CSB is important for cell survival and in vivo after damage coupled with active transcription. (A) UVs1KOSV and HA-CSB expressing UVs1KOSV cells (Left) and CS1AN and HA-CSB expressing CS1AN cells (Right) were pretreated with or without DRB (20 µM) for 24 h, then irradiated with IR at the indicated dose. Colony forming assays were performed, and the survival fraction is shown. (B) Persistent γH2AX and 53BP1 signals at sites of TA-KR after damage without HR and/or CSB. U2OS TRE cells were treated with the indicated siRNA, then transfected with TA-KR. Cells were exposed to white light for 10 min followed by 48 h of postlight incubation, then fixed and immunostained with anti-γH2AX and anti-53BP1. The percentage of γH2AX and 53BP1 foci postive cells (defined as the fold increase of foci at TA-KR sites compared with background intensity being >2) was quantified. (C) Zebrafish embryos injected with CSB-MO/con-MO or WT were irradiated with or without 5 Gy IR at 6 h postfertilization (hpf). Images of three levels of abnormalities (L1, L2, and L3) at 72 hpf are shown. (D) Percentage of embryos in each of the categories. Error bars indicate the SEM of three independent experiments, and the P values were determined by using Student’s two-tailed t test. (E and F) Western blot of CSB and γH2AX in embryos in D (E) or in siCSB treated 293 cells with or without irradiation (F).
|
