Figure 3

The K132E mutation inhibits FGF1 anti-apoptotic activity. (a) Neo, FGF1^WT and FGF1^K132E PC12 cell lines were treated with dexamethasone for 48 h. Then, cell survival after 40 h etoposide treatment was estimated after crystal violet nuclei staining. FGF1^WT protected PC12 cells from p53-dependent apoptosis whereas FGF1^K132E did not (**P<0.01, ***P<0.001, ns P>0.05, n=4). (b) Neo, FGF1^WT and FGF1^K132E PC12 cell lines cultured in the presence of dexamethasone were treated with etoposide for 0, 8 or 16 h. p53 activation (Ser 15 phosphorylation), PUMA expression and caspase-3 cleavage were analyzed by western blot. Actin detection was used as a control. Etoposide induced upregulation of P-p53 (Ser 15), PUMA and cleaved caspase-3 in all cells. However, these levels were lower in FGF1^WT PC12 cells compared with FGF1^K132E and Neo PC12 cells. (c) Noxa (left panel) and p21 (right panel) mRNA levels were analyzed by RT-PCR in native, FGF1^WT and FGF1^K132E PC12 cell lines after 0, 8 or 16 h of etoposide treatment in the presence of dexamethasone. The 18 S rRNA levels were used as a control for the quantifications. In contrast to FGF1^K132E, FGF1^WT decreased p53-dependent up-regulation of noxa mRNA levels (*P<0.5, n=3)