Fig. 8: BMP9 inhibits osteoblast senescence through Smad1-Stat1-P21 axis.

A Relative Stat1 promotor luciferase activity after treated with vehicle or BMP9 for 24 h (n = 3 biological replicates). B qPCR analysis of mRNA levels of Smad1, Smad5, and Smad9 in vertebrae of 20-month-old mice treated with AAV-CON or AAV-BMP9 (n = 6). C MC3T3-E1 cells were co-transfected with Stat1 promoter reporter plasmid and empty vector or Smad1-expressing vector for 36 h. The relative Stat1 promotor luciferase was examined (n = 3 biological replicates). D, E qPCR analysis of mRNA levels of Stat1 (D) and P21 (E) of control and H₂O₂-induced MC3T3-E1 cells treated with BMP9 or BMP9 + LDN193189 for 3 days (n = 3 biological replicates). F Western blot analysis of protein levels of Stat1, p-Smad1/5/9, and total Smad1 in control and H₂O₂-induced MC3T3-E1 cells treated with BMP9 or BMP9 + LDN193189 (n = 3 biological replicates). G Immunofluorescence analysis of P21(+) Stat1 (+) cells (Scale bar, 50 μm). H, I qPCR analysis of mRNA levels of SASPs in control and H₂O₂-induced MC3T3-E1 cells treated with BMP9 or BMP9 + LDN193189 (n = 3 biological replicates). J qPCR analysis of mRNA levels of P21 and Stat1 in vertebrae (n = 5). K qPCR analysis of mRNA levels of SASPs in vertebrae (n = 5). L qPCR analysis of mRNA levels of osteoblastic markers in vertebrae (n = 5). M, N Representative images derived from micro-CT analysis, including 2D image construction of distal femur (M) and 3D image reconstruction of trabecular bone of distal femur (N). O–S Quantitative analysis of the vBMD (O) and microarchitecture analysis of trabecular bone by micro-CT: Trabecular BV/TV (P), Tb.N (Q), Tb.Sp (R) and SMI (S) (n = 5). Data presented as mean ± SD. One-way ANOVA was used for comparison among multiple groups. *P < 0.05; **P < 0.01; ***P < 0.001. Ns, no significance.