Reintroduction of MLK4-WT reduces anchorage-independent colony growth, proliferation and tumor growth in vivo. A, HCT15 and HT115 were seeded in soft agar. Expression of MLK4-WT was induced by tetracycline and after 2 weeks cells were stained with crystal violet. B, HCT15 and HT115 were seeded in 96-well plates. Expression of MLK4-WT was induced by tetracycline for 2 days. Plates were subjected to BrdU assay. Error bars indicate ±SEM from three independent experiments performed in triplicate (n=9). C, Expression of MLK4-WT in HCT15 and HT115 was induced by tetracycline for 4 days. After that time cells were lysed and WCL were analyzed by western blot. D, HCT15 and HT115 were seeded in 96-well plates. Expression of MLK4-WT was induced by tetracycline for 4 days. Plates were subjected to caspase3/7 activity apoptosis assay. Error bars indicate ±SEM from three independent experiments performed in triplicate (n=9). E and F, HCT15 and HT115 were engrafted in nude mice. MLK4-WT expression by doxycycline induction started on the day of injection. Error bars indicate ±SEM (n = 8 mice/group).