miR-566 regulated the formation of the β-catenin/HIF-1α complex and sensitized glioma cells to nimotuzumab therapy. (A) Freshly isolated cell lysates (U87 and LN229 either infected with lenti-AS-566 or left untreated) were used to immunoprecipitate β-catenin or HIF-1α with specific rabbit antibodies. Rabbit whole immunoglobulin (IgG) was used as a control antibody for immunoprecipitation assays. The immunoprecipitated complexes were subjected to Western blot analysis with specific antibodies against β-catenin and HIF-1α as indicated. (B) A schematic diagram showed the miR-566 modulated EGFR pathway through VHL/β-catenin/HIF-1α. (C-F) U87 and LN229 cells were treated with nimotuzumab (100 μg/ml) as indicated, and after 24 h, cells were infected with lenti-AS-566 or were untreated. Cell proliferation (C), cell cycle distribution (D), in vitro invasion (E) and apoptosis (F) were evaluated 4 d after lentiviral infection. The data in all panels represent the mean ± SD. *, P < 0.05.