Fig. 5: BCgel facilitates osteogenesis by activating mechanical Itgb1-mediated focal adhesion signaling and the subsequent force-sensitive transcription factor Runx2.

A, B Immunofluorescent staining of Fak&Itgb1 in MSCs incubated with or without BCgel for 24 h was analyzed by CLSM. Double positive signals were quantified by Image J with colocalization factor (Pearson’s R value) (B). (n = 6 independent replicates, 10 counts per sample, means ± SD, p value calculated by two-tailed Student’s t test). C The quantification of western blotting analysis of Itgb1, Fak, active Rhoa (ARhoa), Rock2 and c-Jun in MSCs incubated with or without BCgel for 24 hours. (n = 3 independent replicates, means ± SD). D, E Gene Set Enrichment Analysis (GSEA) was performed to compare the Ctrl and BCgel-treated MSCs in terms of the focal adhesion assembly signaling pathway (D), integrin-mediated signaling pathway, cell adhesion related to integrin signaling pathway, osteoblast development signaling pathway, and Runx2 bone development signaling pathway (E). (n = 3 biological replicates in each group). F Runx2 expression in the nucleus and cytoplasm of MSCs incubated with vary concentrations of BCgel were measured by western blotting analysis. G, H The gray value of the gel is utilized for quantitative analysis of Runx2 in both the nucleus and karyoplasm. (n = 3 independent replicates, means ± SD, p value calculated by two-tailed Student’s t test, **p = 0.0012, ***p < 0.001, ****p < 0.001). I, J MSCs with or without Itgb1 knockdown were incubated with BCgel for 24 hours. Runx2 expressions were subsequently detected by western blotting analysis and quantification (I). The quantification of western blotting analysis of Fak, active Rhoa (ARhoa), Rock2 and c-Jun was shown in (J). (n = 3 independent replicates, means ± SD, p value calculated by two-tailed Student’s t test). K–M The osteogenic differentiation of MSCs was assessed on day 7 after osteogenic induction by Alp staining (K), Alp activity analysis (L), and western blotting (M). (n = 6 independent replicates for Alp activity analysis, means ± SD, p value calculated by two-tailed Student’s t test; n = 3 independent replicates for western blotting). N MSCs with or without Itgb1 knockdown were incubated with BCgel, and western blotting analysis was performed on day 7 after osteogenic induction. (n = 3 independent replicates).