Knockdown of the identified replication stress response genes causes hypersensitivity to replication stress and ATR pathway inhibitors. (A) Flow-chart of the sensitivity screen assay. U2OS cells were transfected with siRNAs in 384-well plates. Seventy-two hours post-transfection cells were split 1:4 and were left untreated (mock) or treated with 0.2mM HU, 0.1µM ATR inhibitor, or 0.05µM CHK inhibitor for 72 hours before assaying for cell viability. (B) The number and percentage of genes required for resistance to drug treatments is indicated. Data from supplemental figures 2, 3, and 4, which include gemcitabine, camptothecin, and PARP inhibitor treatments, are included in the total count. (C) Results from validation and drug sensitivity screens are presented with each of the four siRNAs per gene as a row. The genes are grouped based on how many siRNAs validated in the RRS secondary screen (black squares in first column). Black squares in the remaining columns depict siRNAs causing sensitivity to the indicated drugs. Grey squares: not determined.