Recognition and repair of DNA double-strand breaks were inhibited <i>via</i> DEC2 in hypoxic pre-conditioned cells.

<p>Phosphorylate histone H2AX (γH2AX) levels after bleomycin or irradiation treatment in HSC-2 cells with or without pre-conditioning under hypoxia were evaluated with immunoblotting analysis. (A), (B) HSC-2 cells were cultured under normoxic or hypoxic conditions for 24 hours; afterwards, the cells were treated with 500 ng/mL of bleomycin or 5 Gy of γ-ray irradiation, and then the cells were cultured for the indicated lengths of time under normoxic conditions. (C), (D) HSC-2 cells were transfected with either non-specific (siNS) or DEC2-specific (siDEC2) siRNA, and then incubated under hypoxia for 24 hours before irradiation. After re-oxygenation, cells were treated with 500 ng/mL of bleomycin or 5 Gy of γ-ray irradiation. Thereafter, the cells were cultured for the indicated length of time under normoxic conditions. Anti-γH2AX, anti-H2AX and anti-β-actin were used for specific detection of each protein, and representative images are shown. Relative expression levels of γH2AX were calculated with H2AX expression as the denominator for each time point, and are shown in bottom panels. Columns are the mean of three independent experiments; bars, SD.