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. 2021 Feb 15;23:1345–1359. doi: 10.1016/j.omtn.2021.02.008

Figure 5.

Figure 5

PI3K/AKT signaling is involved in IBSP-regulated osteoblast differentiation

(A) Transfection of agomiRNA-92a-3p, antagomiRNA-92a-3p, agomiRNA-NC, antagomiRNA-NC, and Lipofectamine 3000 control (200 μm) in MC3T3-E1 cells for 48 h. Western blot detection of the expression of PI3K and AKT after transfection. (B) Western blot detection of IBSP, p-PI3K, and p-AKT levels after transfection with siRNA-NC, siRNA-IBSP, and controls for 48 h. (C–G) Transfection with Lipofectamine 3000, siRNA-NC, siRNA-PI3K, or siRNA-AKT for 48 h. (C) Protein-expression levels of p-AKT, AKT, p-PI3K, and p-PI3K using western blotting. (D–G) qPCR analysis of Col1a1 (D), ALP (E), OCN (F), and Runx2 (G) expression in bone precursor cells. (H) Western blot analysis of Col1a1, ALP, OCN, and Runx2 levels 48 h after transfection. (I) Alizarin red staining of MC3T3-E1 cells 3 weeks after transfection. (J) ALP staining of MC3T3-E1 cells 2 weeks after transfection. n = 3; data are presented as mean ± SD (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; # no significance).