Figure 6
Down-regulation of the p38 MAPK pathway decreases BafA1-induced cell death.
(A) BEL7402 and HepG2 cells were treated with vehicle controls or with 5 nM BafA1 for 24, 48 and 72 h. IB analysis for p-ERK1/2, p-JNK, p-p38, total-ERK1/2, total-JNK, and total-p38 was used to examine protein expression, using β-actin as a loading control. (B) HCC cells were pre-treated with inhibitors to either ERK (PD98059), JNK (SP600125), or p38 (SB202190) and subsequently treated with BafA1 alone, or in combination, for 24 h, double stained with Annexin V/PI and analyzed by FACS. (C) HCC cells were transfected with a stable knockdown for p38 (BEL7402/shp38 and HepG2/shp38), or control cells (BEL7402/shControl and HepG2/shControl) and were treated with vehicle control or 5 nM BafA1 for 24, 48, and 72 h. At each time-point, cell viability was examined using the MTT assay. (D) BEL7402 and HepG2 cells were pre-treated SB202190, vehicle control or 5 nM BafA1 for 24 h. IB analysis for protein expression of Puma was carried out, using β-actin as a loading control. (E) BEL7402 and HepG2 cells were infected with adenoviruses expressing GFP-Puma or vector control at a multiplicity of infection of 250. Cells were treated with BafA1 and cell viability determined using the MTT assay. Data are presented as mean ± SEM from three independent experiments, (BafA1 means Bafilomycin A1).
