Fig 2: Dermaseptin‐PS1 induced U‐251 MG cell death through intrinsic apoptosis signalling. A, Protein expression of caspase 9/cleaved caspase 9, Apaf‐1, Bcl‐2, Bax, Bak, p‐Bad, Bad, p‐p53 and p53 were analysed by Western blot in U‐251 MG cells treated for 4‐24 h with 10−6 M Dermaseptin‐PS1 or 16‐24 h with 20 μmol/L etoposide. The detection of GAPDH protein was used as an internal control. The signal intensity was quantified by Image Lab software and GraphPad Prism 5 software was used for statistical comparison. NC, negative control. *or # P < 0.01, ** or ## P < 0.05 and *** or ### P < 0.001 vs NC. B, Cytochrome c release from the mitochondria into the cytosol was measured via Western blot. COX IV was used as a loading control for mitochondrial fractions. The signal intensity was quantified by Image Lab software and GraphPad Prism 5 software was used for statistical comparison. NC, negative control; PS1, Dermaseptin‐PS1; Eto, etoposide. *P < 0.05 vs protein expression in cytosol after etoposide treatment; ### P < 0.001 vs protein expression in cytosol after Dermaseptin‐PS1 treatment

Fig 3: Effects of TRIAP1-siRNA on INS-1 cell viability and apoptosis. TRIAP1-siRNA and control-siRNA were transfected into INS-1 cells for 48 h. Cell viability and apoptosis of INS-1 cells were evaluated by (A) MTT and (B and C) flow cytometry assays. (D) Western blot showing the expression levels of apoptosis-associated genes. reverse transcription-quantitative PCR results showing the expression levels of apoptosis-associated genes (E) APAF1, (F) caspase-3, (G) caspase-7 and (H) caspase-9. Note: The blots of APAF1 and its internal control GAPDH were from the different parts of the same gel; and the blots of caspase-3, caspase-7 and caspase-9 and the internal control GAPDH were from the different parts of the same gel. All experiments were conducted in triplicate. The results are presented as the mean ± standard deviation. **P<0.01 vs. control-siRNA. TRIAP1, TP53-regulated inhibitor of apoptosis 1; siRNA, small interfering RNA; APAF1, apoptotic protease-activating factor 1.

Fig 4: Mito-priming as a method of induced Bcl-2 addiction.(a) Method outline. (b) SVEC cells expressing eGFP-tBID 2A BCL-xL or other constructs were co-stained with MitoTracker Deep Red. Colocalization was quantified using the Pearson's coefficient. Representative images show eGFP-tBID expression and MitoTracker Deep Red staining from SVEC cells stably expressing eGFP-tBID 2A BCL-xL. Scale bar, 10 μm. (c) SVEC cells expressing eGFP-tBID 2A BCL-xL were treated with ABT-737 and analysed for cell viability using an IncuCyte imager and SYTOX Green exclusion. Percentage cell death was calculated by normalizing against maximal cell death (13-h treatment with 10 μmol l−1 ABT-737). Error bars represent the standard error of the mean (s.e.m.) from three independent experiments. (d) SVEC cells stably expressing the indicated constructs were treated with increasing concentrations of ABT-737 (μmol l−1; 24 h) and assayed for clonogenic survival. Error bars represent the s.d. of triplicate samples from a representative experiment carried out twice independently. (e) SVEC cells stably expressing indicated constructs were treated with increasing concentration of ABT-737 (μmol l−1) for 6 h with 10 μmol l−1 ABT-737 in presence of Q-VD-OPh. Cells were quantified for cytochrome c release by confocal microscopy. Error bars represent the s.e.m. of three independent experiments. (f) SVEC cells stably expressing the indicated constructs were treated for 6 h with ABT-737 (μmol l−1). PARP and caspase-3 cleavage was determined by western blot. Actin was probed as a loading control. (g) SVEC cells stably expressing the indicated constructs were treated for 13 h with 10 μmol l−1 ABT-737 or enantiomer (ENA). Cell viability was determined as in c. Percentage cell death was calculated by normalizing against 100% cell death (24-h treatment with 1 μmol l−1 actinomycin D). (h) Cell lines stably expressing control vector (pLKO1) or two independent shRNA sequences targeting APAF-1 were treated for 16 h with enantiomer (ENA) or ABT-737. 100% death: 24-h treatment with 1 μmol l−1 actinomycin D (for each line). Cell viability was determined as in c. Percentage cell death was calculated by normalizing against 100% cell death (24-h treatment with 1 μmol l−1 actinomycin D). In g,h error bars represent the s.e.m. of three independent experiments. See also Supplementary Fig. 1 and Supplementary Movies 1–3. Q-VD-OPh, quinolyl-valyl-O-methylaspartyl-(2,6-difluoro- phenoxy)-methyl ketone.

Fig 5: (A) Immunoblot analysis of Bax and Bcl2 in response to tBHP and mangiferin exposure, in NKE cells. (B) Immunoblot analysis of Caspase 8, tBid, Bax, cytochrome C, Apaf-1, Caspase 9, Caspase 3 and PARP. NKE cells were exposed to tBHP (50 µM) and mangiferin (20 µM) according to the defined protocol. β actin was used as an internal control Control: untreated cells, M: exposed to mangiferin, tBHP: exposed to tBHP for 18 h, M+tBHP: Mangiferin treated for 2 h followed by 18 h of tBHP exposure. For inhibitor studies, M+T+I: cells were treated with LY294002(I) follwed by Mangiferin(M) and Tbhp(T). All data are mean±SD, of 3 independent experiments and were analyzed by one-way ANOVA. “*” represents the significant difference between the normal control and tBHP exposed cells and “#” represents the significant difference between the tBHP exposed and tBHP exposed antioxidant treated cells (P*<0.05, P#<0.05).