FIG. 4.

Decrease in homologous recombination frequency after WR-1065 treatment is not caused by WR-1065 preventing DNA transfection (panel A), interfering with I-SceI induction of double-strand breaks in substrate DNA (panel B), or changes in reactive oxygen species levels (panel C). Panel A: S31WT cells were treated with WR-1065 for 30 min at 4 mM (light gray bars) or 24 h at 40 μM (dark gray bars) or were sham-treated with PBS (white bars) and were transfected with GFP plasmid as outlined in the Materials and Methods for the I-SceI transient transfection. After 24 h recovery, the GFP-positive cells were counted by flow cytometry. Results of two independent experiments (±SEM) are shown. Panel B: SCneo supercoiled plasmid (1 μg, lanes 2 and 9, untreated control) was preincubated with WR-1065 (4 μM, lane 5; 40 μM, lane 6; 400 μM, lane 7; 4 mM, lanes 4 and 8) for 5 min followed by 16 h incubation with I-SceI enzyme (1 U, lanes 3 and 5–8). The digestion products were resolved using agarose gel electrophoresis (SC, supercoiled circular; OC, open circular; LIN, linear). Panel C: Changes in cellular reactive oxygen species (ROS) levels after WR-1065 treatment (at 4 mM for 30 min, light gray bars; 40 μM for 24 h, dark gray bars; or sham-treated, white bars) and I-SceI transfection were measured using the specific probe CM-H2DCFDA and flow cytometry as described in the Materials and Methods. H₂O₂-treated cells serve as an experimental positive control. Results represent means of two independent experiments ± SEM.