Figure 6. Apoptosis induced by combination of BSO and HCH is mediated by JNK dependent ERK1/2 activation.

(A) K562 cells were treated as indicated for different time periods and cell lysates were subjected to western blot analysis with indicated antibodies. (B) K562 cells were pretreated with JNK inhibitor SP600125 (20 µM), p38 inhibitor SB203580 (20 µM), ERK inhibitor PD98059 (40 µM) for 1 h before treatment with BSO (100 µM) and HCH (10 µM) in combination. After 36 h of incubation, cells were subjected to Annexin V/PI binding assay by flow cytometry. Data represent mean ± SD of three experiments. *** p<0.001 compared to treatment in absence of MAPK inhibitors. (C) Knockdown of JNK by specific JNK1 siRNA attenuates apoptosis. JNK1 protein level was shown after knockdown (upper panel). Annexin V/PI binding assay by flow cytometry in K562 cells after transfection with indicated siRNAs (lower panel). Dot plots are representative of two similar experiments. (D) Knockdown of ERK by specific ERK2 siRNA attenuates apoptosis. ERK2 protein level was shown after knockdown (upper panel). Annexin V/PI binding assay by flow cytometry after knocking down ERK2 (lower panel). Dot plots are representative of two similar experiments (E) K562 cells were transfected with indicated siRNAs for 48 h and then treated with BSO plus HCH for 18 h. Protein expression and phosphorylation of JNK and ERK1/2 were analysed by western blot on whole cell lysates. (F) K562 cells were pre-incubated with or without 2 mM GME for 1 h and further incubated with BSO (100 µM) and HCH (10 µM) in combination for 18 h. The level of each protein and phosphorylation status was analyzed as indicated by western blot. (G) K562 cells were transfected with indicated siRNAs for 48 h and then treated with BSO plus HCH for 24 h. Analysis of intracellular NO was done by flow cytometry after staining with DAF-FM. Histograms are representative of two similar experiments.