Fig 2: DFO induced autophagy in p53S MEFs. (A) Western blot analysis of Atg 7, Atg 12, and LC 3-I and -II in WT, p53S/+, and p53S/S cells with DFO treatment. (B) Western blot analysis of WT, p53S/+, and p53S/S cells with CQ and DFO treatment. (C) MDC staining of autophagic cells with DFO treatment. (D) Quantification of C. (E) Western blot analysis of AMPK and AKT/mTOR in WT, p53S/+, and p53S/S cells with/without 200 µM DFO exposure. (F) Quantification of the relative protein expression of p-mTOR, p-AKT, and p-AMPK in p53S/S cells. (G) The location of p53 was detected by immunofluorescence analysis (red) in cells treated with or without DFO. Nuclei were stained with DAPI (blue). Values represent means ± SD of at least three independent experiments. Statistical significance was evaluated using Student’s t-test. * p < 0.05, *** p < 0.001. ns means no significance.

Fig 3: p53S cell response to hypoxia stress. (A) Expression of hypoxia-induced proteins was analyzed using Western blot with DFO treatment or hypoxia chamber culture. (B) Western blot analysis of HIF-1a, p53, and Pser15-p53 over a 24 h treatment with DFO. (C) The cell morphology was monitored using a phase-contrast microscope after a certain time of DFO treatment. (D) Western blot analysis of PARP and caspase-3 cleavage was carried out in WT, p53S/S, and p53S/+ cells with DFO treatment. (E) Apoptosis induced by DFO treatment was monitored by flow cytometry analysis. (F) Quantification of E. Values represent means ± SD of at least three independent experiments. Statistical significance was evaluated using Student’s t-test. * p < 0.05. ns means no significance.

Fig 4: RS-induced MCM2-7 downregulation and cellular senescence is dependent upon TRP53.(A) RS treatment in primary Trp53-/- MEFs upregulates senescence-associated transcripts similarly to WT MEFs. Trp53-/- primary MEFS were treated (or not) with 200µM HU for 72h. Primers specific to the p16Ink4a and p19ARF, products of the Cdkn2a locus, as well as the primary transcript of Cdkn2a were quantified by qRT-PCR and normalized to ß-actin levels. (B) TRP53 deficiency prevents cellular senescence induction in response to chronic RS. Percentage of cells positive for SA-ß-gal after 72 hours of exposure to 200µM HU is plotted. Error bar = SEM. (C) HU-induced RS causes Mcm2-4 up-regulation in P2 Trp53 null primary MEFs. Cells were cultured in the indicated concentrations of HU for 72h. Plotted are mRNA levels as assessed by qRT-PCR. Control cells received no HU, and these mRNA levels are considered to be 100%. Error bar = SEM. (D) RS induces DNA damage and increases MCM protein in Trp53-null primary MEFs. Cells were treated with 200µM HU for 72h, and then total protein was extracted for immunoblotting with antibodies against the indicated proteins. The phosphorylated form of H2AX (?H2AX) is an indicator of double strand breaks and replication fork stalling.

Fig 5: ADH5 prevents formaldehyde toxicity in human cancer cells.a Viability assay for Nalm6 cells exposed to formaldehyde (FA) (mean ± SEM, n = 6, one-way ANOVA for multiple comparison using a Tukey-corrected test between ?ADH5 or ?FANCB and Wild type (WT) cell lines, **P = 0.0011 (WT vs ?FANCB 15 µmol L-1 FA), **P = 0.004 (WT vs ?ADH5 22.5 µmol L-1 FA), ****P < 0.0001). b Western blots depicting ?-H2AX and p-p53 signals after 48 h of exposure to the indicated concentrations of FA, or after 24 h to 8 µmol L-1 cisplatin (CPt) in Nalm6 cells. c Quantification of ?-H2AX western blots (n = 2, mean relative to same cell line untreated). d Viability assay for HCT116 cells. Data are presented as mean (% of untreated) ± SEM, n = 6, two-way ANOVA for multiple comparison using a Tukey-corrected test between WT and ?ADH5 HCT116 cells, ****P < 0.0001. e Representative images of 3D-sphere formation for WT, ?ADH5 and ?ADH5/pADH5 (pA5) HCT116 cells (scale bar 0.5 mm). µM refers to µmol L-1 and mM refers to mmol L-1. f Western blots showing the induction of p-p53 and ?-H2AX in HCT116 cell lines (48 h for FA, 24 h for Cpt (4 µmol L-1), hydroxyurea (HU, 1 mmol L-1) and mitomycin C (MMC, 1.5 µmol L-1). g Viability assay in the indicated HCT116 cell lines in the presence of FA (% of untreated, n = 8, mean ± SEM, n.s. not significant between ?ADH5 and ?TP53 ?ADH5, two-way ANOVA using a Tukey-corrected for multiple comparisons). h Representative images of metaphases obtained from WT and ?ADH5 HCT116 cells exposed to 150 µmol L-1 FA (scale bar 1 µm) and the quantification (MMC (1.5 µmol L-1) or FA (150 µmol L-1). Data are presented as mean ± SEM, n = 49, one-way ANOVA, Tukey´s multiple comparison test, ****P < 0.0001, n.s.: not significant).