RANKL overexpression induces integrin α
expression, which mediates FAK and Akt phosphorylation. (A) LNCaPRANKL cells expressed increased α2 integrin as determined with qRT-PCR. The expression was the highest when the cells were grown in the 3-D suspension that contained ColI. (B) Increased α2 integrin expression, appearing in a RANKL-dependent manner, was confirmed with western blotting. (C) In the upper panels, cell surface expression of α1, α2 and β1 integrins was detected with FACS. Results indicate significantly higher α2 integrin expression in RANKL-overexpressing cells grown in 3-D suspension. In the lower panel, the histogram represents the ratio of the surface integrin protein level of LNCaPRANKL/LNCaPNeo cells quantified as median fluorescence intensity. Each value is the mean ± SD of two independent experiments. (D) Increased α2 integrin is accompanied by higher p-FAK and p-Akt levels as determined by western blotting. For each group, the ratio of p-FAK/FAk and p-Akt/Akt normalized to LNCaPNeo is shown. Blots were cropped to emphasize the relevant bands.