FIG. 5.
Suppression of endogenous Bim expression inhibits Gadd45a-induced apoptosis. (A) A total of 5 × 105 HeLa Gadd45a-inducible cells were plated into 100-cm dishes and grown in DMEM containing tetracycline. Upon removal of the tetracycline, the cells were transfected with 21-nucleotide siRNA duplexes that target Bim mRNA transcript via oligofectamine (see Materials and Methods). As a control, nonspecific 21-nucleotide siRNA duplexes were included in the experiments. Cells were collected at the indicated times for preparation of whole-cell lysates or mitochondrial protein. The protein levels of Bim in both whole lysates and mitochondria were analyzed by Western immunoblot assays. In addition, cleavages of PARP and caspase-3 were examined in cells treated with Bim siRNA. (B) Detection of Bim mitochondrial induction in cells with disrupted Gadd45a. MEFs derived from Gadd45a knockouts and RKO-AS45 that expresses antisense Gadd45a were treated with UV radiation at a dose of 10 J/m2 or MMS at a concentration of 50 μg/ml. After 12 h, cells were harvested for the preparation of both whole-cell lysates and mitochondrial protein. Immunoblotting assays were performed to examine the Bim protein levels of mitochondria.