Fig. 2.

Pharmacologically inhibiting MAPK blocks BITC-mediated G₂/M cell cycle arrest and apoptosis. (A) Capan-2 cells were pre-treated with 50 μM MEK-1 inhibitor PD98059 for 1 h followed by treatment with or without 2.5–10 μM BITC for 24 h and its effect on the cell cycle distribution and (B) apoptosis was evaluated by flow cytometry as described in Materials and Methods. Capan-2 cells were pre-treated with (C) 30 μM JNK inhibitor (SP600125) or (D) 20 μM p38 inhibitor (SB202190) and then treated with 10 μM BITC for 24 h. The phosphorylation of JNK and p38 was evaluated by western blotting. (E) Immunoblotting against p-ERK (Thr202/Tyr204), ERK, Cdc25C, Cdk1, cyclin B1 and the cleaved caspase-3 and PARP. Each blot was stripped and reprobed with anti-β-actin antibody to ensure equal protein loading. (F) The effects of MEK-1 (PD98059; 50 μM), JNK (SP600125; 30 μM) and P38 (SB202190; 20 μM) inhibitors on apoptosis were also evaluated by Cell Death Detection Apoptosis ELISA assay as described in Materials and Methods. Values are means ± standard errors of the mean of three independent experiments (each conducted in triplicate). Data were analyzed by non-parametric one-way analysis of variance followed by Bonferroni's post hoc multiple comparison test. Differences were considered statistically significant at *P < 0.05, **P < 0.01 and ***P < 0.001 when compared with control and #P < 0.05, ##P < 0.01 and ###P < 0.001 when compared with BITC treatment.