Fig. 5

Role of caspases in AT2R-induced apoptosis in EJ bladder cancer cells. a AT2R-induced increase in caspase-3 activity in EJ cells. Cells underwent transduction with Ad-CMV-eGFP or Ad-G-AT2R-eGFP (100 ifu/cell) or were mock transduced for 2 d. Following this, cells were washed and lysed, and total cellular protein was assayed for caspase-3–like activity by measuring the release of pNA from the colorimetric caspase-3 substrate Ac-DEVD-pNA. Columns, mean A405 nm from three experiments; bars, SD. * P < 0.01 vs Ad-CMV-eGFP–transduced or mock-transduced cells. b Cells underwent transduction with Ad-CMV-eGFP or Ad-G-AT2R-eGFP (100 ifu/cell) or were mock transduced for 48 h. Western blot analysis for expression level of cleaved caspase-8 in cell lysates. c AT2R-induced increase in cleaved caspase-3 generation in EJ cells. Cells were infected with either 200, 100 or 50 ifu/cell of Ad-G-AT2R-eGFP (lanes 1, 3 and 5, respectively), 200, 100 or 50 ifu/cell of Ad-CMV-eGFP (lanes 2,4 and 6, respectively), or were mock transduced (lane 7). At 48 h after transduction, cell protein extracts were subjected to Western blot analysis. Representative blots indicating cleaved caspase-3 and β-actin under each treatment condition. d effects of caspase inhibitors on AT2R-mediated apoptosis in EJ cells. Cells were transduced with Ad-G-AT2R-eGFP (100 ifu/cell) for 6 h followed by treatment with either the caspase-3 inhibitor Ac-DEVD-CMK (20 μmol/L), the caspase-8 inhibitor Z-IETD-FMK (20 μmol/L) or control solvent (DMSO) for 24 h. Green fluorescent cells exhibiting apoptotic morphology were counted from 10 fields per well. Columns, mean of three experiments; bars, SD. * P < 0.01 vs control (DMSO-treated) cells