Figure 2
PLX4720 upregulates Bim. (a) Upper panel: overexpression of Bcl-2 in Mel-RMu and Mel-CV cells stably transfected with cDNA encoding Bcl-2. Whole cell lysates were subjected to western blot analysis of Bcl-2 and GAPDH (as a loading control). Lower panel: Mel-RMu and Mel-CV cells overexpressing Bcl-2 were treated with PLX4720 (10 μM) for 72 h before apoptosis was quantitated by the propidium iodide (PI) method. The data shown are either representative (upper panel) or mean±S.E. (lower panel) of three individual experiments. (b) Whole cell lysates from Mel-RM and Mel-RMu cells treated with PLX4720 (10 μM) for indicated time periods were subjected to western blot analysis of Bim, Bid, PUMA, Noxa, Bax, Bak, Mcl-1, Bcl-2, and GAPDH (as a loading control). The data shown are representative of three individual western blot analyses. (c) Left panel: total RNA from Mel-RM and Mel-RMu cells treated with PLX4720 (10 μM) for indicated time periods was isolated and subjected to real-time PCR analysis for Bim mRNA expression. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. Right panel: total RNA from Mel-RM and Mel-RMu cells treated with PLX4720 (10 μM) for 16 h with or without pretreatment with actinomycin D (Act-D) (3 μg/ml) for 1 h were subjected to real-time PCR analysis. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. The data shown are the mean±S.E. of three individual experiments
