Fig 2: PEDV infection activates ATM-dependent DNA damage response. (A) Western blots were prepared with extracts from Vero-E6 cells uninfected or infected with CV777 (0.1 MOI) for 12, 24, 36, and 48 h. The levels of p-ATM (S1981), total ATM, p-Chk2(T68), total Chk2, p-ATR(T1989), total ATR, p-DNA-PK(S2056), total DNA-PK, ?H2AX, PEDV N, and actin were determined using their respective antibodies. Vero-E6 cells infected with UV-inactivated CV777 (0.1 MOI) for 48 h were used as the negative control. (B) Western blots were prepared with extracts from Vero-E6 cells uninfected or infected with HLJBY (0.1 MOI) for 12, 24, and 36 h. (C) Western blots were prepared with extracts from Marc145 cells uninfected or infected with CV777 (0.1 MOI) for 24, 36, and 48 h at 0.1 MOI.

Fig 3: CHEK2 deficient fibroblasts enhance the proliferation of epithelial cells in a paracrine manner. SFCM were collected from TCF-169-CHEK2-siRNA cells and their controls, and then were added to MCF10A (a) or MDA-MB-231 (b) cells that were seeded on 96-well plates. The proliferation was measured at the indicated times using the WTS-1 assay. Error bars represent ± SD, p value < 0.0001

Fig 4: The DNA damage response was activated in monocytes co-cultured with activated macrophages for 24 h.(A) Monocytes displayed pATM immunostaining when they were co-cultured with activated macrophages (Mph-PMA), but not with non-activated macrophages (Mph-Solvent). (B) Monocytes showed increased 53BP1 foci formation after co-culture with activated macrophages. (C) There was stabilisation of p53 protein as well as phosphorylation of p53 at position Ser46 and phosphorylation of CHK2 in monocytes exposed to activated macrophages (Mph-PMA).

Fig 5: The ATR mutation sensitizes cells to SN-38 ± VE-822 through caspase-3 dependent apoptosis linked with increased DNA damage. (A) Cells were challenged with 1.5 nM SN-38 ± 50 nM VE-822 and assessed for caspase activity (Nexcelom) on the Celigo imager. The percentages of apoptotic cells were calculated. Error bars represent s.e.m from 5 independent replicates. ? F (24 h) and ? B-E (48 h) refers to the time points used in the experiments presented in (B)–(F). Mann–Whitney tests between MUT and WT/REV were applied on the 48 h timepoint. (B) Cleaved caspase-3 and ?H2AX assessed by western blotting (48 h). GAPDH served as a loading control for each individual blot (see [Fig. S5]). A representative GAPDH panel is shown. The GAPDH-normalized quantifications relative to WT (SN-38 + VE-822) are calculated from 3 independent replicates. Mann–Whitney tests were applied. Full blots: [Fig. S5] (C) Immunofluorescence analysis of cleaved caspase-3 (green) and ?H2AX (red) in cells treated with 1.5 nM SN-38 ± 50 nM VE-822 for 48 h. Cells were observed with a Zeiss Axio Imager (× 20), scale bar: 100 µm. An arbitrary threshold was set and percentage of cleaved caspase-3 positive cells was scored (n > 300 cells/sample). Error bars represent s.e.m from 3 experiments and Mann–Whitney tests were applied. (D) Flow cytometry assessment of ?H2AX in cells challenged with SN-38 + VE-822. An arbitrary threshold was set (orange gates) and the percentage of positive cells was plotted. Error bars represent s.e.m from 3 independent experiments and Mann–Whitney tests were applied. (E) Western Blot analysis of total and phosphorylated ATR and CHK1 (ATR pathway) and KAP1 and CHK2 (ATM pathway) in cells challenged with 1.5 nM SN-38 ± 50 nM VE-822 (48 h). GAPDH served as a loading control for each individual blot (see [Fig. S5]). A representative GAPDH panel is shown. The GAPDH-normalized quantifications relative to WT (SN-38 or SN-38 + VE-822) are calculated from at least 3 independent replicates. Mann–Whitney tests were applied. ATR was re-stripped on the ATPpT1989 membrane. Full blots: [Fig. S5] (F) Cells were treated with 1.5 nM SN-38 for 24 h and pulsed with BrdU. Cells were processed on a Gallios (Beckman coulter). Debris and doublets were excluded, and 20,000 cells were analyzed per sample. Error bars are s.e.m from 3 independent experiments. Mann–Whitney tests were applied.