The Polθ inhibitor ART558 radiosensitizes tumor cells. A and B, Clonogenic survival of HCT116, H460, and T24 cells treated with ART558 and/or IR. A, Plating efficiency for unirradiated cells. B, Surviving fractions as a function of the irradiation dose. Representative wells for 0 Gy and 6 Gy ± 1 μmol/L ART558 are shown for each cell line. C, Clonogenic survival of U2OS WT and Polθ KO cells treated with 3 μmol/L ART558 and 6 Gy IR. Bar graphs show the surviving fraction at 6 Gy. The Western blot insets confirm the lack of Polθ expression in the U2OS Polθ KO cells. D, Clonogenic survival of HCT116 and H460 cells transfected with either a control, nontargeting siRNA (siNT) or an siRNA targeted against POLQ (siPOLQ) and treated with 3 μmol/L ART558 and 6 Gy IR. Bar graphs show the surviving fraction at 6 Gy. Western blots show Polθ expression at the time of irradiation. E, Clonogenic survival of H460 and HCT116 treated with 3 μmol/L ART558 and 5×2 Gy (2 Gy once per day for 1 to 5 days). F and G, Clonogenic survival following irradiation and treatment with 3 μmol/L ART558 of synchronized HeLa cells. F, Representative histograms showing the cell-cycle distribution at the time of IR (synchronized in G1 by DT block or after 6 hours release from DT block, compared with asynchronous cultures). G, Degree of radiosensitization estimated by the ratio between the surviving fraction of DMSO- and ART558-treated cells after IR (SF DMSO / SF ART558). Data correspond to average ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).