<p>(A) The effects of the cell signaling inhibitors H-89, XAV939 and PAC1 dimerization inhibitor NAC on the remaining cell viabilities of PAC1-CHO, M-PAC1-CHO and pcDNA-CHO 24 h after serum withdrawal. All the data were plotted as the fold changes in the data of pcDNA-CHO because the changes in pcDNA-CHO had no correlation with PAC1. It was shown that the β-catenin signal inhibitor XAV939 (10 µM) and PAC1 dimerization inhibitor NAC (10 nM) significantly decreased the viabilities of PAC1-CHO cells (*, P<0.01, PAC1-CHO/XAV939 and PAC1-CHO/NAC vs. PAC1-CHO/no inhibitors), whereas the PKA inhibitor H-89 (100 µM) did not exert significant effect on the cells viability. The effects of H-89, XAV939 and NAC on the caspase3 activities (B) and Bcl-2 levels (C) in PAC1-CHO, M-PAC1-CHO and pcDNA-CHO 24 h after serum withdrawal showed that XAV939 (10 µM) and NAC (10 nM) exerted significant inhibitory effects on the anti-apoptotic activity of PAC1-CHO by increasing the caspase3 activity and decreasing the Bcl-2 level in the PAC1-CHO cells significantly (*, P<0.01, PAC1-CHO/XAV939 and PAC1-CHO/NAC vs. PAC1-CHO/no inhibitors), whereas H-89 (100 µM) did not have the similar inhibitory effects. (D)Top-flash assays. PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells were transfected with Top-flash + pRluc and Fop-flash + pRluc respectively. pRluc was used here as the internal control for transfection efficiency. After the transfection, cells were submitted to serum-withdraw induced apoptosis with or without the signal inhibitors H-89 (100 µM), XAV-939 (10 µM), and acetylcysteine (10 nM) for 24 h. And then cells were lysed and luciferase activities were measured. Relative luciferase activities were expressed as the ratio of TOP-flash/FOP-flash luciferase activity and the data were plotted as fold changes in the data from pcDNA-CHO cells. It was found that without inhibitors the relative luciferase activity in PAC1-CHO cells was significantly higher than that in M-PAC1-CHO and pcDNA-CHO (#, P<0.01, PAC1-CHO/no inhibitors vs. M-PAC1-CHO/no inhibitors and pcDNA-CHO/no inhibitors) after serum withdrawal. The addition of Wnt/β-catenin signal inhibitor XAV939 (10 µM) and PAC1 dimerization inhibitor NAC (10 nM) significantly decreased the relative luciferase activity in PAC1-CHO (*, P<0.01, PAC1-CHO/XAV939 and PAC1-CHO/NAC vs. PAC1-CHO/no inhibitors), indicating that the basal activity of PAC1 was Wnt/β-catenin involved and dimer dependent. The data were represented as the means ± S.E. of three independent experiments. (D) Western blotting showed XAV939 (10 µM) and NAC (10 nM) significantly decreased the β-catenin, cyclin D1 and c-myc levels in PAC1-CHO cells. (*, P<0.01, PAC1-CHO/XAV939 and PAC1-CHO/NAC vs. PAC1-CHO/no inhibitors), whereas H-89 (100 µM) did not have the similar inhibitory effects. The data were represented as the means ± S.E. of three independent experiments.</p
