Figure 6.

BMI1 is required for GBM cell radioresistance. A, B, Scramble or shBMI1#3 virus-infected GBM cells were irradiated (3 Gy) and analyzed by FACS 1, 3, or 16 h later. Single-cell suspensions were processed for PH3 labeling (A) or Ki67 labeling and BrdU incorporation (B). In A, PI was added just before FACS acquisition. Values are the percentages of PH3-positive cells. The cell lines used were GBM2802 and GBM0611. In B, BrdU was added to cell cultures 90 min before analysis. The cell line used was GBM2802. Values are the percentages of cells in the respective quadrants. Data are representative of two independent experiments. C–F, GBM cells infected with scramble or shBMI1#3 lentivirus were irradiated with five different radiation doses. BMI1 depleted GBM cells show increased radiation sensitivity as determined by MTT assay (cell lines used: GBM2802, GBM0410, and GBM0811) (C), population doubling (cell lines used: GBM2802 and GBM0811) (D), and CFU assay (E, F). In E (top middle panel), arrowheads indicate surviving colonies. Arrowheads in Eii indicate senescent cells. The EC₅₀ of each cell line was determined using various radiation doses and represented in F when the number of colonies with more than five cells was equivalent to 50% of the total number of colonies in nonirradiated controls. Data are mean ± SEM (n = 3, *p < 0.05, **p < 0.01). Scale bars in E: 1 mm (left and middle panels); 50 μm (right panels).