The effect of COX-2 expression on radiosensitization by gefitinib. Western blot analysis was done using anti-COX-2 antibody in NCI-H460, VMRC-LCD and A549 cells (A) or H460-Mock, -COX2, AN or AS cells (B). β-Actin was used as a loading control. C, Clonogenic cell survival assays after gefitinib plus ionizing radiation (IR) treatment in H460-Mock and -COX2, or AN and AS cells. Cells were treated with gefitinib for 24 h, irradiated, incubated in drug-containing media for another 48 h, and allowed to form colonies in drug-free medium. Surviving fractions for radiation + gefitinib were normalized by dividing by the surviving fraction for gefitinib alone. Error Bars, SE of three independent experiments in triplicate. *, P < 0.05 compared with IR alone group. D, Multinucleated cell induction after gefitinib plus ionizing radiation (IR) treatment in H460-Mock and -COX2, or AN and AS cells. Cells were grown on coverslips and treated with 15 μmol/L gefitinib for 48 h, and then exposed to 6 Gy IR. After 72 h, cells were fixed, stained with DAPI, and then observed under confocal microscopy (X400). Cells with multinuclei were indicated with arrow. The number of multinucleated cells was counted and plotted. At least 100 cells were counted and each column represents the means of three independent experiments. Error bars, SE.