Fig. 4: Hyperthermia sensitized WEE1 inhibitor by augmenting replication stress, deteriorating DNA damage, and inducing mitotic catastrophe.

a Functionalities and trade names of cell cycle-related inhibitors in synergistic drug screening with hyperthermia (HT). b A flow diagram of drug screening is presented. Cells seeded into 96-well plates were treated with increasing doses of inhibitors for 48 h in the presence or absence of HT (42 °C) for 90 min, and cell viability was estimated using the CCK8. c The sensitizing effect of HT on drugs is defined as ΔAUC% = AUC(Ctrl) − AUC(HT)/AUC(Ctrl). d The ΔAUC% for the combination of HT with various drugs was calculated (n = 4 per group in ATMi, n = 3 in others). e Representative western blot results for the combination of HT with AZD1775. OVCAR8, A2780, and ID8 cell lines were treated with AZD1775 (250, 800, and 800 nM, respectively) for 48 h, with or without a 90 min HT treatment during the initial drug exposure. f γH2AX, EdU, and PI staining were analyzed by flow cytometry in A2780 cells treated as in (e) (n = 3 per group). g OVCAR8 and A2780 cells, treated as in (e), underwent DNA fiber analysis with 100 fibers collected from three independent experimental replicates for each group. Scale bar, 10 μm. h Representative images and quantification of BrdU and γH2AX positive OVCAR8 cells treated as in (e) are shown (n = 3 per group). Scale bar, 25 μm. i Representative images and quantification of pH3 and γH2AX positive OVCAR8 cells treated as in e are shown (n = 3 per group). Scale bar, 25 μm. j Representative western blot results depict the combination of HT with AZD1775 in five primary HGSOC samples across five independent experiments. k Cell cycle flow cytometric analysis was performed on five primary HGSOC samples across five independent experiments, as described in (j). Comparisons were performed by unpaired two-tailed Student’s t test in (d) and one-way ANOVA followed by Tukey’s multiple comparisons test in (f, g, h, i). Data are presented as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns., not significant. All analytical data are derived from a minimum of three biologically independent experiments, with “n” indicating the specific number of replicates. Source data are provided as a Source Data file.