Fig 2: Uncoupled elevation of DNA damage and genomic replication in ovarian cancer cells. a, Indirect immunofluorescent (IF) staining of endogenous foci formation of indicated repair factors in OVCAR-8TR and RPE1 cells (upper panel) and quantifications for positive cells (lower panel). 53BP1, RPA32-pSerine 33 and BRCA1-pSerine1524 were monitored by specific antibodies. ***: p < 0.001, two-tailed unpaired t-test. Three biological replicates were analyzed. b, Representative images (upper) and quantification (lower) for 53BP1-positive cells (> 3 per cell) in primary cells isolated from three independent OvCa biopsies after culturing in RPMI1640/Matrigel for 48 h. BrdU-positive cells were quantified in parallel. Data showed as means ± SEM of 3 independent assays

Fig 3: Nucleoside supplementation restores proliferation of PFKFB3 inhibited cells. a Effects on DNA synthesis of U2OS cells treated with DMSO or indicated concentrations of KAN757 (4 h), pulsed with 10 µM EdU for 40 min, followed by fixation, fluorescent labeling, and quantification in CellProfiler, >100 cells were analyzed per condition, n = 2. **P < 0.01; one-way ANOVA analysis. b Schematic representation of the DNA fiber assay and representative images of DNA strands incorporating CldU (red) and IdU (green) from U2OS cells that have received treatment with 10 µM KAN757 supplemented with or without 30 µM nucleosides. Graphs show the distribution of average fork speed and calculated mean fork speed for the indicated treatments (n > 100 forks per condition). Data are displayed as means ± SEM, n = 3. ***P < 0.001; Student’s t-test. c dNTP measurements of U2OS cells treated as indicated. Relative levels of dNTPs were calculated relative to DMSO. Data are displayed as mean ± SEM, n = 2. *P < 0.05; Student’s t-test. d Metabolic rescue of the proliferation of U2OS cells following 10 nM siControl or siPFKFB3 treatments and supplementation with or without 30 µM nucleosides was assessed by DAPI staining and counting nuclei using CellProfiler. siControl cells are set as reference cells. Data are displayed as means ± SEM, n = 2. *P < 0.05; one-way ANOVA analysis. e Scatter dot plot representing the nuclear intensity of the fluorescence levels of RPA32 in U2OS cells following treatment with DMSO or indicated inhibitors as quantified using CellProfiler. Lines represent mean fluorescence, n > 500 cells/treatment. f Representative immunoblots (of n = 2) of U2OS cells treated with DMSO, KAN757 (10, 30 µM) and/or HU (2 mM) for indicated time points. Bands correspond to whole cell extracts, ß-actin was used as loading control. g Bar charts representing relative levels of phosphorylated ATR and ATM, p53 and ?H2AX in (f). Densitometric analysis was performed using Image StudioTM Lite Software. Signal intensity data were normalized against ß-actin and then normalized to DMSO 24 h. Data are displayed as means ± SD, n = 2 independent experiments. KAN757 = KAN0438757

Fig 4: PFKFB3 promotes homologous recombination repair. a Confocal analysis of nuclear localization of PFKFB3 in U2OS cells following treatment with siRNAs (24 h) as indicated, subjected to IR (6 Gy, 2 h recovery), or left untreated, n = 3 independent experiments. Scale bar, 10 µm. To the right, bar chart showing PFKFB3 foci as quantified using CellProfiler, n > 100 cells/treatment. Data are displayed as means ± SEM. ***P < 0.001; one-way ANOVA analysis. b Confocal images of co-localization between PFKFB3 and indicated proteins in U2OS cells upon IR (6 Gy, 2 h recovery). White arrows indicate co-localized foci, n = 2 independent experiments. Scale bar, 10 µm. To the right, bar graph representing the percentage of PFKFB3 foci that co-localized with indicated proteins as quantified using CellProfiler. n > 100 cells/condition. Data displayed as means ± SEM. ***P < 0.001, *P < 0.05; Student’s t-test. c U2OS cells were treated with indicated siRNAs for 24 h, subjected to IR at 6 Gy (2 h recovery) and immunostained for indicated proteins, n = 3 independent experiments. Scale bar, 10 µm. d Scatter dot plot representing the RAD51 or RPA32 nuclear intensity in (c) as quantified using CellProfiler. To the right, bars showing RAD51 or RPA foci per nuclei in (c). n > 500 cells/treatment. Data displayed as means ± SEM. ***P < 0.001; Student’s t-test. e HR activity after treatment of U2OS DR-GFP cells with indicated siRNAs as assessed by FACS analysis, whereby the siControl cells are set as reference cells (100% activity). Data are displayed as means ± SEM, n = 3. **P < 0.01, ***P < 0.001; one-way ANOVA analysis. f DNA histograms of U2OS cells treated with indicated siRNAs for 24 h then subjected to IR at 2 Gy (24 h recovery) or left untreated for 48 and 72 h. Cells were fixed and DNA content was assessed using propidium iodide staining and flow cytometry. Data are displayed as means ± SEM, n = 3. **P < 0.01, ***P < 0.001; one-way ANOVA analysis. g Colony formation of U2OS cells 10 days after treatment with 10 nM siControl or 10 nM siPFKFB3 in combination with 2 Gy or left untreated, was assessed by staining with 4% methylene blue in methanol and counting colonies. Data are displayed as means fold over siControl no IR ± SEM, n > 5. ***P < 0.001; one-way ANOVA analysis

Fig 5: Impact of PARP inhibitor and FEN1 deletion on RPA foci and phosphorylation.(a) ScanR quantification of RPA2 foci in wild type U2OS cells and the indicated FEN1-/- clones treated or not (NT) for 8 h with 10 µM PARP inhibitor (PARPi). S phase cells were distinguished by PCNA staining and DAPI intensity. Data represent the average (±1 SD) RPA2 foci number from three independent experiments with individual data points plotted. Statistical significance was assessed by 1-way ANOVA with post-hoc Sidak’s multiple comparisons test. (b) Western blotting analysis of whole cell extracts from wild type U2OS cells and the indicated FEN1-/- clones treated or not (NT) for 4–8 h with 10 µM PARP inhibitor (PARPi)(n = 2). Source data