Fig 2: The combined use of ZSTK474 and TMZ reduced HR repair efficiency. (a)–(d) SF295 and U87 cells were treated with ZSTK474 and TMZ as single agents or in combination for 48 h. (a) The formation of RAD51 foci was assessed using immunofluorescence. Cell nuclei were stained with DAPI. Scale bar, 20 μm. Representative images are shown. (b) Quantification of the number of RAD51-positive SF295 and U87 cells. (c) The cells were collected for western blotting detection of NBS1, p-ATM, BRCA1, BRCA2, DNA-PKcs, and Ku80. (d) Western blotting analysis of PI3K-p110β, p-Akt, p-mTOR, and c-Myc. All data are presented as the mean ± SD (n = 3). ∗P < 0.05; ∗∗P < 0.01.

Fig 3: Interactions of nGRB2 with DNA damage repair factors.(A) MS identifies DDR protein associated with GRB2. Mean of coverage and unique peptides from three independent data sets are shown. (B) Strep-GRB2 precipitated from HEK293T cells followed by immunoblotting with indicated antibodies. (C) Strep-GRB2 expressed HEK293T cells treated with or without 5-Gy IR were lysed immediately followed by Strep-Tactin precipitation and immunoblotting with indicated antibodies. (D) HEK293T cells were cotransfected with red fluorescent protein (RFP)–GRB2 and green fluorescent protein (GFP)–NBS1 or GFP alone as control, precipitated with GFP-trap beads, and immunoblotted with indicated antibodies. (E) HEK293T cells expressing Flag-RAD50 or Flag-Ctrl were cotransfected with RFP-GRB2 for 24 hours. Unperturbed cells were immunoprecipitated with Flag-M2 beads and immunoblotted with indicated antibodies. (F) HEK293T cells expressing Flag-MRE11 were cotransfected with Strep-GRB2 or Strep-Ctrl for 24 hours. Unperturbed or IR-treated (5 Gy) cells were immunoprecipitated with Flag-M2 beads and immunoblotted with indicated antibodies. (G) Strep-Tactin precipitation of WT and K109R mutant Strep-GRB2 from HEK293T cells that were unperturbed or IR treated (5 Gy) and lysed, followed by Western blot detection with the indicated antibodies. (H) MST isotherms of 100 nM Atto488–labeled WT, K109R, or K109A mutant to titrating concentrations of human MRE11 (residues 1 to 411). All data are representative of three independent experiments.

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; ΔIDR1, MRNIP-ΔIDR1-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 (ΔIDR1). i IF assays were performed to analyze the impact of MRNIP on radiation-induced γ-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 (ΔIDR1); IR 10 min: n = 124 (EV), n = 122 (MRNIP-GFP), n = 119 (ΔIDR1); IR 20 min: n = 111 (EV), n = 96 (MRNIP-GFP), n = 112 (ΔIDR1). Two-tailed unpaired Student’s t test. For (c–i), HeLa-KO-MRNIP cells stably expressing sgRNA-resistant MRNIP-GFP, MRNIP-ΔIDR1-GFP (ΔIDR1) and empty vector (EV) were used.

Fig 5: miR-494 targets the MRN complex in ECs.a miR-TRAP immunoprecipitation. Bars depict mean + SEM of mRNAs bound to the RISC complex quantified by qRT-PCR in HEK-293T cells transfected with miR-494 for 24 h. b qRT-PCR depicting target mRNA levels in HUVECs transfected with either miR-negative control or miR-494. Bars show mean + SEM of the three MRN complex members. c Representative western blot of MRE11a, RAD50, and NBN after 48 h transfection of indicated miRs in HUVECs. d Luminescence from 3’-UTR-luciferase constructs for MRE11a, RAD50, and NBN 24 h after transfection with miR-494. Graph represents mean + SEM of three independent experiments. e HUVECs were plated on glass coverslips and 24 h later transfected with either miR-494 or miR-negative control. After 48 h, cells were fixed and stained with MRE11a antibody. Scale bar represents 10 µm. Bar graph depicts average foci/ cell + SEM. f β-Gal assay and g telomerase activity in HUVECs transfected for 48 h with specific siRNAs against MRE11a, RAD50, and NBN, or an siRNA-negative control. Bars depict % mean + SEM. *P < 0.05; two-tailed Student’s T-test