Fig 2: Extracellular FGF1 and etoposide increase endogenous fgf1 expression in SH-SY5Y cells, in contrast to N2a cells. SH-SY5Y and N2a cells were treated or not with rFGF1 for 72 h (a–c) or etoposide for 16 h (b–d). The levels of all fgf1 mRNAs (a,b) or of the alternative 1B fgf1 mRNA (c,d) were analyzed by RT-PCR. The 18S rRNA levels were used as a control for quantifications. The graphs represent the mean ±S.E.M. of three independent experiments. Student’s t-tests were performed (n=3; n.s.: P>0.05; *P?0.05; **P?0.01; ***P?0.001)

Fig 3: FGF1 phosphorylation inhibits its anti-apoptotic activity in SH-SY5Y cells. SH-SY5Y cells were stably transfected with FGF1WT, FGF1K132E, FGF1S130A, FGFS130D or empty (mock) expression vectors. (a) FGF1 levels were examined by western blot after heparin-sepharose concentration in cell lysates and conditioned media from SH-SY5Y cells expressing FGF1WT, FGF1S130A, FGF1S130D or transfected with an empty vector (mock). (b) Apoptosis in FGF1WT, FGF1K132E, FGF1S130A, FGFS130D or mock stably transfected SH-SY5Y cells after 48 h of etoposide treatment was analyzed by flow cytometry after DiOC6(3) and PI staining. Apoptotic cells are the DIOC-, PI- and size- cells. The graph represents the mean±S.E.M. of three independent experiments. Student’s t-tests were performed relative to the control mock, except where indicated (n=3; n.s.: P>0.05; *: P?0.05; ***: P?0.001). (c,d) PUMA, cleaved caspase-3 and PARP (full-length and cleaved forms) levels were analyzed by western blot in FGF1WT, FGF1K132E, FGF1S130A, FGFS130D or mock stably transfected SH-SY5Y cells after 0, 4, 8 or 16 h of etoposide treatment. Actin detection was used as control

Fig 4: FGF1 phosphorylation inhibits its anti-apoptotic activity. (a and b) Neo, FGF1WT, FGF1S130A and FGF1S130D PC12 cell lines were treated with dexamethasone for 48 h. p53-dependent apoptosis was then induced by etoposide treatment during 40 h. (a) Cell survival was estimated by crystal violet nuclei staining (**P<0.01, ***P<0.001, ns P>0.05, n=6). (b) Apoptosis (percentage of apoptotic nuclei) was estimated after Hoechst nuclei staining (*P<0.05, **P<0.01, ns P>0.05, n=3). FGF1WT and FGF1S130A protected PC12 cells from p53-dependent apoptosis in contrast to FGF1S130D. (c) Neo, FGF1WT, FGF1S130A and FGF1S130D PC12 cells cultured in the presence of dexamethasone were treated with etoposide for 0, 8 or 16 h. The levels of P-p53 (Ser 15), PUMA and cleaved caspase-3 were detected by western blot. Actin was used as a loading control. FGF1WT, FGF1S130A and FGF1S130D decreased etoposide-induced upregulation of these different apoptotic markers. The stronger effect was observed for the unphosphorylable FGF1

Fig 5: The K132E mutation inhibits FGF1 anti-apoptotic activity. (a) Neo, FGF1WT and FGF1K132E 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. FGF1WT protected PC12 cells from p53-dependent apoptosis whereas FGF1K132E did not (**P<0.01, ***P<0.001, ns P>0.05, n=4). (b) Neo, FGF1WT and FGF1K132E 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 FGF1WT PC12 cells compared with FGF1K132E and Neo PC12 cells. (c) Noxa (left panel) and p21 (right panel) mRNA levels were analyzed by RT-PCR in native, FGF1WT and FGF1K132E 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 FGF1K132E, FGF1WT decreased p53-dependent up-regulation of noxa mRNA levels (*P<0.5, n=3)