Fig. 5

Antitumour effect of the combination of trametinib and simvastatin. a Cell proliferation assays at day 3 for the HCT116 and SW480 cells cultured with simvastatin (10 μM) or DMSO in the presence or absence of 50 nM trametinib. b, c Clonogenic assays and qualitative analysis of the HCT116 and SW480 cells cultured with DMSO or 10 μM simvastatin (or/and 50 nM trametinib) at day 7. d Quantitative real-time PCR analysis of ERRα and IDH3A, c-Myc, cyclin D1 in the HCT116 cells treated with 10 μM simvastatin (or/and 50 nM trametinib) for 48 h. GAPDH was used as a control. e WB for IDH3A, c-Myc, cyclin D1 and Bax in the HCT116 and SW480 cells treated with 10 μM simvastatin (or/and 50 nM trametinib) for 48 h. f Dual luciferase reporter gene assay of the SW480 cells treated with 10 μM simvastatin (or/and 50 nM trametinib) for 48 h. g, h Flow cytometric analysis of the cell cycle of the HCT116 and SW480 cells treated with DMSO or 10 μM simvastatin (or/and 50 nM trametinib) for 48 h. i, j Tumour formation assays in the nude mice subcutaneously injected with HCT116 cells (1× 10^6). When the tumours reached 3 mm in diameter, the mice were orally with DMSO, simvastatin (30 mg/kg) or/and trametinib (1.5 mg/kg) daily. The tumour sizes were measured after 2 weeks. The graph shows the relative tumour volume and weight of each group (n=6 animals for each group). The data presented as the mean±SD, n=3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 using Student’s t test (two-tailed). k Representative immunohistochemical staining results for ERRα, IDH3A, c-Myc and Cyclin D1 in xenograft tumour tissues. l The graph shows the immunoreactivity scores of ERRα, IDH3A, c-Myc and Cyclin D1 in each group (n=6 animals for each group)