Figure 4

Vascular endothelial growth factor production is regulated by Smad4. Knockdown of Smad4 by RNAi increased vascular endothelial growth factor (VEGF) expression at the protein level. Chondrocytes were transfected with siRNA specific for Smad4 or with control (Ctrl) siRNA. (A) siRNA targeting Smad4 efficiently reduced endogenous Smad4 expression at the RNA level. Values are the mean ± standard deviation (SD) of at least three independent experiments. **P < 0.01. (B) Inhibition of Smad4 increased the protein level of VEGF. (C) Densitometric analysis of immunoblot band intensities for Smad4 normalized by GAPDH. Data are mean ± SD of three independent experiments. (D) Densitometric analysis of immunoblot band intensities for VEGF normalized by GAPDH. Data are mean ± SD of three independent experiments. (E) Co-transfection of miR-146a and Smad4 expression plasmid reversed miR-146a-mediated upregulation of VEGF. Relative expression levels of protein shown at the bottom of the bands as normalized by GAPDH level. NC, negative control. (F) Densitometric analysis of immunoblot band intensities for Smad4 normalized by GAPDH. Data are mean ± SD of three independent experiments. (G) Densitometric analysis of immunoblot band intensities for VEGF normalized by GAPDH. Data are mean ± SD of three independent experiments.