Figure 5. EPB41L3 suppresses EMT and decreases the protein expression of Snai1. (A) Western blot results of EMT markers in U2OS and MG63 cells stably expressing shNC or shEPB41L3. (B) Quantitative RT-PCR analysis suggested that EPB41L3 knockdown did not increase EMT-TFs mRNA expression including Snai1, Slug, and Twist1. (C) Spearman’s correlation analysis showed an inverse correlation between Snai1 and EPB41L3. (D) Western blot results showed that EPB41L3 knockdown increased Snai1 protein expression. (E) EPB41L3 knockdown enhanced Snai1 protein stability. U2OS shNC and shEPB41L3 cells were treated with 20 μM of CHX and harvested at indicated time points for immunoblotting analysis. The graph showed the relative Snai1 protein expression level (Snai1/β-actin) based on the band intensity from the gels. Snai1 protein level at 0 hour time point of CHX treatment was set as 1. (F) Schematic diagram of the proposed mechanism for regulatory roles of EPB41L3 on metastasis in osteosarcoma. CHX, cycloheximide.