Fig. 5.
G0S2 promotes 53BP1 stability in glioma cells in response to radiation. a WB analyses of effect of G0S2 overexpression on γ-H2AX, Rad51, 53BP1 and PARP expression in response to IR. Arrow, cleaved PARP. b Representative images of 53BP1 staining of glioma LN229 with or without overexpression of G0S2 at 8 h post-IR with 10 Gy. Scale bars: 50 μm. c Quantitative 53BP1 staining of B. Error bars, SD. *, p < 0.05. d G0S2 increased 53BP1 ubiquitination in response to IR. His-Ub was transiently transfected into glioma U87 and LN229 cells with or without overexpression of G0S2. Proteins were pulled down with Ni-NTA beads. 53BP1(Ub)n, polyubiquitinated 53BP1. e The half-life of 53BP1 in LN229 cells with overexpression of G0S2 was prolonged compared with the control. After 8-h post-IR, cells were treated with CHX (10 μg/ml) for indicated times with or without the proteasome inhibitor MG132 (10 μM). f Quantitative 53BP1 protein levels of panel e using image j software. Error bars, SD. *, p < 0.05. g Depletion of 53BP1 with two different shRNAs (sh53BP1–1 and sh53BP1–2) increased G0S2-attenuated γ-H2AX expression in response to radiation in glioma LN229 and U87 cells. h Representative images of γ-H2AX staining of g. Scale bars: 50 μm. i Quantitative γ-H2AX staining of h. Error bars, SD. *, p < 0.05. j Clonogenic survival assay of U87/G0S2 cells transduced with control shRNA (shC) or 53BP1 shRNAs (sh53BP1–1 and sh53BP1–2). Colonies formed by surviving cells 26 days after IR are shown. Error bars, SD. *, p < 0.05. k Survival curves for mice implantated with 5 × 105 cells of U87/G0S2/shC or U87 /G0S2/sh53BP1 and left untreated or given daily dosed 2.5 Gy IR from day 14 to 17 after cell implantation. Arrows, IR treatment times. Data represent two independent experiments with 6 mice per group with similar results. Data in (a to j) represent two to three independent experiments with similar results