Fig. 1: Hyperthermia resulted in CDK1-dependent cell cycle perturbation, replication stress, and DNA damage.

a Experimental scheme of the transcriptomic, proteomic, and phosphoproteomic profiling. LC-MS/MS: Liquid Chromatography-Tandem Mass Spectrometry (n = 3 per group). b Bar plot showing differential phosphosites when comparing hyperthermia (42 °C, HT) to normothermia (37 °C, Ctrl) treated OVCAR8 cells, as analyzed by PTM-SEA. c Heat map showcasing the differentially regulated phosphorylation sites associated with kinases enriched in part (b) (left). Protein-protein interaction network of differential substrates of CDK1, as sourced from the STRING database (right). d Overview of the changes in protein and phosphorylation levels associated with CDK1-dependent cell cycle induction by HT. e OVCAR8, A2780, and ID8 cells were treated with HT for 30, 60, and 90 min. A representative western blot image is shown with indicated proteins. f Cell cycle distribution after exposure to HT for 90 min in OVCAR8, A2780, and ID8 cell lines (n = 3 per group). g DNA fiber analysis of OVCAR8 cells treated as in part (f) yielded representative replication fork images with a scale bar of 10 μm. Fork velocity distribution analysis was based on 100 ongoing replication forks pooled from three independent experiments. h Cells were pretreated with or without a CDK1 inhibitor (CDK1i, RO-3306, 5 μM) for 48 h prior to exposure to HT for 90 min. γH2AX, EdU, and PI staining were analyzed by flow cytometry in OVCAR8 cells (n = 3 per group). The quantification of γH2AX positive cells in various cell cycle phases is shown. i To detect ssDNA, cells were cultured in a medium with 10 μM BrdU throughout the experiment; DNA was not denatured before staining against BrdU. Representative images and quantification of OVCAR8 cells stained with BrdU, γH2AX, and DAPI are shown (n = 3 per group). Scale bar, 25 μm. Comparisons were performed by unpaired two-tailed Student’s t test in (g), one-way ANOVA followed by Dunnett’s multiple comparisons test in (h), and one-way ANOVA followed by Tukey’s multiple comparisons test in (i). Data are presented as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01. 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.