Fig. 5

AnxA6 SUMOylation impedes cell proliferation and migration in EGFR-overexpressing cells by inactivating EGFR-ERK phosphorylation. A The the silencing of AnxA6 weakened the binding of PKCα with EGFR, thereby promoting the EGFR-ERK phosphorylation in HeLa cells. AnxA6-knockdown cell model in HeLa were generated by infecting with lentiviral expressing shRNA targeting to AnxA6, and cells were subjected to serum deprivation for overnight and followed with 100 ng/ml EGF treatment for 3 min. Cell lysates were IP to capture EGFR and subsequently immunoblotted with indicated antibodies. B-D AnxA6 knockdown promoted HeLa cell proliferation and migration.Cell proliferation (B-C) and migration (D) were respectively measured on HeLa cells and HeLa-AnxA6(KD) cells. Cells were grown in DEME media containing 0.1% (v/v) FBS and 100 ng/ml EGF. Representative colony formation images (left) and quantification of colonies (right) that compared with HeLa. Representative wound healing images (left) and the calculated cell migration distances (right). E The K299R mutation of AnxA6 impaired AnxA6 SUMOylation level, which weakened the binding of AnxA6 with EGFR and promoted the EGFR-ERK phosphorylation in A431 cells. A431 stable cells were generated by using lentiviral infections with indicated plasmids, and cells were subjected to serum deprivation for overnight and followed with 100 ng/ml EGF treatment for 3 min. Cell lysates were IP to capture Flag-tagging AnxA6 and subsequently immunoblotted with indicated antibodies. F–H The K299 mutation of AnxA6 impaired its suppression activity on A431 cell proliferation and migration. Cell proliferation (F-G) and cell migration (H) were respectively measured on A431 cells, A431-AnxA6 cells and A431-AnxA6^K299R cells. Cells were grown in RPMI-1640 media containing 0.1% (v/v) FBS and 100 ng/ml EGF. Representative colony formation images (left) and quantification of colonies (right) that compared with A431. Representative wound healing images (left) and the calculated cell migration distances (right). I The K299R mutation of AnxA6 impaired its activity in suppressing EGF-induced cyclin D1 expression. Cells (A431, A431-AnxA6, and A431-AnxA6^K299R) were starved overnight and then treated with 100 ng/ml EGF for 0–6 h as indicated. Ordinate value, relative cyclin D1 expression (fold of t min/0 min) means the ratio of cyclin D1 between t min and 0 min. Data were represented as the mean ± SD of three separate experiments. *p < 0.05;**p < 0.01; ***p < 0.001. S-Flag-AnxA6: SUMOylated Flag-tagging AnxA6, IP: immunoprecipitation, IB: immunoblot, Input: same account of cell lysate to load