Fig. 3. Regulatory effect of melatonin on phosphorylation of PKC and JNK.

a Phosphorylation of pan-PKC is shown n = 3. **p < 0.01 vs. control. b Phosphorylation of pan-PKC in cells treated with melatonin (1 µM) for 30 min prior to rVvhA exposure for 60 min is shown. n = 4. *p < 0.01 vs. control. ^#p < 0.05 vs. rVvhA alone. c Cells transfected with siRNAs for MT₂ and Gαq were incubated with melatonin (1 µM) for 30 min prior to rVvhA exposure for 60 min. The level of PKC phosphorylation is shown. Data represent the mean ± S.E. n = 4. **p < 0.01 vs. nt siRNA. ^#p < 0.01 vs. rVvhA+ melatonin+ nt siRNA. d The effect of rVvhA on the expression of MAPK was determined by western blot. n = 3. *p < 0.05 vs. 0 min. **p < 0.01 vs. 0 min. e Cells transfected with siRNA for PKCα were incubated with melatonin (1 µM) for 30 min prior to rVvhA exposure for 60 min. The level of JNK phosphorylation is shown. Data represent the mean ± S.E. n = 4. *p < 0.01 vs. control. ^#p < 0.05 vs. rVvhA alone. f Cells transfected with siRNAs for MT₂ and Gαq were incubated with melatonin (1 µM) for 30 min prior to rVvhA exposure for 60 min. The level of JNK phosphorylation is shown. Data represent the mean ± S.E. n = 4. *p < 0.01 vs. nt siRNA. ^#p < 0.01 vs. rVvhA+ melatonin+ nt siRNA. g, h Cells were pretreated with SP600125 (10 μM) or melatonin (1 μM) for 30 min prior to rVvhA exposure for 24 h. Bcl-2, Bax, caspase-9 and cleaved caspase-3 were detected by western blot. Data represent the mean ± S.E. n = 4. *p < 0.05 vs. control. ^#p < 0.01 vs. rVvhA alone. h Quantitative analysis of the percentage of apoptotic cells by flow cytometer analysis is shown. n = 4. *p < 0.01 vs. nt siRNA alone. ^#p < 0.01 vs. rVvhA+ nt siRNA