The combination of SHP099 and AZD6244 exhibited synergistic effects in DTC cells with various genetic backgrounds. (A) and (B) Four DTC cell lines were treated with DMSO (ctrl), SHP099 (10 µM), AZD6244 (1 µM), or both drugs (Comb). The colony formation (A) and cell viability (B) assays were performed on days 10 and 0-8, respectively. *P<0.05, **P<0.01, ***P<0.001, compared with the Ctrl group, @P<0.05, @@P<0.01, @@@P<0.001, compared with the Comb group, ANOVA. All data are presented as the mean ± SD of three independent experiments. Compared with the single agent, the combination of SHP099 and AZD6244 exhibited a more significant inhibitory effect on the progression of the four DTC cell lines. (C) The combination of SHP099 and AZD6244 significantly induced G1 phase arrest in the four DTC cell lines. Cell cycle distribution was assessed by flow cytometry. The percentage of cells in the G1, S and G2/M phases was plotted. (D-F) Colony formation assays (D), cell viability assays (E) and cell cycle distribution analysis (F) revealed that knockdown of SHP2 expression exhibited a similar effect as SHP099 treatment after combination with AZD6244.