FIGURE 4.
ITC-induced γ-H2AX signal is Top2β-dependent in primary MEFs. Quiescent primary Top2β+/+ and top2β−/− MEFs were treated with PEITC (A), BITC (B), SFN (C), and selenite (D) for 2 h followed by immunoblotting analysis using anti-γ-H2AX and anti-α-tubulin antibodies. As controls, quiescent MEFs were also treated with VP-16 (100 μm) or hydrogen peroxide (150 μm) under the same conditions. E, quiescent primary Top2β+/+ MEFs were treated with dexrazozane (ICRF-187) for 24 h to induce Top2β down-regulation. Cells were then treated with BITC (5 μm), PEITC (10 μm), SFN (20 μm), VP-16 (100 μm), or H2O2 (250 μm) for 2 h. The levels of γ-H2AX and Top2β were determined by immunoblotting using anti-γ-H2AX and anti-mouse Top2β antibodies, respectively. c-PARP, cleaved PARP.