International Journal of Cell Biology / 2010 / Article / Fig 6

Research Article

Mechanisms of ER Stress-Mediated Mitochondrial Membrane Permeabilization

Figure 6

Regulation of ER stress-induced mitochondrial changes by Bcl-2 family proteins. (a) H9c2 cells were treated with (2  M) Tg alone or pretreated for 30 minutes with BH4-Tat peptide (200 nM) prior to treatment with Tg, for the indicated time periods. Bcl-2 overexpressing H9c2 cells were treated with Tg (2  M) for the indicated time periods. (b) H9c2 cells were treated with Tg (2 μM) alone or pretreated for 30 minutes with bongkrekic acid (BA) (10  M); cyclosporine A (CsA) (2  M) and aristolochic acid (ArA, Calbiochem) at 25 μM prior to treatment with Tg, for the indicated time periods. (a)-(b) Following treatment, cells were incubated with TMRE (100 nM). Mitochondrial membrane potential was monitored by measuring the fluorescence intensity at 582 nm (FL2). As a positive control for depletion of membrane potential, cells were treated with 10  M CCCP for 45 minutes. The data is a representative of at least three independent experiments. (c) H9c2 cells were treated with (2  M) Tg alone or pretreated for 30 minutes with BH4-Tat peptide (200 nM) prior to treatment with Tg, for 48 hours and reduction in cell viability was determined by MTT assay. Error bars represent mean SD from three independent experiments performed in triplicates. (d) Bcl-2 overexpressing H9c2 cells (Bcl-2) and control cells (neo) were treated with Tm (2  g/ml) for 48 hours. Increase in cell death was measured by annexin V staining. The data is representative of at least 3 independent experiments. Protein extracts from Bcl-2 overexpressing H9c2 cells (Bcl-2) and control cells (neo) were subjected to western blot analysis using the indicated antibodies. (e)-(f) H9c2 cells were treated with Tg (2  M) alone or pretreated for 30 minutes with (e) cyclosporine A (CsA) (2  M) and aristolochic acid (ArA) at 25  M; (f) bongkrekic acid (BA) (10  M); prior to treatment with Tg, for 48 hours and reduction in cell viability was determined by MTT assay. Error bars represent mean SD from three independent experiments performed in triplicates.
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