Fig. 3

Synergistic inhibition of OC Cell Proliferation in vitro by RC48 and CM. A A2780, OVCAR-3, SK-OV-3 and HO-8910PM cells were treated with various concentrations of RC48, CM, or their combination (COM) for 72 h. Cell viability was detected using Cell Titer-Glo cytotoxicity assays. B, C The proliferation of A2780, OVCAR-3 cells treated with vehicle control (VEH), 4 μg/mL RC48, 2.5 μM CM, or COM for 60 h was observed using the Incucyte real-time cell analysis system. Images captured at 100 × magnification, respectively. Scale bars = 400 µm. D, E A2780 and OVCAR-3 cells were treated with 4 μg/mL RC48, 2.5 μM CM, or COM for 24 h, and cell proliferation was determined using the EdU assay. Images were captured using a laser scanning confocal microscope. Images were captured at 100 × magnification (C) and 400 × magnification (D), with scale bars = 400 µm and scale bars = 20 µm, respectively. F, G The anti-proliferative effects were determined by assessing the area of colonies stained with crystal violet. H, I A2780 and OVCAR-3 cells were treated with 4 μg/mL RC48, 2.5 μM CM, or COM for 48 h. Apoptosis was analyzed by staining the cells with Annexin V-FITC/PI and flow cytometry. J, K Western blotting was performed to detect c-Myc, and MCL-1 levels as indicators of apoptotic cell death. GAPDH was used as loading control. The experiments were conducted in triplicate. Data represent the mean ± SEM of three independent experiments. Statistical significance was assessed using one-way ANOVA with Bonferroni post hoc test (*p < 0.05; **p < 0.01; ***p < 0.001)