Fig 2: Folic acid (FA) activates ph-ATR-Chk1-NER pathway through increasing expression of Menin. A–B WB analysis revealed that supplemented with 10/20 mg/L FA in 1 mM HTL-treated NE4C cells can effectively attenuate DNA damage, elevating Menin protein expression and H3K4me3 modification level and enhancing P-Atr-Chk1 activation (n = 3). The P values were calculated with one-way ANOVA plus post-hoc test. C The down-regulation of NER pathway genes RNA expression caused by high HTL can be counteracted by supplementing with 20 mg/L FA (n = 3). The P values were calculated with unpaired t test

Fig 3: ROS generation and DNA damage analysis. (a) Generation of ROS in MCF7 and Cx43-MCF7 cells were assessed by measuring DCF fluorescence using flow cytometer. Untreated MCF7 and Cx43-MCF7 cells were taken as control and cells were treated in presence or absence of antioxidant NAC (5 mM). (b) Cell viability assay of MCF7 cells after treatment with ART in presence or absence of catalase. (c) ART induced DNA damage response in MCF7 cells upon exposure. Phosphorylation of ATM, ATR, Chk1, Chk2, histone H2A.X, BRCA1, and p53 was determined 8 h after the onset of treatment. β-actin was used as an endogenous control and to ensure equal proteins levels. The band intensity of the respective proteins were quantitated by densitometric analysis (supplementary Fig. 4).

Fig 4: Effect of Octa post-treatment on UVB-induced DNA damage. (a) Immunofluorescence analysis of CPDs and 8-oxodG (green signal) in NHKs irradiated with UVB 25 mJ/cm2 or UVA 10 J/cm2 and post-treated with 90 µM Octa for 6 h and 24 h. Scale bar: 20 µm. ELISA analysis of the amount of CPDs and 8-oxodG remaining in NHKs irradiated with UVB 25 mJ/cm2 or UVA 10 J/cm2 and post-treated with 90 µM Octa for 6 and 24 h. The values reported represent means ± SD of three independent experiments performed in duplicate. *p < 0,001 (vs UVB-irradiated cells), *p < 0,005 (vs UVA-irradiated cells). (b) Western blot analysis of p53, phospho-p53, GADD45a, phospho-γH2AX, phosho-ATM, phospho-ATR, DDB-2, phospho-Chk1 and phosphor-Chk2 protein expression on cell lysate of NHKs irradiated with UVB 25 mJ/cm2 and post-treated with 90 µM Octa for 24 h. GAPDH and HSP70 were used as a loading control. Results refer to three independent experiments. Representative blots are shown. Densitometric scanning of band intensities was performed to quantify the change of protein expression (control value taken as one fold in each case). (c) Immunofluorescence analysis of phosphorylated histone H2AX expression (red signal) in NHKs irradiated with UVB 25 mJ/cm2 and post-treated with 90 µM Octa for 24 h. Arrows indicate super-bright positive cells which results reduced in Octa post-treated cells respect to UVB irradiated cells. Quantitative analysis of the phosphorylated histone H2AX fluorescence signal evaluated by counting the number of positive cells/total cells. Nuclei are counterstained with DAPI (blue). Scale bar: 20 µm. The values reported represent means ± SD of two distinct experiments. *p < 0,001 (vs non-irradiated cells), °p < 0,005 (vs UVB-irradiated cells) (d) Time course analysis of the effect of 90 µM Octa post-incubation on the protein expression levels of p53, phospho-p53 and GADD45a in UVB (25 mJ/cm2)-exposed NHKs. GAPDH was used as a loading control. Results refer to three independent experiments. Representative blots are shown.

Fig 5: RT up-regulates CD73 in MC38 cells, which depends on the DNA damage repair pathway.Flow cytometric analysis of CD73 expression on MC38 cells at different time points after RT. Images (A), representative histograms of percentage and MFI (B) of CD73 on MC38 cells 48 h after 8 Gy or 12 Gy irradiation. C, D Immunofluorescence staining analysis of CD73 and γ-H2AX on MC38 cells 48 h after 8 Gy irradiation. Representative images (C) and MFI (D) of CD73 and γ-H2AX. Scale bars: 100 μm. Western blot analysis of the expression of CD73 and ATR-Chk1-STAT3 pathway in MC38 cells treated with or without 8 Gy RT (E), and ± Chk1i (50 nM, SCH900776) (F), ±Stat3i (500 nM, Angoline) (G), and harvested 24 h later. Flow cytometric analysis of CD73 expression on the surface of MC38 cells treated ± 8 Gy RT, ±Chk1i (H), ±Stat3i (J). Percentage and MFI (I, K) of CD73 on MC38 cells 48 h after RT. Statistical variations were analyzed utilizing the unpaired t-test. Data are expressed as mean ± SEM (n = 3 per group).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.