Fig. 4

CD133 regulates intercellular ROS via upregulation of GSH levels and leads to resistance to anti-cancer therapies. a Huh7 was transfected with siCont and siCD133, and their efficiency was examined by western blot analysis. b For the measurement of ROS levels, Huh7-siCont and Huh7-siCD133 cells were treated with the indicated concentrations of MTX and cisplatin for 12 h and 5 Gy of radiation. c Huh7-siCont and Huh7-siCD133 cells were treated with the indicated concentrations of MTX and cisplatin for cell survival analysis. After treatment for 48 h, nuclei were stained with Hoechst33342 and counted. For the colony-forming assay, Huh7-siCont and Huh7-siCD133 cells were treated with 1, 2, or 4 Gy of radiation. Cells were allowed to grow for 14 days, stained with trypan blue, and scored. Data represent values ± SD from three independent experiments relative to the value for control. d LCSC spheroids were reattached on a 384-well culture plate and GSH levels were determined by ThiolTracker™ Violet staining using the HCS System. e CD133 levels, ROS accumulation, and GSH were investigated by flow cytometry in Huh7-siCont and Huh7-siCD133. Each value of Huh7-siCD133 was normalized to Huh7-siCont. *p < 0.05. **p < 0.005