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Augmenting MEK inhibitor efficacy in BRAF wild-type melanoma: synergistic effects of disulfiram combination therapy.

In J Exp Clin Cancer Res on 23 January 2024 by Meraz-Torres, F., Niessner, H., et al.

Fig.3.D

Apoptosis induction by trametinib plus CuET in BRAF WT melanoma cells. A BRAF WT melanoma cells were treated with trametinib (10 nM), CuET (125 nM) or their combination in the presence or absence (+/-) of the pan-Caspase inhibitor Z-VAD-FMK (10 µM). Cell cycle analysis at 24, 48 and 72 h was perf... more

Apoptosis induction by trametinib plus CuET in BRAF WT melanoma cells. A BRAF WT melanoma cells were treated with trametinib (10 nM), CuET (125 nM) or their combination in the presence or absence (+/-) of the pan-Caspase inhibitor Z-VAD-FMK (10 µM). Cell cycle analysis at 24, 48 and 72 h was performed, and the cell cycle distribution was plotted with apoptotic cells (sub-G1 fraction) shown as red bars. Three independent experiments were performed (mean ± SD, n = 3 independent experiments). B Cell viability assays (MUH assay) of BRAF WT melanoma cells (SKMEL23 and SKMEL113) after 72 hours of treatment with increasing concentrations of trametinib (up to 2 µM), CuET (up to 500 nM) or their combination with and without (+/-) the pan-Caspase inhibitor Z-VAD-FMK (10 μM). Signals were normalized to untreated controls (n = 3 independent experiments; mean ± SD). Dotted lines represent the effect of 10 nM trametinib on cellular viability. C BRAF WT melanoma cells were treated with trametinib (10 nM), CuET (125 nM) or their combination for 12 and 24 hours. Immunoblots (cropped) of whole cell lysates showing cleavage of Caspase-3 and PARP compared to β-Actin. A representative result of two independent experiments is shown. D Lysates at 12 hours after treatment were prepared, and immunoblot detections for JUN, BIM isoforms (extra-long: EL, long; L, short: S), BAX, BCL-2, p53, p21 and phosphorylated phospho-H2A.X were performed. β-Actin served as a loading control. A representative result of two independent experiments is shown less

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Apoptotic stress induces Bax-dependent, caspase-independent redistribution of LINC complex nesprins.

In Cell Death Discov on 8 October 2020 by Lindenboim, L., Grozki, D., et al.

Fig.5.C

Bax can interact with nesprin-2; Duolink PLA approach.a–d Inducible Bax interacts with nesprin-2. Bax/Bak C16 DKO MEFs were untreated (Con) or treated for 24 h with doxycycline to induce Bax expression (Dox), or treated for 24 h with doxycycline followed by addition of 25 µM cisplatin (Dox + Cis)... more

Bax can interact with nesprin-2; Duolink PLA approach.a–d Inducible Bax interacts with nesprin-2. Bax/Bak C16 DKO MEFs were untreated (Con) or treated for 24 h with doxycycline to induce Bax expression (Dox), or treated for 24 h with doxycycline followed by addition of 25 µM cisplatin (Dox + Cis) or 100 nM staurosporine (Dox + STS) in the presence of Q-VD-OPH for additional 24 h and 17 h, respectively. Duolink-PLA was preformed using anti-Bax 6A7 Ab together with anti-nesprin-2 MBS or anti-nesprin-2G Ab. Cells were visualized using fluorescence microscopy. a Representative photomicrographs of cells showing Duolink signals using anti-nesprin-2 MBS. The images represent the same field visualized separately for detecting Duolink signal (left panel) and nuclei (DAPI) (right panel) staining. Arrows indicate cells which exhibit Duolink signal that accumulates in aggregates. Bar = 50 µm. b Quantification of the percentage of cells exhibiting Duolink signal using anti-nesprin-2 MBS from total number of cells. Cells were detected by their nuclei and at least 200 cells were analyzed in each treatment. The values are represented as mean ± SEM (error bars) (n = 3). (*p < 0.05, ***p < 0.002; one tailed student’s t test compared to control and **p < 0.002 compared to doxycycline treatment). (c) Representative photomicrographs of cells showing Duolink signals using anti-nesprin-2G Ab. The images are shown as described in a. d Quantification of the percentage of cells exhibiting Duolink signal using anti-nesprin-2G Ab from total number of cells. The results are presented as described in b. e, f Interaction between endogenous Bax and nesprin-2. Caspase-9−/− MEFs were untreated (Con) or treated with 25 µM cisplatin (Cis). Duolink signal was detected using anti-active Bax Ab (6A7) and pan anti-nesprin-2 (MBS375177) Ab. e Duolink signal in each treatment was visualized using fluorescence microscopy. The photomicrographs shown for each treatment are from the same field visualized separately for detecting Duolink signal and nuclei. Bar = 50 µm. f Quantification of the Duolink signal. The presented results are expressed as the number of Duolink dots (Duolink signal) per captured field after normalization to the number of nuclei in the field (12-19 fields per experiment; n = 2). Two-way ANOVA after log transformation for normal distribution show significant difference between the two treatments (p < 0.0001). g, h Identification of Bax domains needed for Bax/nesprin-2 interaction. Bax/Bak DKO MEFs were transiently transfected with control GFP (g), or His-tagged (h) expression vectors or with expression vectors for WT Bax or Bax mutants, in the presence of Q-VD-OPH. Duolink PLA assay was preformed using the anti-Bax (6A7) Ab [for GFP-Bax (g)] or anti-His Ab [for His-Bax (h)] and anti-nesprin-2G Ab (N2G) or pan anti-nesprin-2 K2 (N2K2) Ab. Cells exhibiting Duolink signal were visualized using fluorescence microscopy. The number of Duolink dots displayed by the transfected cells (at least 20 cells for each treatment) were normalized to cell size, expressed relative to GFP-transfected cells and presented as mean ± SEM (error bars) (n = 3). (*p < 0.05, **p < 0.02, two tailed student’s t test comparing GFP-Bax or His-Bax to GFP, or His-Bax to His ΔN-Bax and one sample one tailed student’s t test comparing GFP-Bax to GFP-Bax P168A). i Localization of Bax and nesprin-2 interaction site. Bax/Bak DKO MEFs were transiently transfected with GFP-Bax, stained with MitoTracker Red, fixed and assessed for interaction between GFP-Bax and endogenous nesprin-2G by Duolink-PLA using anti-Bax (6A7) and anti-nesprin-2G Ab. Photomicrographs were captured by confocal microscopy. The results shown are from a representative cell (out of 29 cells from 6 independent experiments). Bar = 25 µm. The same field was visualized separately for detection of GFP-Bax (green), Duolink signal (Magenta), and mitochondria (red) fluorescence. Higher magnification of a representative area (denoted by a box in the merge image) in the merge image illustrates close proximity of Duolink dots with GFP-Bax and mitochondria. Bar = 5 µm. less

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Gypenosides Prevent H2O2-Induced Retinal Ganglion Cell Apoptosis by Concurrently Suppressing the Neuronal Oxidative Stress and Inflammatory Response.

In J Mol Neurosci on 1 April 2020 by Zhang, H. K., Ye, Y., et al.

Fig.9.A

The relative protein expressions of Bcl-2, Bax, and cleaved caspase-3 were detected by protein immunoblotting. *p < 0.05 when compared with the control group; #p < 0.05 when compared with the H2O2 group. Data were presented as mean ± SE (n = 3) more

The relative protein expressions of Bcl-2, Bax, and cleaved caspase-3 were detected by protein immunoblotting. *p < 0.05 when compared with the control group; #p < 0.05 when compared with the H2O2 group. Data were presented as mean ± SE (n = 3) less

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Confounding off-target effects of BH3 mimetics at commonly used concentrations: MIM1, UMI-77, and A-1210477.

In Cell Death Dis on 22 February 2019 by Mallick, D. J., Soderquist, R. S., et al.

Fig.6.E

A-1210477 induces cytochrome c release and apoptosis in the absence of BAX and BAK.a Jurkat wt, Jurkat c3, Jurkat c4, and ML-1 cells were analyzed for BAX/BAK expression. b Jurkat wt, Jurkat c3, and Jurkat c4 cells were incubated with either ABT-737 or ABT-199 for 0–24 h as a positive control sho... more

A-1210477 induces cytochrome c release and apoptosis in the absence of BAX and BAK.a Jurkat wt, Jurkat c3, Jurkat c4, and ML-1 cells were analyzed for BAX/BAK expression. b Jurkat wt, Jurkat c3, and Jurkat c4 cells were incubated with either ABT-737 or ABT-199 for 0–24 h as a positive control showing the need for BAX/BAK during apoptosis in these cells. c Jurkat c3 and c4 cells were incubated with 0–20 µM A-1210477 for 6 h. d Jurkat wt, c3, and c4 cells were incubated with 0–20 µM A-1210477 for 6 h. Digitonin permeabilization was performed to assess cytochrome c release. All incubations included 10 µM QVD to prevent loss of cytochrome c from apoptotic cells. PARP, MEK1/2, and Tom20 were used as fraction controls. e HCT116 wt and HCT116 BAX/BAK DKO were incubated with 0–20 µM A-1210477 for 6 h. A 20 µM gossypol was used as a positive control for NOXA induction. f Six cell lines were incubated with 20 µM A-1210477 for 0–6 h and analyzed for PARP cleavage less

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Confounding off-target effects of BH3 mimetics at commonly used concentrations: MIM1, UMI-77, and A-1210477.

In Cell Death Dis on 22 February 2019 by Mallick, D. J., Soderquist, R. S., et al.

Fig.6.A

A-1210477 induces cytochrome c release and apoptosis in the absence of BAX and BAK.a Jurkat wt, Jurkat c3, Jurkat c4, and ML-1 cells were analyzed for BAX/BAK expression. b Jurkat wt, Jurkat c3, and Jurkat c4 cells were incubated with either ABT-737 or ABT-199 for 0–24 h as a positive control sho... more

A-1210477 induces cytochrome c release and apoptosis in the absence of BAX and BAK.a Jurkat wt, Jurkat c3, Jurkat c4, and ML-1 cells were analyzed for BAX/BAK expression. b Jurkat wt, Jurkat c3, and Jurkat c4 cells were incubated with either ABT-737 or ABT-199 for 0–24 h as a positive control showing the need for BAX/BAK during apoptosis in these cells. c Jurkat c3 and c4 cells were incubated with 0–20 µM A-1210477 for 6 h. d Jurkat wt, c3, and c4 cells were incubated with 0–20 µM A-1210477 for 6 h. Digitonin permeabilization was performed to assess cytochrome c release. All incubations included 10 µM QVD to prevent loss of cytochrome c from apoptotic cells. PARP, MEK1/2, and Tom20 were used as fraction controls. e HCT116 wt and HCT116 BAX/BAK DKO were incubated with 0–20 µM A-1210477 for 6 h. A 20 µM gossypol was used as a positive control for NOXA induction. f Six cell lines were incubated with 20 µM A-1210477 for 0–6 h and analyzed for PARP cleavage less

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Adenovirus-mediated decorin expression induces cancer cell death through activation of p53 and mitochondrial apoptosis.

In Oncotarget on 29 September 2017 by Yoon, A. R., Hong, J., et al.

Fig.5.A

Activation of mitochondrial control of apoptosis by DCN-expressing Ad(A) Western blot; U343 and A549 cells were infected with dE1 or dE1/DCN at an MOI of 20. Cell lysates were probed with antibodies against PUMA, BAX, Bcl-2, and cytochrome C. β-actin was used as an internal control to show equal ... more

Activation of mitochondrial control of apoptosis by DCN-expressing Ad(A) Western blot; U343 and A549 cells were infected with dE1 or dE1/DCN at an MOI of 20. Cell lysates were probed with antibodies against PUMA, BAX, Bcl-2, and cytochrome C. β-actin was used as an internal control to show equal protein loading. The expression levels of proteins were semi-quantitatively analyzed with the ImageJ software. (B) Western blot; The U343 and A549 cells were infected with dE1, or dE1/DCN at an MOI of 20. Cell lysates were probed with antibodies against cleaved caspase and PARP. β-actin was used as an internal control to show equal protein loading. The expression levels of proteins were semi-quantitatively analyzed with the ImageJ software. (C) Immunocytochemistry; U343 cells were infected with dE1, or dE1/DCN at an MOI of 20. At 2 days post infection, fixed cells were probed with antibodies against cytochrome C using Alexa Fluor 488 anti-cleaved caspase-3 (green), and the nuclei were counterstained with DAPI (blue). (D) Western blot; U343 and A549 cells were infected with dE1 or dE1/DCN at an MOI of 20. Cell lysates were probed with antibodies against cleaved caspase and PARP. β-actin was used as an internal control to show equal protein loading. The expression levels of proteins were semi-quantitatively analyzed with the ImageJ software. less

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Small Heat Shock Protein Beta-1 (HSPB1) Is Upregulated and Regulates Autophagy and Apoptosis of Renal Tubular Cells in Acute Kidney Injury.

In PLoS One on 12 May 2015 by Matsumoto, T., Urushido, M., et al.

Fig.10.E

BAX activation and cytochrome c release in NRK-52E cells are reduced by overexpression of HSPB1 after H2O2 treatment.(A, B) Determination of active BAX content assessed with a conformation-specific antibody directed against the 6A7 epitope, total BAX content, and cytochrome c of cytosol fractions... more

BAX activation and cytochrome c release in NRK-52E cells are reduced by overexpression of HSPB1 after H2O2 treatment.(A, B) Determination of active BAX content assessed with a conformation-specific antibody directed against the 6A7 epitope, total BAX content, and cytochrome c of cytosol fractions in control NRK-52E cells and NRK-52E cells transfected with pcDNA or the HSPB1 overexpression vector and treated with 400 and 600 μM H2O2 for 4 h. (C, D) Quantitative analysis with a densitometer showed a marked reduction in BAX activation when cells were transfected with the HSPB1 expression vector. Data are presented as means ± SEM, n = 6; *P < 0.05. (E, F) NRK-52E cells were transfected with control scrambled siRNA or HSPB1 siRNA, and the active BAX, total BAX, and cytochrome c of cytosol fractions were examined in control cells and cells exposed to 400 and 600 μM H2O2 for 4 h. (G, H) Quantitative analysis with a densitometer demonstrated that HSPB1 regulated BAX activation in NRK-52E cells. Data are presented as means ± SEM, n = 6; *P < 0.05 vs. control. less

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Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6.

In Cell Death Dis on 12 March 2015 by Schüll, S., Günther, S. D., et al.

Fig.2.C

H2O2 induces mitochondrial apoptosis in COX-deficient cells. (a) HeLa cells pretreated with KCN (1 mM) for 24 h, 143BΔCOX cybrid cells and COX10−/− fibroblasts were subjected to H2O2 in the absence (gray column) or presence of pan-caspase inhibitor zVAD-fmk (20 μM), caspase-8 inhibitor zIETD-fmk ... more

H2O2 induces mitochondrial apoptosis in COX-deficient cells. (a) HeLa cells pretreated with KCN (1 mM) for 24 h, 143BΔCOX cybrid cells and COX10−/− fibroblasts were subjected to H2O2 in the absence (gray column) or presence of pan-caspase inhibitor zVAD-fmk (20 μM), caspase-8 inhibitor zIETD-fmk (2 μM) or caspase-9 inhibitor zLEHD-fmk (2 μM). Cell death was measured by trypan-blue exclusion after 20 h. (b) Cytchrome c and caspase-9 were detected in the cytosol of cells by WB after treatment with H2O2 for 6 h (HeLa), 12 h (143BΔCOX cybrid cells) or 16 h (COX10−/−), respectively. The arrowheads indicate caspase-9 cleavage bands p35 and p37. Activated Bax was immunoprecipitated by 6A7 anti-Bax antibody in total lysates of HeLa cells and detected by WB after 4 h. (c) Wild-type (wt) and Bax/Bak double-knockout MEFs (Bax−/−Bak−/−) were pretreated with KCN (200 μM) for 24 h or left untreated. Cell death upon treatment with H2O2 was measured after 18 h by trypan-blue exclusion. The release of cytochrome c was detected by WB after 6 h. (d) HeLa and HeLa Bcl-2 overexpressing cells were pretreated with KCN (1 mM) for 24 h or left untreated. Cell death upon treatment with H2O2 was measured after 18 h by trypan-blue exclusion and release of cytochrome c was detected by WB after 6 h. The error bars in panel a represent the mean±S.D. (n=4), the error bars in panel c and d represent the mean±S.D. (n=3), two-tailed unpaired t-test. *P<0.05, **P <0.01, ***P<0.001 less

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The expression of apoptosis inducing factor (AIF) is associated with aging-related cell death in the cortex but not in the hippocampus in the TgCRND8 mouse model of Alzheimer's disease.

In BMC Neurosci on 10 June 2014 by Yu, W., Bonnet, M., et al.

Fig.4.B

Cell death markers expression in cortical, hippocampal and cerebellar extracts from Tg mice and age-matched non-Tg littermates. (A): Bax quantification in the studied brain areas as determined by Western blotting. Equal protein loading was ascertained by the parallel assessment of β-actin. (B): B... more

Cell death markers expression in cortical, hippocampal and cerebellar extracts from Tg mice and age-matched non-Tg littermates. (A): Bax quantification in the studied brain areas as determined by Western blotting. Equal protein loading was ascertained by the parallel assessment of β-actin. (B): Bax translocation as assessed by western blot probing of Bax in mitochondrial versus total Bax expression. The percentage of translocation was calculated as indicated in Materials and Methods after normalization for the equal loading over GAPDH and Cox IVb O.D. for cytoplasmic and mitochondrial fractions, respectively. (C): Cyclin D1 quantification by Western blotting in the same brain regions as in (A). Results are expressed as the ratio of the values calculated for the transgenic over non-Tg mice. *P < 0.05; **P < 0.01; #P < 0.01; ##P < 0.001; ###P < 0.0001. less

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The expression of apoptosis inducing factor (AIF) is associated with aging-related cell death in the cortex but not in the hippocampus in the TgCRND8 mouse model of Alzheimer's disease.

In BMC Neurosci on 10 June 2014 by Yu, W., Bonnet, M., et al.

Fig.4.A

Cell death markers expression in cortical, hippocampal and cerebellar extracts from Tg mice and age-matched non-Tg littermates. (A): Bax quantification in the studied brain areas as determined by Western blotting. Equal protein loading was ascertained by the parallel assessment of β-actin. (B): B... more

Cell death markers expression in cortical, hippocampal and cerebellar extracts from Tg mice and age-matched non-Tg littermates. (A): Bax quantification in the studied brain areas as determined by Western blotting. Equal protein loading was ascertained by the parallel assessment of β-actin. (B): Bax translocation as assessed by western blot probing of Bax in mitochondrial versus total Bax expression. The percentage of translocation was calculated as indicated in Materials and Methods after normalization for the equal loading over GAPDH and Cox IVb O.D. for cytoplasmic and mitochondrial fractions, respectively. (C): Cyclin D1 quantification by Western blotting in the same brain regions as in (A). Results are expressed as the ratio of the values calculated for the transgenic over non-Tg mice. *P < 0.05; **P < 0.01; #P < 0.01; ##P < 0.001; ###P < 0.0001. less

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Nitric oxide induces cell death by regulating anti-apoptotic BCL-2 family members.

In PLoS One on 22 September 2009 by Snyder, C. M., Shroff, E. H., et al.

Fig.2.B

BAX and BAK mediate nitric oxide-indeced cell death.Bax−/−/Bak−/− MEFs were infected with either Bax, Bak or GFP as a control. BAX and BAK expression was verified by Western analysis (A,B). Bax−/−/Bak−/− MEFs expressing GFP, BAK or BAX were treated with 0, 100, 200 and 400 µM DETA-NO for 48 hours... more

BAX and BAK mediate nitric oxide-indeced cell death.Bax−/−/Bak−/− MEFs were infected with either Bax, Bak or GFP as a control. BAX and BAK expression was verified by Western analysis (A,B). Bax−/−/Bak−/− MEFs expressing GFP, BAK or BAX were treated with 0, 100, 200 and 400 µM DETA-NO for 48 hours and cell death was measured by LDH release (C). less

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