Figure 7

Activation of the FGF15 and FGF8 downstream cytosolic effectors. Comparison of phosphorylation levels of four proteins that are modified in response to FGF15 and FGF8. E12.5 primary cortical cultures were starved for 24 hours and then treated with recombinant FGF15 or FGF8. Cell lysates were analyzed after 0, 5, 15, 30 and 60 minutes by immunoblottingblotting to detect phosphorylated forms of pERK 42/44 (a, b, top panels), pAKT (a, b, middle panels) and pS6 (c, d, top panels), and pGSK3 (c, d, middle panels). The α-Tubulin (α-Tub) antibody (a-d, bottom panels) was used for normalization (these results are characteristic of what we observed the three times these experiments performed). The numbers under the bands indicate the fold-induction or reduction, with respect to T = 0, and are normalized with respect to the α-Tubulin level. (e-j) Immunofluorescence for β-III-Tubulin (red), phosphohistone-3 (PH3) (green) and Hoechst (blue) on E12.5 primary cortical cultures that were either not treated, treated with 50 ng/ml recombinant FGF8 or FGF15 for 24 and 48 hours (n = 4 for each experiment; p = 0.01, Student's t-test). (k) Graph showing the mitotic index calculated before starting the treatment (T0) and after 24 and 48 hours of treatment with recombinant FGF8 and FGF15. Non-treated cells were used as a control. The mitotic index was calculated by dividing the number of PH3⁺ cells with the total number of cells (Hoechst labeled nuclei). Bars in the graph represent the standard deviation. The average number of nuclei and PH3⁺ cells for each sample/350 μm² were as follow. T0: 113.5 ± 5 nuclei and 6.25 ± 2.5 PH3⁺ cells. At 24 hours: control, 226.75 ± 4 nuclei and 20.5 ± 3.8 PH3⁺ cells; FGF8, 297.5 ± 12.5 nuclei and 48.3 ± 1.5 PH3⁺ cells; FGF15, 113 ± 10 nuclei and 8.25 ± 2.5 PH3⁺ cells. At 48 hours: control, 79.6 ± 3 nuclei and 8.25 ± 1 PH3⁺ cells; FGF8, 164 ± 11 nuclei and 29 ± 3 PH3⁺ cells; FGF15, 44 ± 3 nuclei and 3.4 ± 0.5 PH3⁺ cells.