Fig. 5

MLN8237 treatment induced IMR32 cell “stemness” enhancement. a IMR32 cells were treated with 0, 0.5 μmol/l, and 2 μmol/l of MLN8237, respectively. Cell samples were collected at 6 h, 12 h, 24 h, 48 h, and 72 h time points. Total proteins were extracted for western blot analysis for Bmi1 protein expression. Gray values were calculated by imageJ software. The data were shown as the mean ± SEM of three independent experiments. *P < 0.05. b CD133⁺ cells were separated from IMR32 by using CD133 MicroBead Kit (Miltenyi Biotec). CD133⁺ cells and CD133⁻ cells were seed in ultra-low adhesion culture dishes to culture for 12 days, respectively. At 5 days, 8 days, 10 days, and 12 days time points, microsphere formation were observed by microscope photography. c At 12 days, cell microsphere were collected and digested to seed into 6-well plate. MLN8237 treated for 48 h and total RNA were extracted. The mRNA levels of Bmi1 and CIAP2 were determined by real time RT-PCR. The data were shown as the mean ± SEM of three independent experiments. *P < 0.05