Figure 4.

Knockdown Sirt1 inhibits BMP14 induced tenogenic differentiation of BMSCs. (A) Sirt1 protein and mRNA levels were determined by western blot analysis and RT-qPCR at 48 h post infection with shNC or shSirt1 lentivirus. β-actin was used as the loading control. (B) The mRNA expression collagen I, collagen III and Scx and (C) the protein expression of collagen I, TNMD and Scx were detected in BMSCs infected with shNC or shSirt1 using RT-qPCR and western blot analysis, respectively with or without 50 ng/ml BMP14 treatment for 48 h. (D) Fixed BMSCs were stained with Scx antibodies and Alexa Fluor 488 goat anti-rabbit secondary antibodies (green) and DAPI (blue). Scale bar, 200 µm. (E) PPARγ acetylation in BMSCs was analyzed using immunoprecipitation with anti-PPARγ antibodies and immunoblotting with anti-Acetyl-Lys and anti-PPARγ antibodies. The total cell lysate was immunoblotted with anti-sirt1 and anti-β-actin antibodies. Each bar represents the mean ± standard error of the mean. The results were repeated in three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. the control. BMSC, bone marrow mesenchymal stem cell; Sirt1, sirtuin 1; NC, negative control; sh, short hairpin; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; BMP, bone morphogenetic protein; Sirt1, Sirtuin1; Scx, scleraxis; TNMD, tenomodulin; PPARγ; peroxisome proliferator-activated receptor γ; IP, immunoprecipitation; IB, immunoblotting.