ), caspase-9, PARP, and GAPDH (B) in J/Neo cells treated with NOC, 2-MeO-E 2 , or CPT in the absence and presence of CMEP-NQ for 17 h. After pretreatment with 7.5 μM CMEP-NQ for 1 h, J/Neo cells (5 × 10 5 /mL) were treated with 0.3 μM NOC, 1.0 μM 2-MeO-E2, or 0.02 μM CPT for 17 h and then collected for the preparation of total cell lysates. The levels of individual proteins in the cell lysates were examined by western blotting as described in the Materials and Methods. Symbol: ←*, the phosphorylated form of CDC25C and BIM. A representative result is shown; two additional experiments yielded similar results.

    Article Snippet: Anti-poly (ADP-ribose) polymerase (PARP), anti-BAK, anti-BIM, anti-BAG3, anti-BCL-2, anti-MCL-1, anti-CDK1, anti-cyclin B1, and anti-p53 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Western Blot, Cycling Probe Technology

    XIAP protected against PEITC-mediated sensitization to Docetaxel. A, Immunoblotting for Bcl-2, Bax, Bak, and XIAP proteins using lysates from PC-3 cells treated for 24 h with PEITC and/or Docetaxel. Numbers on top of the bands represent change in protein level relative to DMSO-treated control (first lane). B, Flow cytometric analysis of mitochondrial membrane potential (monomeric JC-1 associated fluorescence) in PC-3 cells treated for 6 h with 2 μM PEITC and/or 1 nM Docetaxel (mean ± SE, n = 3). C, Cytoplasmic histone-associated DNA fragmentation, and D, cell viability in parental HCT-116 cells and its XIAP−/− variant (HCT-116/XIAP−/−) following 24 h treatment with DMSO, 2 μM PEITC (P), 1 nM Docetaxel (D), or the combination of PEITC and Docetaxel (P+D) Results shown are mean ± SE ( n = 3). Significantly different ( P

    Journal: Pharmaceutical research

    Article Title: Phenethyl Isothiocyanate Sensitizes Androgen-independent Human Prostate Cancer Cells to Docetaxel-Induced Apoptosis In Vitro and In Vivo

    doi: 10.1007/s11095-010-0079-9

    Figure Lengend Snippet: XIAP protected against PEITC-mediated sensitization to Docetaxel. A, Immunoblotting for Bcl-2, Bax, Bak, and XIAP proteins using lysates from PC-3 cells treated for 24 h with PEITC and/or Docetaxel. Numbers on top of the bands represent change in protein level relative to DMSO-treated control (first lane). B, Flow cytometric analysis of mitochondrial membrane potential (monomeric JC-1 associated fluorescence) in PC-3 cells treated for 6 h with 2 μM PEITC and/or 1 nM Docetaxel (mean ± SE, n = 3). C, Cytoplasmic histone-associated DNA fragmentation, and D, cell viability in parental HCT-116 cells and its XIAP−/− variant (HCT-116/XIAP−/−) following 24 h treatment with DMSO, 2 μM PEITC (P), 1 nM Docetaxel (D), or the combination of PEITC and Docetaxel (P+D) Results shown are mean ± SE ( n = 3). Significantly different ( P

    Article Snippet: The antibodies against Bak and Bax were from Santa Cruz Biotechnology (Santa Cruz, CA); the antibodies against Bcl-2 and proliferating cell nuclear antigen (PCNA) were from DakoCytomation (Carpinteria, CA); and the anti-XIAP antibody was from BD Biosciences Pharmingen (San Diego, CA).

    Techniques: Flow Cytometry, Fluorescence, Variant Assay

    Expression of Bax, Bak, Bcl-2, and XIAP proteins in tumor supernatants. A, Immunoblotting for Bax, Bak, Bcl-2, and XIAP using tumor supernatants from control mice and those treated with PEITC and/or Docetaxel. B, Quantitation of Bax, Bak, Bcl-2, and XIAP protein levels by densitometric scanning of the immunoreactive bands from control group (c); PEITC alone group (P); Docetaxel alone group (D); and the PEITC-Docetaxel combination group (P+D). Tumor tissues from 3 mice of each group were used for immunoblotting. Results shown are mean ± SE ( n = 3). Significantly different ( P

    Journal: Pharmaceutical research

    Article Title: Phenethyl Isothiocyanate Sensitizes Androgen-independent Human Prostate Cancer Cells to Docetaxel-Induced Apoptosis In Vitro and In Vivo

    doi: 10.1007/s11095-010-0079-9

    Figure Lengend Snippet: Expression of Bax, Bak, Bcl-2, and XIAP proteins in tumor supernatants. A, Immunoblotting for Bax, Bak, Bcl-2, and XIAP using tumor supernatants from control mice and those treated with PEITC and/or Docetaxel. B, Quantitation of Bax, Bak, Bcl-2, and XIAP protein levels by densitometric scanning of the immunoreactive bands from control group (c); PEITC alone group (P); Docetaxel alone group (D); and the PEITC-Docetaxel combination group (P+D). Tumor tissues from 3 mice of each group were used for immunoblotting. Results shown are mean ± SE ( n = 3). Significantly different ( P

    Article Snippet: The antibodies against Bak and Bax were from Santa Cruz Biotechnology (Santa Cruz, CA); the antibodies against Bcl-2 and proliferating cell nuclear antigen (PCNA) were from DakoCytomation (Carpinteria, CA); and the anti-XIAP antibody was from BD Biosciences Pharmingen (San Diego, CA).

    Techniques: Expressing, Mouse Assay, Quantitation Assay

    Bcl-X L protein levels are selectively regulated by hemopoietins. ( A ) Levels of Bcl-2 family members were assessed by immunoblot analyses with antibodies that detect murine Bcl-2, Bcl-X L , Mcl-1, Bax, Bad, and Bak proteins. Protein extracts (50 μg each) were prepared from 32D.3 ( left ) and FDC-P1.2 ( right ) myeloid cells growing in IL-3 or deprived of IL-3 for the indicated intervals and analyzed by immunoblots. Results shown are representative of six independent experiments. ( B ) Fetal liver-derived myeloid cells were grown in IL-3 (20 U/ml), IL-6 (10 ng/ml), and SCF (10 ng/ml) and then were deprived of all hemopoietins. Protein extracts (40 μg each) were prepared from cells after the indicated times after removal of hemopoietins and analyzed by immunoblot. Results shown are representative of three independent experiments. ( C ) The rates of death of 32D.3, FDC-P1.2, and primary myeloid cells were assessed following withdrawal of cytokines by trypan blue dye exclusion. Cell death was always apoptotic based on morphological criteria, and AnnexinV and TUNEL assays (data not shown). Results shown are representative of 10 independent experiments for 32D.3 and FDC-P1 cells and three for primary myeloid cells.

    Journal: Genes & Development

    Article Title: Selective regulation of Bcl-XL by a Jak kinase-dependent pathway is bypassed in murine hematopoietic malignancies

    doi:

    Figure Lengend Snippet: Bcl-X L protein levels are selectively regulated by hemopoietins. ( A ) Levels of Bcl-2 family members were assessed by immunoblot analyses with antibodies that detect murine Bcl-2, Bcl-X L , Mcl-1, Bax, Bad, and Bak proteins. Protein extracts (50 μg each) were prepared from 32D.3 ( left ) and FDC-P1.2 ( right ) myeloid cells growing in IL-3 or deprived of IL-3 for the indicated intervals and analyzed by immunoblots. Results shown are representative of six independent experiments. ( B ) Fetal liver-derived myeloid cells were grown in IL-3 (20 U/ml), IL-6 (10 ng/ml), and SCF (10 ng/ml) and then were deprived of all hemopoietins. Protein extracts (40 μg each) were prepared from cells after the indicated times after removal of hemopoietins and analyzed by immunoblot. Results shown are representative of three independent experiments. ( C ) The rates of death of 32D.3, FDC-P1.2, and primary myeloid cells were assessed following withdrawal of cytokines by trypan blue dye exclusion. Cell death was always apoptotic based on morphological criteria, and AnnexinV and TUNEL assays (data not shown). Results shown are representative of 10 independent experiments for 32D.3 and FDC-P1 cells and three for primary myeloid cells.

    Article Snippet: Antibodies used were as follows: mouse Bcl-2 (15021, PharMingen, 1:250); mouse Bcl-X (B2260, Transduction Labs, 1:250); mouse Bax (13686E, PharMingen, 1:500); Mcl-1 ( , Transduction Labs, 1:500); Bak (G-23, Santa Cruz, 1:100); Bad ( , Transduction Labs, 1:250) and Bag-1 ( ).

    Techniques: Western Blot, Derivative Assay, TUNEL Assay

    Carvedilol induces the expression of miR-125b-5p in simulated ischemia/reperfusion in both atrial and ventricular CMs, and inhibits the expression of its targets bak1 or klf13 in NRVCs A–B , The expression of mature miR-125b-5p in HL-1 cells treated with either vehicle (DMSO) or 1µM of carvedilol (Carv) for 4 hours and subjected to either normoxia (basal) or simulated ischemia/reperfusion (sI/R). C–D , The expression of mature miR-125b-5p in H9c2 cells treated with 1µM of Carv for 4 hours and subjected to either normoxia (basal) or sI/R. N=5 in each group. * P

    Journal: Journal of molecular and cellular cardiology

    Article Title: A carvedilol-responsive microRNA, miR-125b-5p protects the heart from acute myocardial infarction by repressing pro-apoptotic bak1 and klf13 in cardiomyocytes

    doi: 10.1016/j.yjmcc.2017.11.003

    Figure Lengend Snippet: Carvedilol induces the expression of miR-125b-5p in simulated ischemia/reperfusion in both atrial and ventricular CMs, and inhibits the expression of its targets bak1 or klf13 in NRVCs A–B , The expression of mature miR-125b-5p in HL-1 cells treated with either vehicle (DMSO) or 1µM of carvedilol (Carv) for 4 hours and subjected to either normoxia (basal) or simulated ischemia/reperfusion (sI/R). C–D , The expression of mature miR-125b-5p in H9c2 cells treated with 1µM of Carv for 4 hours and subjected to either normoxia (basal) or sI/R. N=5 in each group. * P

    Article Snippet: In addition to reporting that bak1 is a functional CM target of miR-125b-5p, we demonstrate the novel finding that klf13 is regulated by miR-125b-5p in CMs, consistent with a report in hematopoietic stem cells [ ].

    Techniques: Expressing

    Bak1 and klf13 are necessary for miR-125b-5p-dependent regulation of cardiomyocyte apoptosis A–E , NRVCs transfected with control scramble siRNA (si-control), bak1 siRNA (si-Bak1), klf13 siRNA (si-Klf13), anti-miR-125b-5p/si-Bak1, or anti-miR-125b-5p/si-Klf13 were subjected to in vitro simulation of I/R (sI/R). TUNEL assays were then performed under both basal and sI/R conditions. The percentage of apoptotic nuclei (green) was calculated by normalizing total nuclei (blue). Knockdown of bak1 or klf13 decreases ventricular cardiomyocyte apoptosis and protects NRVCs from the pro-apoptotic effects of anti-miR-125b-5p ( A–C ). QRT-PCR analyses for bak1 and klf13 ( D ) and miR-125b-5p ( E ) were performed to verify the knockdown efficiency. Data are shown as mean ± SEM for five independently obtained biological samples. * P

    Journal: Journal of molecular and cellular cardiology

    Article Title: A carvedilol-responsive microRNA, miR-125b-5p protects the heart from acute myocardial infarction by repressing pro-apoptotic bak1 and klf13 in cardiomyocytes

    doi: 10.1016/j.yjmcc.2017.11.003

    Figure Lengend Snippet: Bak1 and klf13 are necessary for miR-125b-5p-dependent regulation of cardiomyocyte apoptosis A–E , NRVCs transfected with control scramble siRNA (si-control), bak1 siRNA (si-Bak1), klf13 siRNA (si-Klf13), anti-miR-125b-5p/si-Bak1, or anti-miR-125b-5p/si-Klf13 were subjected to in vitro simulation of I/R (sI/R). TUNEL assays were then performed under both basal and sI/R conditions. The percentage of apoptotic nuclei (green) was calculated by normalizing total nuclei (blue). Knockdown of bak1 or klf13 decreases ventricular cardiomyocyte apoptosis and protects NRVCs from the pro-apoptotic effects of anti-miR-125b-5p ( A–C ). QRT-PCR analyses for bak1 and klf13 ( D ) and miR-125b-5p ( E ) were performed to verify the knockdown efficiency. Data are shown as mean ± SEM for five independently obtained biological samples. * P

    Article Snippet: In addition to reporting that bak1 is a functional CM target of miR-125b-5p, we demonstrate the novel finding that klf13 is regulated by miR-125b-5p in CMs, consistent with a report in hematopoietic stem cells [ ].

    Techniques: Transfection, In Vitro, TUNEL Assay, Quantitative RT-PCR

    MiR-125b-5p represses pro-apoptotic bak1 and klf13 A–C , RNAs isolated from NRVCs transfected with 100nM mir vana miR-125b-5p inhibitor or 15-mer control ( A ) and miR-125b-5p mimic or 15-mer control ( B ) were analyzed by miR-125b-5p-specific RT-PCR and QRT-PCR to access the levels of miR-125b-5p. * P

    Journal: Journal of molecular and cellular cardiology

    Article Title: A carvedilol-responsive microRNA, miR-125b-5p protects the heart from acute myocardial infarction by repressing pro-apoptotic bak1 and klf13 in cardiomyocytes

    doi: 10.1016/j.yjmcc.2017.11.003

    Figure Lengend Snippet: MiR-125b-5p represses pro-apoptotic bak1 and klf13 A–C , RNAs isolated from NRVCs transfected with 100nM mir vana miR-125b-5p inhibitor or 15-mer control ( A ) and miR-125b-5p mimic or 15-mer control ( B ) were analyzed by miR-125b-5p-specific RT-PCR and QRT-PCR to access the levels of miR-125b-5p. * P

    Article Snippet: In addition to reporting that bak1 is a functional CM target of miR-125b-5p, we demonstrate the novel finding that klf13 is regulated by miR-125b-5p in CMs, consistent with a report in hematopoietic stem cells [ ].

    Techniques: Isolation, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    Increased immunogenicity of Bax −/− Bak −/− DC

    Journal: Journal of Immunology (Baltimore, Md. : 1950)

    Article Title: Immune Regulation through Mitochondrion-dependent Dendritic Cell Death Induced by T Regulatory Cells

    doi: 10.4049/jimmunol.1101834

    Figure Lengend Snippet: Increased immunogenicity of Bax −/− Bak −/− DC

    Article Snippet: The following primary antibodies were used: polyclonal rabbit antibodies to caspase-8, caspase-3, Bcl-xL, Bad (Cell Signaling), Bax (Santa Cruz Biotechnology), Bcl-2, Bak (Upstate Biotechnology), Mcl-1 (Fitzgerald), Bim (Stressgen), Bid (Imgenex), Blk or Bmf (Biovision), or mouse monoclonal antibody to caspase-9 (MBL), Noxa (Imgenex) or XIAP (BD Bioscience).

    Techniques:

    Effect of ES, ES-BAX, ES-BAK, and ES-BAX-ES in the ex vivo rat aortic ring assay. Ring sections were cut out from freshly isolated aorta of euthanized rats. The rings were cultivated in Matrigel, in the absence or presence of 10 μ g/ml of each protein for 7 days. The aortic rings and the angiogenic sprouting were stained with MTT and photographed. ( a ) Photographs from representative rings from assays performed in triplicate. Scale bar: 1000 μ m. ( b ) Quantification of microvessel outgrowth in each condition. The mean of the pixels number of the outgrowth of the control group was set as 100%. Error bars: mean±S.E.M. The statistical significance of differences between the groups was assessed by one-way ANOVA with Bonferroni post-test, ** P

    Journal: Cell Death & Disease

    Article Title: Improving the therapeutic potential of endostatin by fusing it with the BAX BH3 death domain

    doi: 10.1038/cddis.2014.309

    Figure Lengend Snippet: Effect of ES, ES-BAX, ES-BAK, and ES-BAX-ES in the ex vivo rat aortic ring assay. Ring sections were cut out from freshly isolated aorta of euthanized rats. The rings were cultivated in Matrigel, in the absence or presence of 10 μ g/ml of each protein for 7 days. The aortic rings and the angiogenic sprouting were stained with MTT and photographed. ( a ) Photographs from representative rings from assays performed in triplicate. Scale bar: 1000 μ m. ( b ) Quantification of microvessel outgrowth in each condition. The mean of the pixels number of the outgrowth of the control group was set as 100%. Error bars: mean±S.E.M. The statistical significance of differences between the groups was assessed by one-way ANOVA with Bonferroni post-test, ** P

    Article Snippet: For the immunoblotting, ES, ES-BAX, and ES-BAK were detected with rabbit anti-ES polyclonal antibody (AB1880, Merck Millipore, diluted 1 : 500), anti-BCL-2 (C-21: sc-783, Santa Cruz Biotechnology, diluted 1 : 1000), anti-BCL-XL (H-62: sc-50, Santa Cruz Biotechnnology, diluted 1 : 1000), anti-BAX (Becton-Dickinson, diluted 1 : 1000), or anti-BAK (Becton-Dickinson, diluted 1 : 1000), followed by incubation with HRP conjugated with secondary antibody (Merck Millipore, diluted 1 : 5000).

    Techniques: Ex Vivo, Aortic Ring Assay, Isolation, Staining, MTT Assay

    Internalization of ES, ES-BAX, ES-BAK, and ES-BAX-ES by endothelial cells. ( a ) C-PAE or NIH 3T3 cells were incubated at 37°C for 2 h with 10 μ g/ml of the biotynilated proteins: ES, ES-BAX, ES-BAK, or ES-BAX-ES. The cells were then fixed, permeabilized, and incubated with Alexa 488 conjugated with streptavidin. The nucleuses were stained with DAPI. The cells were analyzed under a fluorescence microscope. Scale bar: 10 μ m. ( b ) Western blot of endothelial C-PAE cells incubated with 10 μ g/ml of the indicated proteins at 37°C and detected by anti-ES antibody. Control (+): ES; control (−): lysate of cells incubated with ES that were immediately processed for western blot ( t =0 min)

    Journal: Cell Death & Disease

    Article Title: Improving the therapeutic potential of endostatin by fusing it with the BAX BH3 death domain

    doi: 10.1038/cddis.2014.309

    Figure Lengend Snippet: Internalization of ES, ES-BAX, ES-BAK, and ES-BAX-ES by endothelial cells. ( a ) C-PAE or NIH 3T3 cells were incubated at 37°C for 2 h with 10 μ g/ml of the biotynilated proteins: ES, ES-BAX, ES-BAK, or ES-BAX-ES. The cells were then fixed, permeabilized, and incubated with Alexa 488 conjugated with streptavidin. The nucleuses were stained with DAPI. The cells were analyzed under a fluorescence microscope. Scale bar: 10 μ m. ( b ) Western blot of endothelial C-PAE cells incubated with 10 μ g/ml of the indicated proteins at 37°C and detected by anti-ES antibody. Control (+): ES; control (−): lysate of cells incubated with ES that were immediately processed for western blot ( t =0 min)

    Article Snippet: For the immunoblotting, ES, ES-BAX, and ES-BAK were detected with rabbit anti-ES polyclonal antibody (AB1880, Merck Millipore, diluted 1 : 500), anti-BCL-2 (C-21: sc-783, Santa Cruz Biotechnology, diluted 1 : 1000), anti-BCL-XL (H-62: sc-50, Santa Cruz Biotechnnology, diluted 1 : 1000), anti-BAX (Becton-Dickinson, diluted 1 : 1000), or anti-BAK (Becton-Dickinson, diluted 1 : 1000), followed by incubation with HRP conjugated with secondary antibody (Merck Millipore, diluted 1 : 5000).

    Techniques: Incubation, Staining, Fluorescence, Microscopy, Western Blot

    Effect of ES, ES-BAX, ES-BAK, and ES-BAX-ES on endothelial cells. ( a ) C-PAE cells were incubated in the presence or absence of 20 μ g/ml of either protein for 16 h. The cells were trypsinized and stained with propidium iodide. Fluorescence of individual nuclei was measured by flow cytometry. Each sample was analyzed in triplicate. Error bars: mean±S.E.M. The statistical significance of differences between the groups was assessed with one-way ANOVA with Bonferroni post-test, *** P

    Journal: Cell Death & Disease

    Article Title: Improving the therapeutic potential of endostatin by fusing it with the BAX BH3 death domain

    doi: 10.1038/cddis.2014.309

    Figure Lengend Snippet: Effect of ES, ES-BAX, ES-BAK, and ES-BAX-ES on endothelial cells. ( a ) C-PAE cells were incubated in the presence or absence of 20 μ g/ml of either protein for 16 h. The cells were trypsinized and stained with propidium iodide. Fluorescence of individual nuclei was measured by flow cytometry. Each sample was analyzed in triplicate. Error bars: mean±S.E.M. The statistical significance of differences between the groups was assessed with one-way ANOVA with Bonferroni post-test, *** P

    Article Snippet: For the immunoblotting, ES, ES-BAX, and ES-BAK were detected with rabbit anti-ES polyclonal antibody (AB1880, Merck Millipore, diluted 1 : 500), anti-BCL-2 (C-21: sc-783, Santa Cruz Biotechnology, diluted 1 : 1000), anti-BCL-XL (H-62: sc-50, Santa Cruz Biotechnnology, diluted 1 : 1000), anti-BAX (Becton-Dickinson, diluted 1 : 1000), or anti-BAK (Becton-Dickinson, diluted 1 : 1000), followed by incubation with HRP conjugated with secondary antibody (Merck Millipore, diluted 1 : 5000).

    Techniques: Incubation, Staining, Fluorescence, Flow Cytometry, Cytometry

    ES, ES-BAX, ES-BAK, and ES-BAX-ES. ( a ) Alignment of the primary sequences of ES and the hybrid proteins. The ES α -helix 1 is indicated. The segments corresponding to the BAX or BAK BH3 domain are underlined. The cysteine residues involved in disulfide bonds (C33-C173 and C135-C165) are indicated by arrows. ( b ) Cartoon representation of ES and hybrid proteins. ES α -helices are shown in red, BAX BH3 α -helices are shown in blue, β -sheets are shown in yellow, and disulfide bridges are shown in green. ( c ) Refolded and purified ES and hybrid proteins analyzed by SDS-PAGE

    Journal: Cell Death & Disease

    Article Title: Improving the therapeutic potential of endostatin by fusing it with the BAX BH3 death domain

    doi: 10.1038/cddis.2014.309

    Figure Lengend Snippet: ES, ES-BAX, ES-BAK, and ES-BAX-ES. ( a ) Alignment of the primary sequences of ES and the hybrid proteins. The ES α -helix 1 is indicated. The segments corresponding to the BAX or BAK BH3 domain are underlined. The cysteine residues involved in disulfide bonds (C33-C173 and C135-C165) are indicated by arrows. ( b ) Cartoon representation of ES and hybrid proteins. ES α -helices are shown in red, BAX BH3 α -helices are shown in blue, β -sheets are shown in yellow, and disulfide bridges are shown in green. ( c ) Refolded and purified ES and hybrid proteins analyzed by SDS-PAGE

    Article Snippet: For the immunoblotting, ES, ES-BAX, and ES-BAK were detected with rabbit anti-ES polyclonal antibody (AB1880, Merck Millipore, diluted 1 : 500), anti-BCL-2 (C-21: sc-783, Santa Cruz Biotechnology, diluted 1 : 1000), anti-BCL-XL (H-62: sc-50, Santa Cruz Biotechnnology, diluted 1 : 1000), anti-BAX (Becton-Dickinson, diluted 1 : 1000), or anti-BAK (Becton-Dickinson, diluted 1 : 1000), followed by incubation with HRP conjugated with secondary antibody (Merck Millipore, diluted 1 : 5000).

    Techniques: Purification, SDS Page

    Interaction of ES, ES-BAX, and ES-BAK with members of the BCL-2 family. Whole cell lysates of 10 7 endothelial cells (HUV-EC-C) were incubated in the absence or presence of 100 μ g/ml ES, ES-BAX, or ES-BAK. The ES, ES-BAX, or ES-BAK complexes bound to cellular proteins (BCL-2, BCL-X L , BAX, or BAK) were incubated with the antibodies indicated for co-immunoprecipitation, captured on Protein G agarose beads, and subsequently subjected to SDS-PAGE. The complexes were detected by immunoblotting with the indicated antibodies. Similar blots were probed with the same antibodies that were used for co-immunoprecipitation, to ensure that equivalent amounts of proteins were present in each reaction. Cell lysates were applied at equivalent volumes. Non-related monoclonal antibodies were used to control co-immunoprecipitation

    Journal: Cell Death & Disease

    Article Title: Improving the therapeutic potential of endostatin by fusing it with the BAX BH3 death domain

    doi: 10.1038/cddis.2014.309

    Figure Lengend Snippet: Interaction of ES, ES-BAX, and ES-BAK with members of the BCL-2 family. Whole cell lysates of 10 7 endothelial cells (HUV-EC-C) were incubated in the absence or presence of 100 μ g/ml ES, ES-BAX, or ES-BAK. The ES, ES-BAX, or ES-BAK complexes bound to cellular proteins (BCL-2, BCL-X L , BAX, or BAK) were incubated with the antibodies indicated for co-immunoprecipitation, captured on Protein G agarose beads, and subsequently subjected to SDS-PAGE. The complexes were detected by immunoblotting with the indicated antibodies. Similar blots were probed with the same antibodies that were used for co-immunoprecipitation, to ensure that equivalent amounts of proteins were present in each reaction. Cell lysates were applied at equivalent volumes. Non-related monoclonal antibodies were used to control co-immunoprecipitation

    Article Snippet: For the immunoblotting, ES, ES-BAX, and ES-BAK were detected with rabbit anti-ES polyclonal antibody (AB1880, Merck Millipore, diluted 1 : 500), anti-BCL-2 (C-21: sc-783, Santa Cruz Biotechnology, diluted 1 : 1000), anti-BCL-XL (H-62: sc-50, Santa Cruz Biotechnnology, diluted 1 : 1000), anti-BAX (Becton-Dickinson, diluted 1 : 1000), or anti-BAK (Becton-Dickinson, diluted 1 : 1000), followed by incubation with HRP conjugated with secondary antibody (Merck Millipore, diluted 1 : 5000).

    Techniques: Incubation, Immunoprecipitation, SDS Page

    BID and BAK physically interact, and a BAK-blocking Ab prevents cytochrome c release. ( A ) In vitro binding between GST–BAK and IVT BCL-X L , p15 BID, and BIDα345/TM, but not p15 BID mIII.4 or BIDα345/TM mIII.4 (lanes 1–5 ). None of the IVT proteins bound GST itself (data not shown). Preincubation with an anti-BAK Ab inhibits GST–-BAK in vitro binding to p15 BID (lanes 6,7 ). ( B ) Coimmunoprecipitation of BAK and wild-type p15 BID. 35 S-labeled p15 BID wild-type (lane 1 ) and mutant p15 BID mIII.4 (lane 2 ) were targeted to mitochondria in vitro. The mitochondria were then solubilized and immunoprecipitated with an anti-BAK Ab. Coimmunoprecipitated p15 was detected by autoradiography. The anti-BAK Ab did not directly immunoprecipitate p15 BID wild type from solution (lane 3 ). BAK (24 kD) comigrates with light chain (25 kD), precluding detection of BAK by IP Western. Consequently, 35 S-labeled IVT BAK was mixed with IVT p15 wild type or p15 mIII.4 in buffer B. BID was immunoprecipitated with an anti-BID Ab, and coimmunoprecipitated BAK was detected by autoradiography (lanes 4,5 ). ( C ) Inhibition of the p15 BID/BAK interaction prevents cytochrome c release. Wild-type mitochondria were incubated with the indicated amounts of anti-BAK Ab (G-23) or anti-BCL-X L Ab (SC-18) for 20 min at room temperature. A total of 25 ng of recombinant p15 BID was targeted to the mitochondria. The cytochrome c released into the supernatant and the p15 BID targeted to the mitochondrial pellet are shown.

    Journal: Genes & Development

    Article Title: tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c

    doi:

    Figure Lengend Snippet: BID and BAK physically interact, and a BAK-blocking Ab prevents cytochrome c release. ( A ) In vitro binding between GST–BAK and IVT BCL-X L , p15 BID, and BIDα345/TM, but not p15 BID mIII.4 or BIDα345/TM mIII.4 (lanes 1–5 ). None of the IVT proteins bound GST itself (data not shown). Preincubation with an anti-BAK Ab inhibits GST–-BAK in vitro binding to p15 BID (lanes 6,7 ). ( B ) Coimmunoprecipitation of BAK and wild-type p15 BID. 35 S-labeled p15 BID wild-type (lane 1 ) and mutant p15 BID mIII.4 (lane 2 ) were targeted to mitochondria in vitro. The mitochondria were then solubilized and immunoprecipitated with an anti-BAK Ab. Coimmunoprecipitated p15 was detected by autoradiography. The anti-BAK Ab did not directly immunoprecipitate p15 BID wild type from solution (lane 3 ). BAK (24 kD) comigrates with light chain (25 kD), precluding detection of BAK by IP Western. Consequently, 35 S-labeled IVT BAK was mixed with IVT p15 wild type or p15 mIII.4 in buffer B. BID was immunoprecipitated with an anti-BID Ab, and coimmunoprecipitated BAK was detected by autoradiography (lanes 4,5 ). ( C ) Inhibition of the p15 BID/BAK interaction prevents cytochrome c release. Wild-type mitochondria were incubated with the indicated amounts of anti-BAK Ab (G-23) or anti-BCL-X L Ab (SC-18) for 20 min at room temperature. A total of 25 ng of recombinant p15 BID was targeted to the mitochondria. The cytochrome c released into the supernatant and the p15 BID targeted to the mitochondrial pellet are shown.

    Article Snippet: Antibodies included anti-cytochrome c (75981A, Pharmingen), anti-BAK (G-23, Santa Cruz), anti-BAK (Upstate Biotechnology), anti-VDAC (Ab-4, Calbiochem), anti-BCL-XL (Pharmingen), anti-BID , and anti-ANT (generous gift of P. Schmid, University of Minnesota).

    Techniques: Blocking Assay, In Vitro, Binding Assay, Labeling, Mutagenesis, Immunoprecipitation, Autoradiography, Western Blot, Inhibition, Incubation, Recombinant

    mt DNA is released from mitochondria following MOMP in a BAX / BAK ‐dependent manner Fixed super‐resolution Airyscan images of U2OS cells immunostained with anti‐TOM20 (green) and anti‐DNA (red) antibodies. Scale bar = 10 μm. Representative images from three independent experiments. Airyscan images of U2OS cells treated with 10 μM ABT‐737, 1 μM ActD and 20 μM qVD‐OPh for 3 h, immunostained with anti‐TOM20 and anti‐DNA antibodies. Scale bar = 10 μm. Representative images from three independent experiments. Quantification of cells exhibiting > 10% mtDNA release following treatment with 10 μM ABT‐737, 1 μM ActD and 20 μM qVD‐OPh. Data are expressed as mean ± SD from three independent experiments and analysed by Student's t ‐test. Quantification of the extent of mitochondrial DNA (mtDNA) nucleoid release per cell following treatment with 10 μM ABT‐737, 1 μM ActD and 20 μM qVD‐OPh. Data are expressed as mean ± SD from two independent experiments and analysed by Student's t ‐test. Airyscan images of MEF cells untreated or treated with 10 μM ABT‐737 and 20 μM qVD‐OPh for 3 h, immunostained with anti‐TOM20 and anti‐DNA antibodies. Scale bar = 10 μm. Representative images from three independent experiments. BAX and BAK expression levels in U2OS cells with CRISPR‐Cas9‐mediated deletion of BAX, BAK or BAX/BAK. U2OS cells with BAX, BAK or BAX/BAK deletion by CRISPR‐Cas9 treated with 10 μM ABT‐737 and 2 μM S63845 and analysed for cell viability using an IncuCyte live‐cell imager and SYTOX Green exclusion. Data are expressed as mean ± SEM, representative of three independent experiments, and have been normalised to starting confluency. Airyscan images of U2OS (i) control cells or with CRISPR‐Cas9‐mediated deletion of either (ii) BAX, (iii) BAK or (iv) BAX and BAK treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh for 3 h. Scale bar = 10 μm. Representative images from three independent experiments. Quantification of mtDNA nucleoid release per cell in U2OS EMPTY CRISPR , BAX CRISPR , BAK CRISPR and BAX/BAK CRISPR cells. Data are expressed as mean ± SD from three independent experiments and analysed using Student's t ‐test. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Mitochondrial inner membrane permeabilisation enables mt DNA release during apoptosis

    doi: 10.15252/embj.201899238

    Figure Lengend Snippet: mt DNA is released from mitochondria following MOMP in a BAX / BAK ‐dependent manner Fixed super‐resolution Airyscan images of U2OS cells immunostained with anti‐TOM20 (green) and anti‐DNA (red) antibodies. Scale bar = 10 μm. Representative images from three independent experiments. Airyscan images of U2OS cells treated with 10 μM ABT‐737, 1 μM ActD and 20 μM qVD‐OPh for 3 h, immunostained with anti‐TOM20 and anti‐DNA antibodies. Scale bar = 10 μm. Representative images from three independent experiments. Quantification of cells exhibiting > 10% mtDNA release following treatment with 10 μM ABT‐737, 1 μM ActD and 20 μM qVD‐OPh. Data are expressed as mean ± SD from three independent experiments and analysed by Student's t ‐test. Quantification of the extent of mitochondrial DNA (mtDNA) nucleoid release per cell following treatment with 10 μM ABT‐737, 1 μM ActD and 20 μM qVD‐OPh. Data are expressed as mean ± SD from two independent experiments and analysed by Student's t ‐test. Airyscan images of MEF cells untreated or treated with 10 μM ABT‐737 and 20 μM qVD‐OPh for 3 h, immunostained with anti‐TOM20 and anti‐DNA antibodies. Scale bar = 10 μm. Representative images from three independent experiments. BAX and BAK expression levels in U2OS cells with CRISPR‐Cas9‐mediated deletion of BAX, BAK or BAX/BAK. U2OS cells with BAX, BAK or BAX/BAK deletion by CRISPR‐Cas9 treated with 10 μM ABT‐737 and 2 μM S63845 and analysed for cell viability using an IncuCyte live‐cell imager and SYTOX Green exclusion. Data are expressed as mean ± SEM, representative of three independent experiments, and have been normalised to starting confluency. Airyscan images of U2OS (i) control cells or with CRISPR‐Cas9‐mediated deletion of either (ii) BAX, (iii) BAK or (iv) BAX and BAK treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh for 3 h. Scale bar = 10 μm. Representative images from three independent experiments. Quantification of mtDNA nucleoid release per cell in U2OS EMPTY CRISPR , BAX CRISPR , BAK CRISPR and BAX/BAK CRISPR cells. Data are expressed as mean ± SD from three independent experiments and analysed using Student's t ‐test. Source data are available online for this figure.

    Article Snippet: Primary antibodies for Western blotting were as follows: rabbit anti‐BAX, rabbit anti‐BAK, rabbit anti‐DRP1, rabbit anti‐STING (Cell Signaling), mouse MitoProfile Membrane Integrity Cocktail (for cyclophilin D; Abcam) and β‐actin (Sigma).

    Techniques: Expressing, CRISPR

    Mitochondrial inner membrane and mt DNA are extruded through BAX pores on the mitochondrial outer membrane Further examples of stills from live‐cell imaging of U2OS BAX/BAK CRISPR cells expressing JF 646 ‐MOM (magenta) with BAX foci (cyan) and TFAM extrusion (green) from Fig 6 A and Video EV10 . Cells were treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh at t = 0. Numbers indicate time in minutes. Further examples of TFAM extrusion through BAX pores from Fig 6 B of U2OS cells stably expressing JF 646 ‐MOM (magenta), transiently expressing TFAM‐mClover (green) and immunostained for active BAX (6A7, cyan). Cells were treated with 10 μM ABT‐737 and 20 μM qVD‐OPh for 3 h. Further examples of Imaris 3D‐renderings from Fig 6 B of U2OS cells stably expressing JF 646 ‐MOM (green), transiently expressing TFAM‐mClover (red) and immunostained for active BAX (6A7, cyan). Cells were treated with 10 μM ABT‐737 and 20 μM qVD‐OPh for 3 h. Further examples of IMM extrusion through BAX pores from Fig 6 D of U2OS cells stably expressing JF 646 ‐MOM (magenta) and immunostained for AIF (IMM, green) and active BAX (6A7, cyan). Cells were treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh for 3 h. Further examples of Imaris 3D‐renderings from Fig 6 D of U2OS cells stably expressing JF 646 ‐MOM (green) and immunostained for AIF (IMM, red) and active BAX (6A7, cyan). Cells were treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh for 3 h.

    Journal: The EMBO Journal

    Article Title: Mitochondrial inner membrane permeabilisation enables mt DNA release during apoptosis

    doi: 10.15252/embj.201899238

    Figure Lengend Snippet: Mitochondrial inner membrane and mt DNA are extruded through BAX pores on the mitochondrial outer membrane Further examples of stills from live‐cell imaging of U2OS BAX/BAK CRISPR cells expressing JF 646 ‐MOM (magenta) with BAX foci (cyan) and TFAM extrusion (green) from Fig 6 A and Video EV10 . Cells were treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh at t = 0. Numbers indicate time in minutes. Further examples of TFAM extrusion through BAX pores from Fig 6 B of U2OS cells stably expressing JF 646 ‐MOM (magenta), transiently expressing TFAM‐mClover (green) and immunostained for active BAX (6A7, cyan). Cells were treated with 10 μM ABT‐737 and 20 μM qVD‐OPh for 3 h. Further examples of Imaris 3D‐renderings from Fig 6 B of U2OS cells stably expressing JF 646 ‐MOM (green), transiently expressing TFAM‐mClover (red) and immunostained for active BAX (6A7, cyan). Cells were treated with 10 μM ABT‐737 and 20 μM qVD‐OPh for 3 h. Further examples of IMM extrusion through BAX pores from Fig 6 D of U2OS cells stably expressing JF 646 ‐MOM (magenta) and immunostained for AIF (IMM, green) and active BAX (6A7, cyan). Cells were treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh for 3 h. Further examples of Imaris 3D‐renderings from Fig 6 D of U2OS cells stably expressing JF 646 ‐MOM (green) and immunostained for AIF (IMM, red) and active BAX (6A7, cyan). Cells were treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh for 3 h.

    Article Snippet: Primary antibodies for Western blotting were as follows: rabbit anti‐BAX, rabbit anti‐BAK, rabbit anti‐DRP1, rabbit anti‐STING (Cell Signaling), mouse MitoProfile Membrane Integrity Cocktail (for cyclophilin D; Abcam) and β‐actin (Sigma).

    Techniques: Live Cell Imaging, CRISPR, Expressing, Stable Transfection

    MOMP ‐induced mt DNA release initiates a cGAS ‐ STING ‐dependent type I interferon response SVEC cells treated with 10 μM ABT‐737 and 10 μM S63845 ± 20 μM qVD‐OPh. Cell viability was analysed using an IncuCyte live‐cell imager and SYTOX Green exclusion. Data are expressed as mean ± SEM, representative of two independent experiments. Airyscan images of SVEC cells treated with 10 μM ABT‐737, 10 μM S63845 and 20 μM qVD‐OPh for 3 h, immunostained with anti‐TOM20 and anti‐DNA antibodies. Scale bar = 10 μm. Representative images from three independent experiments. Ifnb1 , Irf7 and Oasl1 mRNA expression in SVEC cells treated with 10 μM ABT‐737, 10 μM S63845 and 20 μM qVD‐OPh for 3 h. Data are representative of three independent experiments. Ifnb1 mRNA expression in SVEC cells treated with 10 μM ABT‐737, 10 μM S63845 ± 20 μM qVD‐OPh for 2 h. Data are representative of two independent experiments. STING expression in CRISPR‐Cas9‐mediated STING‐deleted SVEC cells using three independent sgRNA sequences. Ifnb1 mRNA expression in STING CRISPR‐Cas9‐deleted SVEC cells treated with 10 μM ABT‐737, 10 μM S63845 and 20 μM qVD‐OPh for 3 h. Data are representative of two independent experiments. BAX and BAK expression in SVEC cells harbouring CRISPR‐Cas9‐mediated deletion of BAX, BAK or BAX/BAK. Ifnb1 mRNA expression in BAX, BAK or BAX/BAK CRISPR‐Cas9‐deleted SVEC cells treated with 10 μM ABT‐737, 10 μM S63845 and 20 μM qVD‐OPh for 3 h. Data are representative of two independent experiments. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Mitochondrial inner membrane permeabilisation enables mt DNA release during apoptosis

    doi: 10.15252/embj.201899238

    Figure Lengend Snippet: MOMP ‐induced mt DNA release initiates a cGAS ‐ STING ‐dependent type I interferon response SVEC cells treated with 10 μM ABT‐737 and 10 μM S63845 ± 20 μM qVD‐OPh. Cell viability was analysed using an IncuCyte live‐cell imager and SYTOX Green exclusion. Data are expressed as mean ± SEM, representative of two independent experiments. Airyscan images of SVEC cells treated with 10 μM ABT‐737, 10 μM S63845 and 20 μM qVD‐OPh for 3 h, immunostained with anti‐TOM20 and anti‐DNA antibodies. Scale bar = 10 μm. Representative images from three independent experiments. Ifnb1 , Irf7 and Oasl1 mRNA expression in SVEC cells treated with 10 μM ABT‐737, 10 μM S63845 and 20 μM qVD‐OPh for 3 h. Data are representative of three independent experiments. Ifnb1 mRNA expression in SVEC cells treated with 10 μM ABT‐737, 10 μM S63845 ± 20 μM qVD‐OPh for 2 h. Data are representative of two independent experiments. STING expression in CRISPR‐Cas9‐mediated STING‐deleted SVEC cells using three independent sgRNA sequences. Ifnb1 mRNA expression in STING CRISPR‐Cas9‐deleted SVEC cells treated with 10 μM ABT‐737, 10 μM S63845 and 20 μM qVD‐OPh for 3 h. Data are representative of two independent experiments. BAX and BAK expression in SVEC cells harbouring CRISPR‐Cas9‐mediated deletion of BAX, BAK or BAX/BAK. Ifnb1 mRNA expression in BAX, BAK or BAX/BAK CRISPR‐Cas9‐deleted SVEC cells treated with 10 μM ABT‐737, 10 μM S63845 and 20 μM qVD‐OPh for 3 h. Data are representative of two independent experiments. Source data are available online for this figure.

    Article Snippet: Primary antibodies for Western blotting were as follows: rabbit anti‐BAX, rabbit anti‐BAK, rabbit anti‐DRP1, rabbit anti‐STING (Cell Signaling), mouse MitoProfile Membrane Integrity Cocktail (for cyclophilin D; Abcam) and β‐actin (Sigma).

    Techniques: Expressing, CRISPR

    BAX / BAK ‐dependent MOMP commits cells to die U2OS cells treated with 10 μM ABT‐737 and 1 μM ActD ± 20 μM qVD‐OPh. Cell viability was analysed using an IncuCyte live‐cell imager and SYTOX Green exclusion. Data are expressed as mean ± SEM, representative of three independent experiments. Clonogenic survival assay of U2OS cells treated with 10 μM ABT‐737 and 2 μM S63845 ± 20 μM qVD‐OPh or 10 μM ABT‐737 and 1 μM ActD ± 20 μM qVD‐OPh. Representative images from three independent experiments. Airyscan images of U2OS cells treated with 10 μM ABT‐737, 1 μM ActD and 20 μM qVD‐OPh for 3 h, immunostained with anti‐TOM20 (green) and anti‐cytochrome c (red). Scale bar = 10 μm. Representative images from three independent experiments. Quantification of cytochrome c release from mitochondria. Data are expressed as mean ± SD from three independent experiments and analysed using Student's t ‐test. Quantification of cytochrome c release from BAX‐, BAK‐, and BAX/BAK‐deleted cells. Data are expressed as mean ± SD from three independent experiments and analysed using Student's t ‐test. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Mitochondrial inner membrane permeabilisation enables mt DNA release during apoptosis

    doi: 10.15252/embj.201899238

    Figure Lengend Snippet: BAX / BAK ‐dependent MOMP commits cells to die U2OS cells treated with 10 μM ABT‐737 and 1 μM ActD ± 20 μM qVD‐OPh. Cell viability was analysed using an IncuCyte live‐cell imager and SYTOX Green exclusion. Data are expressed as mean ± SEM, representative of three independent experiments. Clonogenic survival assay of U2OS cells treated with 10 μM ABT‐737 and 2 μM S63845 ± 20 μM qVD‐OPh or 10 μM ABT‐737 and 1 μM ActD ± 20 μM qVD‐OPh. Representative images from three independent experiments. Airyscan images of U2OS cells treated with 10 μM ABT‐737, 1 μM ActD and 20 μM qVD‐OPh for 3 h, immunostained with anti‐TOM20 (green) and anti‐cytochrome c (red). Scale bar = 10 μm. Representative images from three independent experiments. Quantification of cytochrome c release from mitochondria. Data are expressed as mean ± SD from three independent experiments and analysed using Student's t ‐test. Quantification of cytochrome c release from BAX‐, BAK‐, and BAX/BAK‐deleted cells. Data are expressed as mean ± SD from three independent experiments and analysed using Student's t ‐test. Source data are available online for this figure.

    Article Snippet: Primary antibodies for Western blotting were as follows: rabbit anti‐BAX, rabbit anti‐BAK, rabbit anti‐DRP1, rabbit anti‐STING (Cell Signaling), mouse MitoProfile Membrane Integrity Cocktail (for cyclophilin D; Abcam) and β‐actin (Sigma).

    Techniques: Clonogenic Cell Survival Assay

    Mitochondrial inner membrane and mt DNA are extruded through BAX pores on the mitochondrial outer membrane Live‐cell Airyscan images of U2OS BAX/BAK CRISPR‐Cas9‐deleted cells stably expressing JF 646 ‐MOM (magenta) and mCherry‐BAX (cyan) and transiently expressing TFAM‐mClover (green). Cells were treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh at t = 0. Scale bar = 10 μm. See Video EV10 . Numbers indicate time in minutes. Airyscan images of U2OS MCL1 CRISPR cells stably expressing JF 646 ‐MOM (magenta) and transiently expressing TFAM‐mClover (green), treated with 10 μM ABT‐737 and 20 μM qVD‐OPh for 3 h, immunostained with anti‐active BAX (6A7, cyan). Images are maximum intensity projections of z ‐stack data. Scale bar = 10 μm. Representative images from three independent experiments. Imaris 3D‐rendering of U2OS cells as in (B) showing MOM (green), TFAM (red) and active BAX (cyan). U2OS cells stably expressing JF 646 ‐MOM (magenta) treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh for 3 h. Cells were immunostained with antibodies for active BAX (6A7, cyan) and AIF (IMM, green). Images are maximum intensity projections of z ‐stack data. Scale bar = 10 μm. Representative images from three independent experiments. Imaris 3D‐rendering of U2OS cells as in (D) showing MOM (green), AIF (red) and active BAX (cyan).

    Journal: The EMBO Journal

    Article Title: Mitochondrial inner membrane permeabilisation enables mt DNA release during apoptosis

    doi: 10.15252/embj.201899238

    Figure Lengend Snippet: Mitochondrial inner membrane and mt DNA are extruded through BAX pores on the mitochondrial outer membrane Live‐cell Airyscan images of U2OS BAX/BAK CRISPR‐Cas9‐deleted cells stably expressing JF 646 ‐MOM (magenta) and mCherry‐BAX (cyan) and transiently expressing TFAM‐mClover (green). Cells were treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh at t = 0. Scale bar = 10 μm. See Video EV10 . Numbers indicate time in minutes. Airyscan images of U2OS MCL1 CRISPR cells stably expressing JF 646 ‐MOM (magenta) and transiently expressing TFAM‐mClover (green), treated with 10 μM ABT‐737 and 20 μM qVD‐OPh for 3 h, immunostained with anti‐active BAX (6A7, cyan). Images are maximum intensity projections of z ‐stack data. Scale bar = 10 μm. Representative images from three independent experiments. Imaris 3D‐rendering of U2OS cells as in (B) showing MOM (green), TFAM (red) and active BAX (cyan). U2OS cells stably expressing JF 646 ‐MOM (magenta) treated with 10 μM ABT‐737, 2 μM S63845 and 20 μM qVD‐OPh for 3 h. Cells were immunostained with antibodies for active BAX (6A7, cyan) and AIF (IMM, green). Images are maximum intensity projections of z ‐stack data. Scale bar = 10 μm. Representative images from three independent experiments. Imaris 3D‐rendering of U2OS cells as in (D) showing MOM (green), AIF (red) and active BAX (cyan).

    Article Snippet: Primary antibodies for Western blotting were as follows: rabbit anti‐BAX, rabbit anti‐BAK, rabbit anti‐DRP1, rabbit anti‐STING (Cell Signaling), mouse MitoProfile Membrane Integrity Cocktail (for cyclophilin D; Abcam) and β‐actin (Sigma).

    Techniques: CRISPR, Stable Transfection, Expressing

    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

    Acetylated Sp1 does not bind at the bak or p21 promoters . Panel A shows the organisation and sequence of the bak (Ai) and p21 (Aii) probes, with hypothetical and proven Sp1/3 binding sites underlined. Sequence numbers refer to distance from the transcriptional start site of each gene. Panel B shows that binding of Sp1 to both target sequences is decreased following butyrate treatment. Panel B: Western of mobility shift assay (WeMSA) analysis of binding to the Bak and p21 probes shows that Sp1 binding decreases following treatment with 10 mM sodium butyrate for 24 h compared to an untreated control (upper panel). Binding of acetyl-Sp1 could not be detected by WeMSA (lower panel). These data are representative of three independent repeats. Panel C: The binding of Sp1 to the bak (panel Ci) and p21 (panel Cii) promoter sequences was determined following treatment of HCT116 cells with a range of butyrate concentrations (0-20 mM). The upper panels show WeMSA gels immunoprobed for Sp1. As a loading control, the same extracts were also separated by SDS page, and immunoprobed with the same antibody (lower panels). Data shown in Ci and Cii are representative of at least two independent repeat experiments. Panel Ciii shows mean (+/- SD) of response at the Bak promoter. Whilst the levels of Sp1 are broadly constant, levels of Sp1 binding for both probes are reduced following treatment with butyrate.

    Journal: Molecular Cancer

    Article Title: Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line

    doi: 10.1186/1476-4598-9-275

    Figure Lengend Snippet: Acetylated Sp1 does not bind at the bak or p21 promoters . Panel A shows the organisation and sequence of the bak (Ai) and p21 (Aii) probes, with hypothetical and proven Sp1/3 binding sites underlined. Sequence numbers refer to distance from the transcriptional start site of each gene. Panel B shows that binding of Sp1 to both target sequences is decreased following butyrate treatment. Panel B: Western of mobility shift assay (WeMSA) analysis of binding to the Bak and p21 probes shows that Sp1 binding decreases following treatment with 10 mM sodium butyrate for 24 h compared to an untreated control (upper panel). Binding of acetyl-Sp1 could not be detected by WeMSA (lower panel). These data are representative of three independent repeats. Panel C: The binding of Sp1 to the bak (panel Ci) and p21 (panel Cii) promoter sequences was determined following treatment of HCT116 cells with a range of butyrate concentrations (0-20 mM). The upper panels show WeMSA gels immunoprobed for Sp1. As a loading control, the same extracts were also separated by SDS page, and immunoprobed with the same antibody (lower panels). Data shown in Ci and Cii are representative of at least two independent repeat experiments. Panel Ciii shows mean (+/- SD) of response at the Bak promoter. Whilst the levels of Sp1 are broadly constant, levels of Sp1 binding for both probes are reduced following treatment with butyrate.

    Article Snippet: Antibodies used were: Sp1 (Cat# 07-645, Millipore), p21 (Cellomics p21 Kit, Thermo Fischer), Bak (Cat# 556396, BD Biosciences) and a custom antibody to acetylated Sp1.

    Techniques: Sequencing, Binding Assay, Western Blot, Mobility Shift, SDS Page

    Sp1 acetylation increases in a co-linear manner with bak and p21 expression following sodium butyrate treatment . HCT116 cells were treated with increasing concentrations of butyrate (0-20 mM) for 24 hr, fixed and fluorescence immunostained either for Sp1 and Bak or Ace-Sp1 and p21. Cellular fluorescence was quantified by High-Content Analysis. Panel A shows protein expression levels of Sp1 (Panel Ai); acetyl-Sp1 (Panel Aii); Bak, (Panel Aiii) and p21 (Panel Aiv). Data are from a single pass experiment with three replicates, with 50 fields per replicate scored. The EC 50 value for each event in response to butyrate is shown in Panel Av. Panel B shows representative images of HCT116 cells following 24 h of 0 or 10 mM sodium butyrate treatment and fluorescence immunostaining for acetyl-Sp1 and p21. Panel Bi shows representative fields from the control (untreated) cells (upper panel) and the treated culture (lower panel). Staining patterns in the treated cells broadly fell into two main types, cell positive for acetyl-Sp1 alone (examples marked by arrows i) and cells positive staining for both acetyl Sp1 and p21 (marked by arrows ii). These subpopulations were distinguished by plotting acetyl-Sp1 vs. p21 fluorescence (see supplementary online data Fig 2 for gating strategy). Panel Bii shows quantitation of data from three independent experiments, showing numbers of cells in gated fractions that were dual stained, as indicated.

    Journal: Molecular Cancer

    Article Title: Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line

    doi: 10.1186/1476-4598-9-275

    Figure Lengend Snippet: Sp1 acetylation increases in a co-linear manner with bak and p21 expression following sodium butyrate treatment . HCT116 cells were treated with increasing concentrations of butyrate (0-20 mM) for 24 hr, fixed and fluorescence immunostained either for Sp1 and Bak or Ace-Sp1 and p21. Cellular fluorescence was quantified by High-Content Analysis. Panel A shows protein expression levels of Sp1 (Panel Ai); acetyl-Sp1 (Panel Aii); Bak, (Panel Aiii) and p21 (Panel Aiv). Data are from a single pass experiment with three replicates, with 50 fields per replicate scored. The EC 50 value for each event in response to butyrate is shown in Panel Av. Panel B shows representative images of HCT116 cells following 24 h of 0 or 10 mM sodium butyrate treatment and fluorescence immunostaining for acetyl-Sp1 and p21. Panel Bi shows representative fields from the control (untreated) cells (upper panel) and the treated culture (lower panel). Staining patterns in the treated cells broadly fell into two main types, cell positive for acetyl-Sp1 alone (examples marked by arrows i) and cells positive staining for both acetyl Sp1 and p21 (marked by arrows ii). These subpopulations were distinguished by plotting acetyl-Sp1 vs. p21 fluorescence (see supplementary online data Fig 2 for gating strategy). Panel Bii shows quantitation of data from three independent experiments, showing numbers of cells in gated fractions that were dual stained, as indicated.

    Article Snippet: Antibodies used were: Sp1 (Cat# 07-645, Millipore), p21 (Cellomics p21 Kit, Thermo Fischer), Bak (Cat# 556396, BD Biosciences) and a custom antibody to acetylated Sp1.

    Techniques: Expressing, Fluorescence, High Content Screening, Immunostaining, Staining, Quantitation Assay

    Effect of HDACi on cell cycle, p21 expression, bak expression and Sp1 expression and acetylation . The extent of the concomitant response of Sp1 acetylation, cell cycle arrest and p21 up-regulation was determined using a high-content biology approach. HCT116 cells were treated with concentration ranges of 0-20 mM sodium butyrate, 0-20 mM valproic acid (VPA), 0-20 μM Oxamflatin, 0-20 μM Scriptaid, 0-20 μM APHA compound 8, 0-20 μM CHAHA. In all cases treatments were carried outfor 24 h. Cells were stained using immunocytochemistry for DNA content (Hoescht), p21, bak, Sp1 and acetyl-Sp1 as described in the methods section. Cells were analysed, on the basis of DNA content, for cell cycle phase and divided into G1 (filled circles), S (filled squares) or G2/M (filled triangles). Levels of protein were calculated from mean total fluorescence and are expressed in terms of fluorophore fluorescence relative to that observed in untreated cells. Sp1 (filled squares) and acetyl-Sp1 (open squares) are shown on the same graph.

    Journal: Molecular Cancer

    Article Title: Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line

    doi: 10.1186/1476-4598-9-275

    Figure Lengend Snippet: Effect of HDACi on cell cycle, p21 expression, bak expression and Sp1 expression and acetylation . The extent of the concomitant response of Sp1 acetylation, cell cycle arrest and p21 up-regulation was determined using a high-content biology approach. HCT116 cells were treated with concentration ranges of 0-20 mM sodium butyrate, 0-20 mM valproic acid (VPA), 0-20 μM Oxamflatin, 0-20 μM Scriptaid, 0-20 μM APHA compound 8, 0-20 μM CHAHA. In all cases treatments were carried outfor 24 h. Cells were stained using immunocytochemistry for DNA content (Hoescht), p21, bak, Sp1 and acetyl-Sp1 as described in the methods section. Cells were analysed, on the basis of DNA content, for cell cycle phase and divided into G1 (filled circles), S (filled squares) or G2/M (filled triangles). Levels of protein were calculated from mean total fluorescence and are expressed in terms of fluorophore fluorescence relative to that observed in untreated cells. Sp1 (filled squares) and acetyl-Sp1 (open squares) are shown on the same graph.

    Article Snippet: Antibodies used were: Sp1 (Cat# 07-645, Millipore), p21 (Cellomics p21 Kit, Thermo Fischer), Bak (Cat# 556396, BD Biosciences) and a custom antibody to acetylated Sp1.

    Techniques: Expressing, Concentration Assay, Staining, Immunocytochemistry, Fluorescence

    Reciprocal expression of miR‐29c and Birc2 and Bak1. ( A ) The expression of miR‐29c in three groups. ( B ) and ( C ) The expression of Birc2 mRNA and Bak1 mRNA in rat brains. ( D ) and ( E ) The expression of Birc2 protein and Bak1 protein in three groups. * P

    Journal: CNS Neuroscience & Therapeutics

    Article Title: Micro RNA‐29c Correlates with Neuroprotection Induced by FNS by Targeting Both Birc2 and Bak1 in Rat Brain after Stroke

    doi: 10.1111/cns.12383

    Figure Lengend Snippet: Reciprocal expression of miR‐29c and Birc2 and Bak1. ( A ) The expression of miR‐29c in three groups. ( B ) and ( C ) The expression of Birc2 mRNA and Bak1 mRNA in rat brains. ( D ) and ( E ) The expression of Birc2 protein and Bak1 protein in three groups. * P

    Article Snippet: These findings show that miR‐29c inhibitor effectively increases, whereas miR‐29c mimic decreases the expression of Birc2 and Bak1 ( in vivo ; Figure B–E).

    Techniques: Expressing

    Luciferase reporter assays showed Birc2 and Bak1 were authentic targets of miR‐29c; ( A ) and ( D ) Predicted binding sites of miR‐29c in the 3’UTR of Birc2 and Bak1. ( B ) and ( E ) Design of a miR‐29c luciferase reporter vector with HSV‐driven expression of a luciferase vector fused to a wild Birc2 and Bak1 3’UTR or mutated Birc2 and Bak1 3’UTR. The underlined nucleotides were subsequently mutated for the 3?UTR‐miRNA binding studies. ( C ) and ( F ) Rno‐miR‐29c mimics decreased expression of luciferase containing a wild‐type miR‐29c binding site (left two columns) but not a mutant binding site (right two columns). * P

    Journal: CNS Neuroscience & Therapeutics

    Article Title: Micro RNA‐29c Correlates with Neuroprotection Induced by FNS by Targeting Both Birc2 and Bak1 in Rat Brain after Stroke

    doi: 10.1111/cns.12383

    Figure Lengend Snippet: Luciferase reporter assays showed Birc2 and Bak1 were authentic targets of miR‐29c; ( A ) and ( D ) Predicted binding sites of miR‐29c in the 3’UTR of Birc2 and Bak1. ( B ) and ( E ) Design of a miR‐29c luciferase reporter vector with HSV‐driven expression of a luciferase vector fused to a wild Birc2 and Bak1 3’UTR or mutated Birc2 and Bak1 3’UTR. The underlined nucleotides were subsequently mutated for the 3?UTR‐miRNA binding studies. ( C ) and ( F ) Rno‐miR‐29c mimics decreased expression of luciferase containing a wild‐type miR‐29c binding site (left two columns) but not a mutant binding site (right two columns). * P

    Article Snippet: These findings show that miR‐29c inhibitor effectively increases, whereas miR‐29c mimic decreases the expression of Birc2 and Bak1 ( in vivo ; Figure B–E).

    Techniques: Luciferase, Binding Assay, Plasmid Preparation, Expressing, Mutagenesis

    The consequences of in vivo experiment. ( A ) The expression of miR‐29c in five groups. ( B ) and ( C ) The expression of Birc2 mRNA and Bak1 mRNA in rat brains. ( D ) and ( E ) The expression of Birc2 protein and Bak1 protein in five groups. MiR‐29c antagomir treatment decreased the infarct volume ( F ), apoptosis ( G ) and improved neurological scores ( H ), while miR‐29c agomir treatment increased the infarct volume ( F ), apoptosis ( G ) and aggravated neurological scores ( H ). * P

    Journal: CNS Neuroscience & Therapeutics

    Article Title: Micro RNA‐29c Correlates with Neuroprotection Induced by FNS by Targeting Both Birc2 and Bak1 in Rat Brain after Stroke

    doi: 10.1111/cns.12383

    Figure Lengend Snippet: The consequences of in vivo experiment. ( A ) The expression of miR‐29c in five groups. ( B ) and ( C ) The expression of Birc2 mRNA and Bak1 mRNA in rat brains. ( D ) and ( E ) The expression of Birc2 protein and Bak1 protein in five groups. MiR‐29c antagomir treatment decreased the infarct volume ( F ), apoptosis ( G ) and improved neurological scores ( H ), while miR‐29c agomir treatment increased the infarct volume ( F ), apoptosis ( G ) and aggravated neurological scores ( H ). * P

    Article Snippet: These findings show that miR‐29c inhibitor effectively increases, whereas miR‐29c mimic decreases the expression of Birc2 and Bak1 ( in vivo ; Figure B–E).

    Techniques: In Vivo, Expressing

    Anti-apoptotic prohibitin shown by knockdown analysis. (A) ARPE-19 cells were transfected by siRNA and random sequence. Anti-apoptotic BCL-xL increased when prohibitin was down-regulated. Pro-apoptotic proteins, including AIF, caspase-9, and BAK, were

    Journal: Biochemistry

    Article Title: Mitochondrial-Nuclear Communication by Prohibitin Shuttling Under Oxidative Stress

    doi: 10.1021/bi2008933

    Figure Lengend Snippet: Anti-apoptotic prohibitin shown by knockdown analysis. (A) ARPE-19 cells were transfected by siRNA and random sequence. Anti-apoptotic BCL-xL increased when prohibitin was down-regulated. Pro-apoptotic proteins, including AIF, caspase-9, and BAK, were

    Article Snippet: Proteins were assayed by Western blot to detect AIF (EMD Millipore), Bcl-xL (sc-634, Santa Cruz Biotechnology), Caspase-9 (AB16969, EMD Millipore), BAK (ab32371, Abcam) and actin (sc-47778, Santa Cruz Biotechnology).

    Techniques: Transfection, Sequencing