Fig. 5

CA activates the PERK-eIF2a-ATF4 pathway leading to the pro-apoptotic CHOP up-regulation in human PCa cells. The PC-3 and DU145 cells were treated with CA at the indicated concentrations (5, 10, 15 μM.) for 24 h. a the phosphorylation of PERK, eIF2α and expression levels of Bip, PERK, eIF2α, ATF4 and CHOP were measured by western blot using corresponding antibodies. b And Protein levels were quantified by grayscale scan and the GAPDH was used as the loading control. c Total RNAs were extracted and subjected to real-time PCR analysis using specific primer sets for CHOP and GAPDH, and the data were normalized to GAPDH expression. The results are presented as mean ± SD and represent three individual experiments. *p < 0.05 and **p < 0.01 as compared with untreated control. d Effects of CA induced-CHOP activation analyzed by immunofluorescence staining. The PC-3 and DU145 cells were transfected with PERK siRNA or control siRNA, respectively and treated with CA for 24 h. e The protein expression of PERK and CHOP was measured by western blotting. f Protein levels were quantified by grayscale scan and the GAPDH was used as the loading control. g The cell viability was determined by MTT assay. The results are presented as mean ± SD and represent three individual experiments. *p < 0.05 and **p < 0.01 as compared with corresponding group. h Overexpression of PERK in PC-3 and DU145 cells that were treated with CA for 24 h. PERK, p-PERK and CHOP protein expressions were determined by western blot. i Protein levels were quantified by grayscale scan and the GAPDH was used as the loading control. j MTT assay demonstrated that PERK overexpression aggravated CA-induced migration inhibition of cell viability in PC-3 and DU145. The results are presented as mean ± SD and represent three individual experiments. *p < 0.05 and **p < 0.01 as compared with corresponding group