Figure 6. PDGF-BB stimulates p-PDGFRβ/p-ERK/RUNX2 signaling in PCs to activate multiple key osteoblast differentiation genes.

(A–D) Primary mouse brain PCs were exposed to recombinant mouse PDGF-BB at a concentration of 30 ng/mL for shorter time periods (A and B) and longer time periods (C and D). Expression of the indicated proteins was detected by Western blot analysis (A and C). The relative intensities of the proteins were quantified using ImageJ (B and D). (E) Primary mouse brain PCs were exposed to recombinant mouse PDGF-BB at a concentration of 30 ng/mL for 4 and 8 hours. Western blot analysis of RUNX2 and OPN expression. (F) Double-immunofluorescence staining of frozen brain tissue sections from 3- and 22-month-old male mice using antibodies against CD31 and p-PDGFRβ. n = 3. (G) Primary mouse brain PCs were subjected to an 8-hour treatment with recombinant mouse PDGF-BB. Double immunocytochemical staining was performed using antibodies against PDGFRβ and OPN. Boxed areas are shown at a higher magnification in the corresponding panels to the right. n = 3. (H) Primary mouse brain PCs were treated with 30 ng/mL PDGF-BB for 4 and 8 hours. qRT-PCR analysis was conducted to assess the expression levels of osteogenic marker genes, including Spp1, Runx2, and Sp7. n = 6. (I) Primary brain astrocytes were treated with 30 ng/mL PDGF-BB for 4, 8, and 16 hours. qRT-PCR was conducted to assess the expression levels of osteogenic markers, including Sp7 and Bglap. n = 3. Scale bars: 100 μm (F and G). Relative fold-change results are shown as the mean ± SD. *P < 0.05, **P < 0.01, and ****P < 0.0001, by ordinary 1-way ANOVA for multiple-group comparisons (H and I).