Fig. 3: Integrated stress response activation by WEE1i is cell cycle independent.

a RPE1 TP53^KO PAC^KO cells were treated with AZD1775 (250 nM) for 6 h in the presence or absence of ISRIB (1 μM), pulse labeled with EdU, and processed for quantitative image-based cytometry. Cell cycle stage was defined by DNA content and EdU positivity. Each point represents a cell. Two independent replicates are defined by two different colors. Bars represent means of each replicate. Experimental set-up (b) and flow cytometry analysis (c) of RPE1 TP53^KO ATF4-mScarlet-NLS reporter cells. Cells were treated overnight with nocodazole. Mitotic cells were isolated and replated in the presence or absence of palbociclib. After 2 h, AZD1775 (1 µM) and/or A92 (1 µM) was added for 20 h. Data represent mean ± SD (n = 3). Flow cytometry gating strategy (d) and analysis of γH2AX in RPE1 TP53^KO cells after treatment with AZD1775 (1 µM) and/or A92 (1 µM) in the absence (e, left) or presence (e, right) of nocodazole. Data represent mean ± SD (n = 4). f Flow cytometry analysis of MPM2-positivity in RPE1 TP53^KO cells after treatment with nocodazole, doxorubicin, AZD1775 (1 µM) and/or A92 (1 µM). Data represent mean ± SD (n = 3). g Flow cytometry analysis of MPM2-positivity in control RPE1 TP53^KO PAC^KO and ATF4^KO #1 or GCN2^KO #1 cells after treatment with nocodazole, doxorubicin and AZD1775 (1 µM). Data represent mean ± SD (parental conditions n = 4, KO conditions n = 3). h Immunoblot of resting PBMCs or PBMCs stimulated with anti-CD3/CD28 beads. PBMCs were treated with DMSO, AZD1775 (500 nM) or thapsigargin (500 nM) for 24 h. Representative blot of n = 3 independent experiments. i RPE1 TP53^KO PAC^KO and GCN2^KO cells were treated with AZD1775 and/or cisplatin for 5 days. Cell survival was analyzed by MTT conversion. Data represent mean ± SD (n = 4). Statistical analysis for c, e, f, g was performed using unpaired t-tests (two-sided), with p ≤ 0.05 considered significant. All replicates are biological replicates unless indicated otherwise. Source data are provided as a Source data file.