AS-Hsp27 induces the release of mitochondrial protein Smac and activates downstream caspase cascade. (A) Ectopic expression of AS-Hsp27 in MM.1R cells restores apoptosis in response to Dex via Smac release. MM.1R cells were transfected with GFP vector alone or GFP-AS-Hsp27. GFP⁺ cells were isolated and treated with Dex (5 μM). Cytosolic extracts from these cells were separated by SDS-PAGE and analyzed by immunoblotting with anti-Smac and anti-cyto-c antibodies (first and second panels, respectively). Expression and functional specificity of AS-Hsp27 was determined by immunoblotting with anti-Hsp27 and anti-Hsp70 antibodies (third and fourth panels, respectively). As a control for equal transfections, filters were reprobed with anti-GFP antibody (fifth panel). Blots are representative of 3 independent experiments with similar results. (B) Dex triggers caspase-9 activation. MM.1R cells were transfected with GFP vector alone or GFP-AS-Hsp27. GFP⁺ cells were isolated and treated with Dex (5 μM) for 48 hours and harvested. Caspase-9 activation was determined by subjecting cytosolic extracts for protease activity using LEHD-pNA as substrate. Results are representative of 3 independent experiments (P < .005; n = 3). Error bars represent standard deviation. (C) Dex triggers caspase-3 and PARP cleavage in AS-Hsp27-transfected MM.1R cells. Cytosolic extracts from the cells were subjected to immunoblot analysis with anti-caspase-3 (upper panel), anti-tubulin (middle panel), or anti-PARP (lower panel) antibodies. Blots are representative of 3 independent experiments with similar results. (D) MM.1S cells treated with Dex (5 μM) for 24 hours; cytosolic extracts were prepared and subjected to immunoblot analysis with anti-Smac (upper panel), anti-cyto-c (middle panel), or anti-tubulin (lower panel) antibodies. Blots are representative of 3 independent experiments with similar results.