Fig. 4: The synergistic antitumor effects of RC48 and/or GEM on HER2-positive CRC cells.

A Cytotoxicity of RC48 combined with GEM in P53R, RKO, HCT116 and COLO205 cells were determined by CellTiter-Glo® cytotoxicity assays. The dose of RC48 monotherapy was set as 0, 0.25, 0.5, 1, 2, 4 and 8 μg/mL in P53R, RKO, HCT116 and COLO205 cells, while GEM was set as 0, 3, 10, 30, 100 and 300 nM in P53R, RKO and HCT116 cells, and GEM was set as 0, 0.03, 0.1, 0.3, 1 and 3 μM in COLO205 cells. B, C P53R, RKO, HCT116 and COLO205 cells were treated with RC48, GEM and their COM for 14 days. The proliferative ability of CRC cells was investigated via colony formation assays. D, E P53R, RKO, HCT116 and COLO205 cells were treated with RC48, GEM, or their COM for 48 h, then cell proliferation was determined by EdU assay. F, G P53R, RKO, HCT116 and COLO205 cells were treated with RC48, GEM, or their COM for 48 h. The cells were stained with an anti-Annexin V-FITC antibody and PI for apoptosis analysis by flow cytometry. Annexin V-FITC-positive cells were defined as apoptotic. H, I P53R, RKO, HCT116 and COLO205 cells were treated with RC48, GEM, or their COM for 48 h.The induction of cell cycle analysis in P53R, RKO, HCT116 and COLO205 cells was detected by flow cytometry. Data represent the mean ± SEM of at least three independent experiments and statistical significance was assessed by unpaired T-test (*p < 0.05; **p < 0.01; ***p < 0.001).