2.3 Clonogenic survival assay

Cells were seeded at densities ranging from 300 to 10,000 in 6 cm dishes and incubated for 12 hr to allow attachment. Cells were then treated with NCS (stock concentration 37 μM diluted to 2 μM in 20 mM sodium citrate buffer, pH 4.0) at concentrations ranging from 0.25 nM to 2 nM for HCT116 cells and 2 nM to 6 nM for HeLa cells for 6 hr. Following treatment, cells were incubated in fresh medium for 8-9 days to form colonies. Colonies were fixed with 100% methanol for 10 min, stained with 0.5% crystal violet in 20% methanol for 10 min, washed under tap water, air dried and counted manually. Plating efficiency (PE) was calculated as the number of colonies formed/number of cells seeded *100 for each dose. Surviving fraction (SF) was calculated as PE of treated/PE of control *100. Dose-modifying factor (DMF) was calculated as IC90 of control/IC90 of the mutant cell line. For experiments using ionizing radiation, cells were irradiated using a MDS Nordion Gammacell 40 research irradiator (ON, Canada), with a 137Cs source. For experiments with KU-60019, NU-7441, olaparib (AZD-2287) and veliparib (ABT-888), the respective inhibitor was added 1 hr prior to NCS treatment and left in the medium during and 24 hr after NCS treatment. Inhibitors were from Selleck Chemicals (Houston, TX).