Figure 5.

Foxo3a overexpression attenuated the activity of Wnt/β-catenin signaling by competing with TCF4 for binding to β-catenin. Chondrocytes were co-transfected with miR-29a mimics and pcDNA3.1(+)-Foxo3a. After treatment with 5 (low) or 20 μg/mL (high) TMF for 24 hrs, the mRNA (A) and protein (B) expression were detected by RT-PCR and Western blot, respectively. (C) was the summary of protein bands intensity. (B) The co-immuno-precipitation assay was conducted for detecting the interaction between β-catenin and Foxo3a or TCF4. 10% of chondrocytes protein extracts were used as the input, which was subjected to Western Blot. The remaining protein extracts were subjected to IP by using control goat IgG or β-catenin antibody, followed by IB with anti-Foxo3a, anti-TCF4, and anti-β-catenin. (D) Chondrocytes were co-transfected with miR-29a mimics, pcDNA3.1(+)-Foxo3a, and Topflash or Fopflash luciferase reporters. Transfected cultures were treated with 5 or 20 μg/mL TMF for 24 hrs. The values were indicated as Firefly/Renilla ratio in the column. Data were presented by mean ± SD of 3 replicates. P<0.05 was considered to be statistically significant (*P<0.05, **P<0.01).