Effect of MAL on cell growth and colony formation of HNSCC cell lines. (A) Exogenous MAL expressed in Tca, Tca-M and Tb cells transfected with the pcDNA3.1 vector. Parental cells with empty vector were used as a control. The growth of these cells was analyzed using the CCK-8 kit, and the symbols represent the mean values of triplicate tests (mean ± SD). Western blot analysis indicated the expression of MAL in these cell lines. A t-test was used to show significant differences between the two groups (P < 0.05). (B) To examine the effect of MAL on colony formation, pcDNA3.1-MAL was transfected into these three cell lines. After transient transfection for 24 hours, the cells were harvested and seeded onto 100 mm dishes and cultured in G418 for 14 days. The representative dishes show the inhibitory effect of MAL on colony formation, and the lower graphics show that colony formation was significantly suppressed by MAL as compared with the empty vector control (P < 0.05). All experiments were repeated at least three times. (C) The cell cycle distribution after transient transfection with pcDNA3.1-MAL or an empty vector as assessed by flow cytometry.