Figure 7.
BMI1 overexpression improves DNA DSB response, DNA repair efficiency and radioresistance of human NSCs. A, B, Human NSCs overexpressing BMI1-GFP (BMI1tg) or GFP (GFPtg) were irradiated (3 Gy), fixed 1 h after IR, and costained with either γH2AX or pATM and H3K27me3 antibodies. γH2AX or p-ATM colocalized with H3K27me3 at discrete foci (arrowheads) only in BMI1tg cells. C, BMI1 overexpression enhances the DNA damage response and inhibits RNF8 focal accumulation. NSCs were treated as in A and B and labeled with γH2AX, pATM, and RNF8 antibodies. Deconvolution images were acquired with a Leica DMRE fluorescence microscope and the OpenLab 3.1.1 software. D, BMI1 overexpression accelerates the kinetic of DNA repair. Cells were isolated at different time points after IR (3 Gy) and protein extracts were immunoblotted with p-ATM, γH2AX, and β-actin antibodies. β-Actin was used as loading control. E, BMI1tg or GFPtg NSCs were irradiated at different doses and analyzed for metabolic activity using the MTT assay 24 h after IR. Data are mean ± SEM (n = 3, *p < 0.05, **p < 0.01). F, BMI1tg cells are radioresistant compared to GFPtg cells. Cell cultures were analyzed for the level of apoptosis using the AnnexinV/7-AAD labeling before and 16 h after IR.