Figure 4.

Loss of Bim_EL–DLC1 binding enhances the proapoptotic activity of Bim_EL. (A) RNAi specific for DLC1 sensitizes MEFs for apoptosis induced by thapsigargin in a predominantly Bim-dependent manner. Cells were transfected with control siRNA or siRNA specific for DLC1 16 h prior to treatment with 200 nM thapsigargin for 24 h. QVD-OPh (10 µM) was added to control samples together with thapsigargin to test for caspase dependency. Data show means/SEM of three independent experiments. (*) P = 0.015 wild type; (*) P = 0.005 Bim^−/−, two-tailed paired t-test. (B) Bim_ELAA has higher proapoptotic activity than wild-type Bim_EL when expressed in MEFs. MEFs carrying a 4HT-inducible construct for the expression of either wild-type Bim_EL or Bim_ELAA were treated with 100 nM 4HT for 24 h. Apoptosis was measured as the percentage of cells positive for active caspase-3. Data show means/SEM of four independent experiments. (**) P = 0.010, two-tailed paired t-test. Both Bim_EL and Bim_ELAA were expressed at comparable levels (see Supplemental Fig. S13). (C,D) RNAi specific for DLC1 can sensitize MEFs to the induction of wild-type Bim_EL but not Bim_ELAA. MEFs carrying a 4HT-inducible construct for the expression of either wild-type Bim_EL (C) or Bim_ELAA (D) were transfected with control or DLC1-specific siRNA 16 h prior to Bim induction with 100 nM 4HT for 24 h. Cells with active caspase-3 were detected by flow cytometry as above. Data show means/SEM of four independent experiments. (*) P = 0.014 (C); (ns) nonsignificant (P = 0.679) (D), two-tailed paired t-test.