Figure 6.

Inhibition of ER stress-induced apoptosis by miR-106b-25 cluster is mediated by repression of Bim. (a) The control (Neo) and Bim-shRNA expressing (Bim-shRNA) PC12 cells were either untreated (Un) or treated with (0.25 μM) Tg, (2.0 μg/ml) Tm, (0.5 mM) tBHQ, and (100 μM) 4-4-HNE for 48 h. Apoptosis was determined with annexin-V/PI staining followed by FACS analysis. Percentages of cells positive for both annexin-V and annexin-V/PI are shown. Average and error bars represent mean±S.D. from three independent experiments. (*P<0.005, two-tailed unpaired t-test as compared with control (Neo) cells). (b) Upper panel, The indicated cells were either untreated (Un) or treated with (0.25 μM) Tg for indicated 24 h. Total protein was isolated and immunoblotting was performed using antibodies against Bim, Chop and β-actin. Lower panel, in the experiment described in (a), induction of Bim and Chop was calculated after densitometric analysis of the autoradiographs with Image J. Average and error bars represent mean±S.D. from three independent experiments.(**P<0.05, two-tailed unpaired t-test as compared with miR-106b-25 expressing (Neo/miR-106b-25) cells). (c) The indicated cells were either untreated or treated with (0.25 μM) Tg, (2.0 μg/ml) Tm, (0.5 mM) tBHQ, and (100 μM) 4-4-HNE for 48 h. Apoptosis was determined with annexin-V/PI staining followed by FACS analysis. Percentages of cells positive for both annexin-V and annexin-V/PI are shown. Average and error bars represent mean±S.D. from three independent experiments. (*P<0.005, two-tailed unpaired t-test as compared with control (Neo/pCDH) cells). (d) The indicated cells were either untreated or treated with (0.25 μM) Tg for 24 h or (2.0 μg/ml) Tm for 24 h, (0.5 mM) tBHQ for 12 h, and (100 μM) 4-4-HNE for 12 h, and DEVDase activity was measured as described in Materials and Methods. Average and error bars represent mean±S.D. from three independent experiments. (*P<0.005, two-tailed unpaired t-test as compared with control (Neo/pCDH) cells)