Figure 4.

Thiostrepton suppresses tumor sphere- and colony-formation potentials of HCT-15 and HT-29 cells as well as the expression of factors involved in stemness maintenance. (a) HCT-15 and HT-29 cells were cultured in defined media supplemented continuously with DMSO (Veh), oxaliplatin (Oxa, 25 μM), or thiostrepton (Thio, 5 μM). Seven days later, spheres stained by MTT were photographed ( × 40) and counted using software. *P<0.05 when compared with the DMSO-treated cells by Student's t-test. (b) Clonogenic assays were performed using cells exposed previously to DMSO (Ctrl), oxaliplatin, or thiostrepton for 6 h as described in ‘Materials and Methods'. Ten days after seeding, colonies were stained by crystal violet and their numbers were counted using Colony 1.1 software. *P<0.05 when compared with the DMSO-treated cells by Student's t-test. Total lysates prepared from (c) Snail OE and (d) r29 cells after being treated with the indicated doses of thiostrepton for 48 h were subjected to western blot analyses using antibodies against CD44, ALDH1, Oct4, Nanog, Bmi1, and Twist as probes. Tubulin signal served as a loading control