Fig. 4: BMP9 attenuates senescence and promotes osteoblast differentiation in MC3T3-E1 cells. | Cell Death Discovery

Fig. 4: BMP9 attenuates senescence and promotes osteoblast differentiation in MC3T3-E1 cells.

From: BMP9 reduces age-related bone loss in mice by inhibiting osteoblast senescence through Smad1-Stat1-P21 axis

Fig. 4

Passage 7 of MC3T3-E1 cells were treated with vehicle as control group. Passage 17 of MC3T3-E1 cells were treated with vehicle or BMP9 for 3 days, respectively. A, B β-galactosidase staining was performed (A) and number of β-gal (+) cells was counted for each group (B) (Scale bar, 100 μm). C Immunofluorescence analysis of γ-H2AX expression (Scale bar, 50 μm). D qPCR analysis of mRNA levels of senescent genes P21 and P53. E, F qPCR analysis of mRNA levels of SASPs. G Alp staining of MC3T3-E1 cells suffered to osteogenic differentiation induction for 7 days with or without BMP9. H, I qPCR analysis of osteoblast differentiation markers in cells suffered to osteogenic differentiation induction for 7 days with or without BMP9. J Western blot analysis of protein levels of Runx2 and Osx in MC3T3-E1 cells suffered to osteogenic differentiation induction for 7 days with or without BMP9. Senescence of MC3T3-E1 cells was also induced by H2O2 treatment for 2 h, then fresh culture medium added with vehicle or BMP9 was replaced for another 3 days. K, L β-galactosidase staining was performed (K) and number of β-gal (+) cells was counted for each group (L). (Scale bar, 100 μm). M Immunofluorescence analysis of γ-H2AX expression (Scale bar, 50 μm). N qPCR analysis of mRNA levels of senescent genes P21 and P53. O, P qPCR analysis of mRNA levels of SASPs. Q ALP staining of control and H2O2-stimulating MC3T3-E1 cells suffered to osteogenic differentiation induction for 7 days with or without BMP9. R, S qPCR analysis of osteoblast differentiation markers in control and H2O2-stimulating MC3T3-E1 cells suffered to osteogenic differentiation induction for 7 days with or without BMP9. T Western blot analysis of protein levels of Runx2 and Osx in control and H2O2-stimulating MC3T3-E1 cells suffered to osteogenic differentiation induction for 7 days with or without BMP9. Data presented as mean ± SD. n = 3 biological replicates. One-way ANOVA was used for comparison among multiple groups. *P < 0.05; **P < 0.01; ***P < 0.001. Ns, no significance.

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