Fig. 6

AnxA6 SUMOylation promotes gefitinib-mediated inhibition of epithelial cancer cell growth and migration. A-C AnxA6 knockdown impaired its ability of enhancing anti-proliferation and anti-migratory properties of gefitinib in HeLa cells. The CCK8 (A) and colony-forming (B) assays were used to measure cell proliferation ability of gefitinib-treated HeLa cells. Cells were grown in DMEM media containing 0.1% (v/v) FBS and 100 ng/ml EGF. Quantification of colonies was shown at right, which was calculated as the proportion of cell clone number relative to HeLa. C Cell motility was determined by wound healing assay in gefitinib-treated HeLa cells. The bar graphs showed cell migration distances, which were calculated relative to the initial distance before migration (right). D-F The K299 mutation of AnxA6 impaired its ability of enhancing anti-proliferation and anti-migratory properties of gefitinib in A431 cells. The CCK8 (D) and colony-forming (E) assays were used to measure cell proliferation ability of gefitinib-treated A431 cells. Cells were grown in RPMI-1640 media containing 0.1% (v/v) FBS and 100 ng/ml EGF. Quantification of colonies was shown at right, which was calculated as the proportion of cell clone number relative to A431. F Cell motility was determined by wound healing assay in gefitinib-treated A431 cells. The bar graphs showed cell migration distances, which were calculated relative to the initial distance before migration (right). G The K299 mutation of AnxA6 impaired its ability of enhancing gefitinib-mediated inhibition of EGFR-ERK signaling. The A431, A431-AnxA6, and A431-AnxA6^K299R cells were treated with 10 μM gefitinib for 24 h, and followed with 100 ng/ml EGF treatment for 3 min. The levels of pY-EGFR, EGFR, pERK1/2, ERK1/2 and AnxA6 were determined by western blotting. Data were represented as the mean ± SD of three separate experiments. ns, no statistical; *p < 0.05;**p < 0.01; ***p < 0.001