Fig 2: Evaluation of DNA damage and repair capacity in the post-chronic Cd-exposed cell line. (A) Intrinsic DNA damage level of CR0 cells (over PM control cells) was measured by comet assay (n = 6); two DNA damage parameters, % tail DNA and tail extent moment, are shown. (B) Representative images of comet assay in PM and CR0 cells stained with 5 µg/mL of propidium iodide after gel electrophoresis. (C) Expression of histone H2AFX and phosphorylated H2AFX (?H2AFX) were determined by Western blot (see Figure S2 for full blot). (D,E) Cell viability of PM and CR0 cells exposed to various concentrations of DNA damage-inducing agent H2O2 for 2 h without or with a 24 h recovery period was measured by MTS assay; results were representative of at least three independent experiments, and error bars represent mean ± SD of at least three technical replicates. (F) Tail extent moment of PM and CR0 cells exposed to 0, 2.5, and 10 µM of H2O2 for 2 h without or with a 24 h recovery period was analyzed by comet assay. * p = 0.05; ** p = 0.01; *** p = 0.001.

Fig 3: MRNIP condensates incorporate the MRN complex and relocalize to damaged DNA.a MRNIP-GFP droplets compartmentalized DNA from solutions. MRNIP-GFP droplets that formed in vitro were incubated with DNA solutions and fractionated by centrifugation. b ?-H2A.X localized within MRNIP droplets in cells after 15 min recovery post irradiation. MRNIP-GFP-expressing HeLa cells were irradiated (2 Gy) and subjected to ?-H2A.X detection at indicated time. c The quantification of ?-H2A.X foci area in (b). Colocalized, ?-H2A.X foci colocalized with MRNIP puncta; Surrounding, ?-H2A.X foci didn’t colocalize with MRNIP puncta. Data are presented as means ± SEM. Colocalized, n = 30; Surrounding, n = 61. Two-tailed unpaired Student’s t test. d MRNIP-GFP condensates moved to the microirradiation-induced DNA damage site. IR, microirradiation; mock, no IR. HeLa cells were transfected with plasmid and incubated with 10 µM BrdU for 24 h before microirradiation. e MRNIP-GFP droplets compartmentalized the MRN complex from the nuclear extract of HeLa cells. The indicated protein was incubated with 10 µg of HeLa nuclear extract in buffer containing 150 mM NaCl (pH 7.4) for 20 min at 20 °C and fractionated by centrifugation. f MRNIP droplets incorporated with MRE11 in cells. Live-cell image of cells co-expressing MRE11-mCherry and MRNIP-GFP. HeLa cells were transfected with indicated plasmids for 24 h before observation. g Co-immunoprecipitation assay showed that IDR1-deleted MRNIP did not interact with MRN complex. h MRNIP-GFP droplets incorporated MRE11 and dsDNA in vitro. The indicated proteins or dsDNA were incubated in buffer containing 150 mM NaCl, pH 7.4, for 20 min at 20 °C. Rho Rhodamine.

Fig 4: MRNIP condensates accelerate the MRN complex loading and DNA damage response.a, b MRNIP condensates incorporated DNA from solutions within 10 s. Ten micromolar MRNIP was incubated with 15 ng/µL plasmid DNA or 0.5 µM Cy5-dsDNA in buffer containing 150 mM NaCl, pH 7.4. c MRNIP depletion reduced radiation-induced MRN complex binding to chromatin. d The impact of wild-type or IDR1-deleted MRNIP on the radiation-induced binding of the MRN complex to DNA. For (c, d), chromatin was fractionated at the indicated time after irradiation (10 Gy). EV, empty vector; ?IDR1, MRNIP-?IDR1-GFP. e, f The impact of MRNIP condensates on radiation-induced ATM and NBS1 phosphorylation. IR: 3 Gy. g, h IF assays were performed to analyse the impact of MRNIP on radiation-induced ATM phosphorylation. HeLa cells were irradiated with 3 Gy. Data are presented as means ± SEM. Two-tailed unpaired Student’s t test. g n = 116 (WT), n = 77 (KO-MRNIP). h n = 107 (EV), n = 93 (MRNIP-GFP), n = 103 (?IDR1). i IF assays were performed to analyze the impact of MRNIP on radiation-induced ?-H2A.X phosphorylation. HeLa cells were exposed to 3 Gy X-ray. Data are presented as means ± SEM. IR-: n = 69 (EV), n = 87 (MRNIP-GFP), n = 87 (?IDR1); IR 10 min: n = 124 (EV), n = 122 (MRNIP-GFP), n = 119 (?IDR1); IR 20 min: n = 111 (EV), n = 96 (MRNIP-GFP), n = 112 (?IDR1). Two-tailed unpaired Student’s t test. For (c–i), HeLa-KO-MRNIP cells stably expressing sgRNA-resistant MRNIP-GFP, MRNIP-?IDR1-GFP (?IDR1) and empty vector (EV) were used.

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.