Fig 2: Inhibition of HSV-1 replication blocks cGAS release from the chromatin.a RAW 264.7 cells were mock-infected or infected with 1 MOI of HSV-1 KOS d109 for 16 h. Cell lysates were fractionated and blotted as indicated. b RAW 264.7 cells were pretreated without or with 8 µg/mL of acyclovir for 16 h, followed by infection with 1 MOI of HSV-1 McKrae for 12 h. Cell lysates were fractionated and blotted as indicated. The arrow indicates nuclear soluble cGAS. c RAW 264.7 macrophages were treated with dimethylformamide (DMF) as a vehicle mock control, 50 µM cisplatin for 4 h, or infected with HSV-1 McKrae for 12 h. Cell lysates were fractionated and blotted as indicated. The arrow indicates nuclear soluble cGAS. STING: membrane marker; a-tubulin: cytosolic marker; H3: nuclear marker; ICP8: HSV-1 viral protein; ?-H2A.X: DNA damage marker. The arrow indicates nuclear soluble cGAS. d RAW 264.7 macrophages were treated with DMF (mock control), 50 µM cisplatin or infected with HSV-1 McKrae for indicated times. Then, cell viability was determined by MTT assays. Data represent means ± s.d. of three independent experiments. The P-value was calculated by two-way ANOVA followed by Sidak’s multiple comparisons test.

Fig 3: METTL3, FTO, and PARP1 regulate m6A RNA at damage sitesa–b, U2OS cells were subjected to UVA laser microirradiation, incubated at 37°C for the indicated time, then stained for ?H2A.X and METTL3 (a) or FTO (b) as indicated. Cells in blue boxes are shown at higher zoom level in the second column. Arrows denote ?H2A.X-positive damage sites with colocalizing METTL3 (a) or FTO (b). The percentage of ?H2A.X-positive cells displaying colocalizing METTL3 or FTO signal is indicated on the right. c, WT or H2A.X KO MEF cells were microirradiated by UVA laser, incubated at 37°C for 2 min, and co-stained for m6A and TFIIH or ?H2A.X as indicated. Where possible, the percentage of ?H2A.X-positive cells showing colocalizing m6A signal is indicated on the right. d, U2OS cells pre-treated with DMSO or PARP inhibitors BYK, PJ-34, or Olaparib were microirradiated by UVA laser, incubated at 37°C for 2 min, and then costained for m6A and ?H2A.X. e, U2OS cells pre-treated with DMSO or PARP inhibitor (Olaparib, 10 µM) were microirradiated by UVA laser, incubated at 37°C for 2 min, and then stained for PARP1, Poly(ADP-ribos)ylation (PAR), m6A, and ?H2A.X as indicated. The percentage of ?H2A.X-positive cells showing colocalizing signal is indicated on the right. f, U2OS cells pre-treated with DMSO or PARP inhibitor (Olaparib, 10 µM) were microirradiated by UVA laser, incubated at 37°C for 2 min, and then costained for ?H2A.X and METTL3. Arrows denote representative ?H2A.X-positive cells displaying colocalizing METTL3 signal. The percentage of ?H2A.X-positive cells showing colocalizing METTL3 is indicated on the right. All images are representative of at least 50 cells in triplicate. Scale bar, 20 µm.

Fig 4: Role of circ-DB and USP7 in regulating proliferation and DNA damage in HCC cells. HCC cells were pretreated with circ-DB overexpressing plasmid (OE.circ-DB) and circ-DB siRNA deleted by circ-DB siRNA (si.circ-DB). a, b The cells were stained with DAPI and EdU, and then, the nuclei were imaged to reveal the proliferation of HCC cells (n = 3). c, d The cells were stained with DAPI and H2AFX, and then, the nuclei were imaged (n = 3). HCC cells were pretreated with USP7-overexpressing plasmid (OE.USP7) and USP7 siRNA (si.USP7). e, f The cells were stained with DAPI and EdU, and then, the nuclei were imaged to reveal the proliferation of HCC cells (n = 3). g, h The cells were stained with DAPI and H2AFX, and then, the nuclei were imaged (n = 3). **p < 0.01

Fig 5: MAPK15 promotes TNF-a-augmented cisplatin-induced DNA damage(A) Treatment same as in Figure 1C. Immunoblotting was performed to detect cisplatin-induced DNA damage using antibodies against p-H2AX (Ser139) and H2AX.(B) Stable MAPK15 knockdown and control H1299 cells were sham-exposed or exposed to 40 µM cisplatin for 24 h. Immunoblotting was performed using antibodies against p-H2AX (Ser139), H2AX, p21, and RAD51.(C) Same treatment as in Figures 4C and 4D. Immunoblot assay was performed using antibodies against p-H2AX (Ser139), H2AX, and RAD51.(D) Same as in Figure 5B, DNA damage was detected by immunoblotting with antibodies against p-H2AX (Ser139), H2AX, p21, and RAD51 (n = 3, mean ± SEM, one-way ANOVA). Significant differences are indicated as *p<0.05, **p<0.01, or ***p<0.001.(E) H1299 cells were sham-exposed or exposed to 20 ng/mL TNF-a for 12 h, then co-treated with 20 ng/mL TNF-a and 40 µM cisplatin or cisplatin alone for 12–24 h. Cells were harvested and lysed, and protein extracts were subjected to immunoblot assay using antibodies against caspase-3, p-H2AX (Ser139), H2AX, p21, and RAD51. ß-Actin was used as the internal control. The gray intensity of the bands was quantified by Gelpro 32 software. The active caspase-3 was normalized to total caspase-3, cleaved RAD51 was normalized to total RAD51, and p-H2AX was normalized to total H2AX.