Fig. 2: Hyperthermia promotes dNTPs depletion which is responsible for replication stress and DNA damage accumulation.

a OVCAR8, A2780, and ID8 cells were treated with hyperthermia (HT, 42 °C) for 30, 60, and 90 min. Representative western blot image illustrates dynamic RRM2 levels at different heating durations. b Cells were cultured with or without dNTPs mix (dNs, 50 μM) for 48 h prior to being treated with HT for 90 min. Changes in cell viability were determined by the CCK8 assay (n = 6 per group). c γH2AX, EdU, and PI staining were analyzed by flow cytometry in OVCAR8 and A2780 cells after exposure to HT with or without dNs (n = 3 per group). Quantification of γH2AX positive cells in various cell cycle phases is presented. d Representative images and quantification of BrdU and γH2AX positive OVCAR8 cells after being treated as in part b are presented (n = 3 per group). Scale bar, 25 μm. Comparisons were performed by one-way ANOVA followed by Dunnett’s multiple comparisons test in (c) and one-way ANOVA followed by Tukey’s multiple comparisons test in (b, d). Data are presented as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. 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.