Fig. 1

RC48 inhibited tumor cell growth in HER2-positive melanoma cells in vitro. A Quantification of HER2 protein expression in A2058 and SK-MEL-28 melanoma cell lines using western blotting. B The subcellular localization of HER2 protein (shown in green) in the A2058 and SK-MEL-28 cells. HER2 is localized to both the plasma membrane and the nucleus. It was determined using immunofluorescence. DAPI (shown in blue) was used to stain nuclei. The images were captured at 200× magnification, with scale bars set at 50 µm. C The A2058 and SK-MEL-28 cells were treated with RC48 and trastuzumab for 72 h. The cell viability was evaluated using the Cell Titer-Glo cytotoxicity assays. D, E The A2058 and SK-MEL-28 cells were treated with indicated concentrations of RC48. The cell confluency (%) was calculated from 0 to 72 h using Incucyte S3 Zoom software based on phase-contrast images. F–G The A2058 and SK-MEL-28 cells were treated with RC48 at concentrations of 0, 1, 3 and 10 μg/mL for 14 days. The area (%) of colonies stained with crystal violet was used to measure the antiproliferative effects. H, I The A2058 and SK-MEL-28 cells were incubated with RC48 at concentrations of 0, 1, 3 and 10 μg/mL for 24 h. Then, the cells were stained with Azide 555 (shown in red) to detect EdU and DAPI (shown in blue). Fluorescence images were obtained and analyzed using a confocal laser scanning microscope at 200× magnification, with scale bars set at 50 µm. The data presented are the means ± SEM from three independent experiments, and statistical significance was determined using an unpaired t-test (*p < 0.05; **p < 0.01; ***p < 0.001 compared with control groups)