Fig. 2

Sequence-dependent synergistic effect on tumor cell metastasis inhibition in EGFR-mutant NSCLC cell lines. A Wound healing assays were performed in H1975 and HCC827 to determine the effects of different drug treatments on cell migration.10 nM pemetrexed and 10 nM aumolertinib were used for H1975. 5 nM pemetrexed and 5 nM aumolertinib were used for HCC827. The crosses of wounding lines and horizontal lines were observed at 0 h, 24 h and 48 h post drug administration. The cell migration rates were quantified and compared (n = 3). B Transwell assays were further performed in H1975 and HCC827 to prove the synergetic effect of P-A on tumor metastasis inhibition. 10 nM pemetrexed and 10 nM aumolertinib were used for H1975. 5 nM pemetrexed and 5 nM aumolertinib were used for HCC827. Representative images were shown and the average number of migrating or invasive cells in each group was counted (n = 3). C The EMT-related proteins, β-catenin, vimentin and snail, were analyzed by western blot after P-A sequence treatment or single drug treatment. Con represents control; P represents pemetrexed; A represents aumolertinib; P + A represents concomitant treatment with pemetrexed and aumolertinib; P-A represents pemetrexed treatment followed by aumolertinib treatment; A-P represents aumolertinib treatment followed by pemetrexed treatment. All of the data were expressed as the mean ± SEM, * p < 0.05, ** p < 0.01 and *** p < 0.001