Figure 3. Knocking-down of Bim expression leads to attenuation of apoptosis induced by UO126 in HS2 cells.

(A) Knocking-down of Bim in 606HS2 and 931HS2 cells. Whole cell extracts were immunoblotted using indicated antibodies. (B) Erk1/2 activation and Bim_EL expression in UO126 treated cells. Whole cell extracts from Bim-shRNA-94-HS2 cells or control ns-shRNA-HS2 cells treated or not (–) during 48hours with UO126 (10 or 20 µM) were immunoblotted using indicated antibodies. (C) For mortality analysis, cells were plated at 2×10⁵ cells/mL. Bim-shRNA-94-HS2 cells or control ns-shRNA-HS2 cells were treated or not (–) during 48hours with UO126 (10 or 20 µM) 24 h after lentiviral infection. The percentage of dead cells was evaluated by trypan blue exclusion. The percentage of apoptotic cells was determined by labeling the cells with cleaved caspase3-antibody and flow cytometry analysis. For both cell death and cell apoptosis, data are mean ±SEM (n = 3). Statistical differences from the value of the control (–) are indicated as follows: * P<0.05;** P<0.01; *** P<0.001 (Student t test). (D) Proteosomal degradation of Bim_EL. Cells were treated or not (–) for 2 h or 4 h with MG132 (10 µM). Whole cell extracts were immunoblotted using indicated antibodies. Data are a representative experiment out of three.