Figure 7

Analyses of the proliferation of osteoblast progenitors in vitro (A) Reductions in Fgfr1, Fgfr2, and Fgfr3 mRNA by the introduction of respective siRNA into wild-type osteoblast progenitors. Each value of Fgfr1–3 in the introduction of control siRNA was set as 1 and the relative levels are shown. n = 3. **p < 0.01. (B) Effects of FGF2 and each siRNA for Fgfr1, Fgfr2, and Fgfr3 on the proliferation of wild-type osteoblast progenitors. The values of the vehicle in the control siRNA were set as 1, and the relative levels are shown. n = 4. * vs. the respective vehicle. ^†vs. the respective experiment in the control siRNA. **^,††p < 0.01. (C) Effects of Runx2 on proliferation and the FGF2-induced proliferation of wild-type osteoblast progenitors and the inhibition by AZD4547 (50 nM). The values of the vehicle in the GFP group were set as 1, and the relative levels are shown. n = 4. * vs. the respective vehicle. †vs. the respective experiment in the GFP group. **^,††,##p < 0.01. (D) Effects of inhibitors on the FGF2-induced proliferation of wild-type osteoblast progenitors. AZD: AZD4547 (50 nM), U: U0126 (50 μM), LY: LY294002 (1 μM), Akt-I: Akt inhibitor (2.5 μM). The values in the vehicle were set as 1, and the relative levels are shown. n = 4. * vs. the respective vehicle. ^†vs. the respective experiment in the vehicle group. ^†p < 0.05, ^**,††p < 0.01. (E,F) Western blots of activated p42/44 MAP kinase (E) and Akt (F). IGF-1 was used as a positive control for Akt activation (F). (G) Effects of FGF2 and AZD4547 (50 nM) on the proliferation of Sp7^−/− osteoblast progenitors. The values in the vehicle of wild-type osteoblast progenitors were set as 1, and relative levels are shown. n = 4. * vs. the respective vehicle. ^†vs. the respective experiment in the wild-type group. *^,†,#p < 0.05, **^,††,##p < 0.01. (H) Real time RT-PCR analysis using RNA from Sp7^−/− osteoblast progenitors. The values for control siRNA were set as 1 and the relative levels are shown. n = 3. **p < 0.01. (I) The effects of siRNA for Runx2 on the FGF2-induced proliferation of Sp7^−/− osteoblast progenitors. The values for the vehicle were set as 1 and the relative levels are shown. n = 4. * vs. the respective vehicle. †vs. the respective experiment in the control siRNA. **^,††p < 0.01. Similar results were obtained in two to four independent experiments and representative data are shown in A–I.