Gls2 suppressed cancer cell growth and induced cell cycle arrest. (A) Ectopic expression of Gls2 in human liver and colon cancer cells. SMMC-7721 and HCT116 cells were transiently transfected with either empty vector or 3 × Flag-tagged Gls2 expressing vector and the expression level of Gls2 was measured by western blot. β-actin served as a loading control. (B) Cell proliferation ability was measured by colony formation assay, which was performed as described in Methods. (C) Overexpression of Gls2 inhibited cancer cell proliferation measured by MTS assay. The values obtained from transfected and control cells represent mean ± SD of three independent experiments. *p < 0.05 between the two groups were calculated using individual Student’s t-test. (D) Changes of the cell cycle profiles in cancer cells after Gls2 overexpression were determined using flow cytometry, with representative histograms shown for each treatment. (E) Changes of cell cycle regulators (phospho-cdc25 (Ser216), p21 and cyclin D1) were determined using western blotting. β-actin was used as the internal control.