Fig 2: CPT induces transcription- and proteasome-dependent apoptosis in quiescent WI38 hTERT cells. (A and B) Serum-starved cells were treated with DMSO or FLV (1 µM) for 1 h before the addition of DMSO or CPT (25 µM) for 16 and 24 h. (A) Percentages of cells that remained attached to culture flasks (means ± SD of quadruplicates). (B) Cell survival was analyzed by a CellTiter-Blue assay (means ± SD of triplicates). (C–G) Western blot of the indicated proteins in serum-starved cells treated for 1 h with DMSO or with FLV (1 µM) (C), lactacystin (10 µM) (D), veliparib (5 µM) or olaparib (10 µM) (E), ATMi (10 µM) (F) or DNA-PKi (10 µM) (G) before the addition of DMSO (‘-’, 24 h in panels C and F) or CPT (25 µM) for the indicated times. Data shown are representatives from two to three experiments. PARPCL: cleaved PARP. H2AX and aTubulin were the loading controls.

Fig 3: In-cell western analysis of histone H2AX phosphorylation after treatment with DNA-damaging agents and silencing of MDM2 gene expression in the SKBR3 cells. The signal from H2AX phosphorylation was normalized to untreated cells. Statistical significance was assessed by comparing siMDM2 with the control (non-specific siRNA) for all tested concentrations of compounds, * p < 0.05, ** p < 0.01, *** p < 0.001. Four independent biological experiments were performed for each chemotherapeutic (n = 4).

Fig 4: p53-TP53-induced glycolysis regulator (TIGAR) ameliorates exhaustion of Fanconi anemia (FA) hematopoietic stem cells (HSCs). (A): Limited dilution assay. Various doses of sorted transduced Lineage-Sca-1+c-kit+ (LSK) cells (25, 75, 150, and 250 mCherry+ cells) expressing mCherry or mCherry-TIGAR were mixed with 2 × 105 protector bone marrow (BM) cells (CD45.1+) and transplanted into irradiated congenic recipients (n = 8–10 per group). Plotted are the percentages of recipient mice containing <1% CD45.2+ blood nucleated cells (negative) at 8 weeks after transplantation. Frequency of functional HSCs was calculated according to Poisson statistics. (B): Overexpression of TIGAR improves hematopoiesis reconstitution capacity of FA HSPCs. One thousand sorted mCherry or mCherry-TIGAR lentiviral vector transduced LSK cells were transplanted into lethally irradiated BoyJ mice. The recipients were subjected to flow cytometry analysis for donor-derived cells 16 weeks after BM transplantation. Representative flow plots (left) and quantification (right) are shown (n = 9 per group). (C): Overexpression of TIGAR improves long-term repopulating activity of FA HSCs. One million BM cells from the primary recipient mice described in (B) were transplanted into sublethally irradiated secondary CD45.1+ recipient mice and donor-derived chimera in secondary recipients were determined by flow cytometry 16 weeks later (n = 9 per group). (D): Overexpression of TIGAR reduces DNA damage in donor cells. CD45.2+ LSK cells from the recipients described in (B) were subjected to flow cytometry analysis for ?-H2AX. Representative plots (upper) and quantification (lower) are shown. (E): Overexpression of TIGAR increases donor HSC quiescence. Percentage of Pyronin Y+ cells in donor HSC compartment (CD45.2+ SLAM) was analyzed by flow cytometry. Representative flow plots (left) and quantification (right) are shown (n = 6 per group); *, p < .05; **, p < .01; ***, p < .001.

Fig 5: The relationships between ovarian patient age and ?-H2A.X level in the whole cohort (a), patients with different FIGO stages (b,c), and patients with different malignant ascites statuses (d,e).