(A) Overview of the HCI AZD1775 screen in breast cancer cell lines and drug response characteristics. Activity Area - AA, EC50 - concentration of compound achieving 50% of the maximum effect, IC50 - concentration of compound achieving 50% reduction in cell number, and cell cycle profile. AZD1775 response profile of SUM149PT cells is shown (see also Figure 1—figure supplement 1A, Figure 6—figure supplement 1G and Supplementary file 1). (B) Comparison of acute AZD1775 sensitivity between basal-like and luminal breast cancer cell lines measured by HCI analysis. The AZD1775 response characteristics (activity area) depicted correspond to the area above the response curve. Boxes are coloured according to AZD1775 response in basal-like and luminal BC cell lines, respectively. * denotes p<0.05 as determined using Student’s t-test. Data are represented as mean ± SD. (C) Acute response to AZD1775, AZD6738 or the combination relative to DMSO-treated control in different BC cell lines. Cell numbers were analysed by crystal violet staining and quantified by colorimetry after 72 hr treatment. Data shown refer to a single concentration of AZD1775 (500 nM), AZD6738 (1 µM) or their combination (fixed ratio, 1:2). Error bars indicate standard deviation calculated from three independent experiments. Drug combination effects were calculated, based on the ratio and concentrations above, using the CImbinator online tool (Flobak et al., 2017). Both AZD1775-sensitive (CI >1.0) and AZD1775-recovering (CI <1.0) BC cell lines are depicted and drug synergy is marked *. The following CI values were calculated; HCC1954 = 0.31; HCC1143 = 0.18; BT20 = 0.45; SUM159PT = 0.34; HCC38 = 2.06; HCC70 = 1.09; HCC1937 = 1.57. (D) Comparison of acute AZD1775 response between recovering basal-like, sensitive basal-like and luminal breast cancer cell lines (recovering) measured by HCI analysis. The AZD1775 response characteristics were set to the area above the response curve (Activity area) left, and EC50, right. Boxes are coloured according to AZD1775 response of the different BC cell lines (recovering-basal, sensitive-basal and recovering-luminal). ** denotes p<0.01 as determined using Student’s t-test. (E) Recovery of proliferation following removal of AZD1775 (500 nM), AZD6738 (1 µM) or their combination was analysed by crystal violet staining and quantified by colorimetry. BC cell lines were treated for three days and allowed to recover for an additional four days without the drugs. Representative images of each cell line (from >three independent experiments) treated with indicated drugs and stained with crystal violet are shown. (F) Representative flow cytometry dot plots (left panels, two independent experiments) showing the distribution of EdU and γH2AX incorporation in recovering BT20, and sensitive HCC38 cells, following 24 hr treatment with AZD1775 (500 nM), AZD6738 (1 μM) or their combination as compared to DMSO control. Labelling of γH2AX-positive cells in the replicating fraction (EdU-positive - red) and γH2AX-positive cells in the non-replicating fraction (EdU-negative - gray) of each cell line is shown (right panels).