Fig. 3

Ubiquitin-mediated AnxA6 degradation in EMT cells. (A) Cell morphology changes of HepG2 cells under TPA exposure. HepG2 cells were treated with 100 ng/mL TPA for 0, 4, 8, 12 days, and the images were captured with a microscope. Scale bars, 20 μm. (B) Cell migration ability was increased in TPA-induced EMT cells. Cell migration of the TPA-induced HepG2 cells was analyzed using transwell assays. Images were captured with a microscope, and three random fields were selected to count the number of migration cells. *p < 0.05, **p < 0.01. Scale bars, 20 μm. (C) EMT biomarkers were dynamically expressed in TPA-induced cells at different treatment days. HepG2 cells were treated with 100 ng/mL TPA for 0, 4, 8, 12d, and cell proteins were collected for western blot with indicated antibodies (left). Quantification of protein levels of EMT markers in TPA-induced HepG2 cells. Data were represented as the mean ± SD of three separate experiments (right). ns, no statistical; *P < 0.05; **P < 0.01; ***P < 0.001. (D) Cell endogenous AnxA6 protein level was a time-dependent decrease in response with cycloheximide (CHX) incubation. The HepG2 cells were incubated with 100 μg/mL CHX for 0, 2, 4 and 8 h, then cell lysates were used to detect target protein level with anti-AnxA6 and anti-β-actin antibodies. *P < 0.05. (E) AnxA6 was validated to degrade via ubiquitination in HepG2 cells under 20 μM MG132 exposure for 12 h. Cell lysates were extracted to concentrate AnxA6 by IP and detected by anti-AnxA6 or anti-ubiquitin antibodies. (F) The levels of AnxA6 ubiquitination were increased in EMT-featured cells. The EMT-featured cells were treated with 20 μM MG132 for 12 h, then the lysates were IP to capture ubiquitin protein and analyzed by immune-blotting with AnxA6 antibody. Ub_(n)-AnxA6, Ubiquitinated AnxA6