Fig. 2

RC48 induced cell cycle arrest, apoptosis and inhibited cell motility in melanoma cells. A The A2058 and SK-MEL-28 cells were treated with RC48 at concentrations of 0, 1, 3, and 10 μg/mL for 48 h. The cells were stained with an anti-Annexin V-FITC antibody and PI for apoptosis analysis using flow cytometry. The percentage of Annexin V⁺ PI⁻ and Annexin V⁺ PI⁺ cells (apoptosis cells) is shown in right bar chart. B The protein levels of apoptosis markers, including poly ADP-ribose polymerase (PARP), cleaved PARP, and Mcl-1 were detected using western blot analysis. β-tubulin was used as a loading control. C A2058 and SK-MEL-28 cells were treated with RC48 at concentrations of 0, 1, 3, and 10 μg/mL. The induction of cell cycle changes was analyzed using flow cytometry. The percentage of cells in each phase of the cell cycle was shown in a bar chart. D The protein levels of cell cycle regulators, CDK2 and CDK4, were detected using western blot. GAPDH was used as a loading control. E, F A2058 and SK-MEL-28 cells were treated with RC48 at concentrations of 0, 1, 3, and 10 μg/mL for 24 h. Cell motility was assessed using Transwell migration (E) and invasion (F) assays. G A2058 and SK-MEL-28 cells were treated with different concentrations of RC48 for 24 h. The expression of epithelial–mesenchymal transition (EMT) markers, including N-cadherin, SNAI1, and ZEB1, was examined using western blot analysis. GAPDH was used as a loading control. The data presented represent the mean ± SEM of at least three independent experiments. The statistical significance was assessed using an unpaired t-test (*p < 0.05, **p < 0.01, and ***p < 0.001 compared with control groups)