TNF-α promotes the malignant transformation of human normal intestinal stem cells NCM460s. NCM460s cells were treated with 1 ng/ml TNF-α for 24 h, and then compared with NCM460 adherent cells. (A) TNF-α increased the survival rates of NCM460s cells in various concentrations of 5-FU by the CCK-8 assay. (B) TNF-α increased the capacity of colony formation of NCM460s cells in (a) colony formation assay and (b) soft agar assay. Scale bar, 100 µm. (C) TNF-α induced changes of the cell cycle distribution in NCM460s cells as revealed by flow cytometry. (D) TNF-α increased the (a) migratory and (b) invasive number of NCM460s cells in the Transwell assay. Scale bar, 50 µm. (E) TNF-α reduced the protein level of E-cadherin and increased the protein levels of N-cadherin, Vimentin and Snail as revealed by western blotting. (F) TNF-α induced an increased proportion of surface marker gene CD133+ in NCM460s cells as revealed by flow cytometry. *P<0.05, **P<0.01, ***P<0.001 compared with NCM460s cells without TNF-α stimulation. TNF, tumor necrosis factor; NCM460s, NCM460 spheroid; ns, not significant.