Figure 5

Effects of inhibitors of MAPKs and siRNAs for CHOP and caspase 12 on ER stress-induced cell death and CHOP expression and phosphorylation in HT22 neuronal cells. (A) HT22 cells were pretreated with vehicle or inhibitors of MAPKs (10 µM SB203580 for p38, 10 µM SP600125 for JNK, 20 µM PD98059 for ERK) for 1 h, and treated with vehicle (Control), 5 µM TG, or 10 µM BFA for 24 h. Cell viability was then determined by MTT assay. ^*P < 0.01 when compared with untreated control cells. ^#P < 0.01 when compared with TG- or BFA-treated cells without inhibitors. (B) HT22 cells were transfected with CHOP, caspase-12, and control scrambled siRNAs for 24 h and treated with TG or BFA for 24 h. Cell viability was then determined by MTT assay. ^*P < 0.01 when compared with untreated control cells. ^#P < 0.01 when compared with TG- or BFA-treated cells with scrambled siRNA transfection. (C, D) Cells were pretreated with 10 µM SB203580 (SB), 10 µM SP600125 (SP), or 20 µM PD98059 (PD) for 1 h, and treated with vehicle, 5 µM TG (C), or 10 µM BFA (D) for 24 h. Cell lysates were separated by SDS-PAGE and the protein levels of CHOP and β-actin were detected by Western blot analysis. The protein level of CHOP in C and D was quantified by densitometry and normalized to the level of β-actin. The fold increase was calculated based on each data of vehicle (first lane). ^*P < 0.05 when compared with untreated control cells. ^#P < 0.05 when compared with TG- or BFA-treated cells in the absence of baicalein at each incubation time. (E) Cells were pretreated with 10 µM SB203580 (SB) for 1 h and then treated with vehicle, 5 µM TG, or 10 µM BFA for 12 h. Cell lysates were immunoprecipitated with anti-CHOP antibody and the immunoprecipitates were analyzed by Western blotting using anti-phospho-serine antibody and anti-CHOP antibody.