Figure 5
Figure 5. FGF2 and PDGF-BB augment PDGFRα expression in PαS-MSCs. (A) Whole-cell extracts of ex vivo–cultured PαS-MSCs derived from vehicle or tPA-treated mice were incubated on mouse receptor tyrosine kinase-phosphorylation antibody array, and phosphorylation status was determined by subsequent incubation with HRP-conjugated antiphosphotyrosine (each RTK spotted in duplicate, positive controls in corners, gene identity in the right panel). Increased tyrosine phosphorylation for PDGFRα (black arrow) and PDGFRβ (red arrow) is shown (n = 2/group). (B) Representative western blot of PDGFRα and PDGFRβ and EGF receptor in cell lysates of cultured PαS-MSCs derived from control PBS- or tPA-treated mice (n = 3/group). α-Actinin was used as a loading control. (C-D) Relative mRNA expression of (C) PDGFRα and (D) PDGFRβ in cultured PαS-MSCs stimulated with recombinant FGF2 at indicated concentrations for 18 hours in serum-reduced medium as determined by RT-PCR. Data are normalized to GAPDH (n = 3/group). (E-F) Relative protein expression of PDGF-Rα and PDGFRβ in PαS-MSCs stimulated with recombinant FGF2 at indicated concentrations for 18 hours in serum-reduced medium as determined by western blot analysis. Relative intensity of each band was measured and normalized to α-actinin (n = 3/group). (G) Relative mRNA expression of PDGFRα in cultured PαS-MSCs treated for 2 days with PBS, tPA, FGF2, PDGF-BB, or FGF2 and PDGF-BB as determined by RT-PCR. Data are normalized to GAPDH (n = 3/group). (H) WT B6 mice were injected daily intraperitoneally with vehicle, tPA, the tyrosine kinase inhibitor Gleevec, and/or FGFR inhibitor. BMNCs were harvested from mice at day 2 and stained for CD45, Ter119, Sca-1, and PDGFRα and analyzed by flow cytometry. Percentage of PαS-MSCs is given (n = 4/group). (I) Absolute numbers of MSCs in the femur of PBS-, tPA-, EGFR inhibitor–, and PDGFR inhibitor–treated mice was determined by FACS analysis (n = 4/group). Mean ± SEM. *P < .05; **P < .01; ***P < .001 by Student t test or ANOVA with Tukey HSD tests for multigroup analysis. Data are representative of 2 independent experiments.

FGF2 and PDGF-BB augment PDGFRα expression in PαS-MSCs. (A) Whole-cell extracts of ex vivo–cultured PαS-MSCs derived from vehicle or tPA-treated mice were incubated on mouse receptor tyrosine kinase-phosphorylation antibody array, and phosphorylation status was determined by subsequent incubation with HRP-conjugated antiphosphotyrosine (each RTK spotted in duplicate, positive controls in corners, gene identity in the right panel). Increased tyrosine phosphorylation for PDGFRα (black arrow) and PDGFRβ (red arrow) is shown (n = 2/group). (B) Representative western blot of PDGFRα and PDGFRβ and EGF receptor in cell lysates of cultured PαS-MSCs derived from control PBS- or tPA-treated mice (n = 3/group). α-Actinin was used as a loading control. (C-D) Relative mRNA expression of (C) PDGFRα and (D) PDGFRβ in cultured PαS-MSCs stimulated with recombinant FGF2 at indicated concentrations for 18 hours in serum-reduced medium as determined by RT-PCR. Data are normalized to GAPDH (n = 3/group). (E-F) Relative protein expression of PDGF-Rα and PDGFRβ in PαS-MSCs stimulated with recombinant FGF2 at indicated concentrations for 18 hours in serum-reduced medium as determined by western blot analysis. Relative intensity of each band was measured and normalized to α-actinin (n = 3/group). (G) Relative mRNA expression of PDGFRα in cultured PαS-MSCs treated for 2 days with PBS, tPA, FGF2, PDGF-BB, or FGF2 and PDGF-BB as determined by RT-PCR. Data are normalized to GAPDH (n = 3/group). (H) WT B6 mice were injected daily intraperitoneally with vehicle, tPA, the tyrosine kinase inhibitor Gleevec, and/or FGFR inhibitor. BMNCs were harvested from mice at day 2 and stained for CD45, Ter119, Sca-1, and PDGFRα and analyzed by flow cytometry. Percentage of PαS-MSCs is given (n = 4/group). (I) Absolute numbers of MSCs in the femur of PBS-, tPA-, EGFR inhibitor–, and PDGFR inhibitor–treated mice was determined by FACS analysis (n = 4/group). Mean ± SEM. *P < .05; **P < .01; ***P < .001 by Student t test or ANOVA with Tukey HSD tests for multigroup analysis. Data are representative of 2 independent experiments.

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