Fig 2: RPLP0 promotes NHEJ-mediated DSB repair.A Knockdown of RPLP0 extends the recovery time of IR-induced DNA damage. Forty-eight hours after siRNA transfection, HCT116 and U2OS cells were irradiated (10 Gy), and cultured for indicated time before western blotting analysis. B Knockdown of RPLP0 increases ?-H2A.X foci in tumor cells after IR. Thirty-six hours after transfection, HCT116 cells were irradiated (2 Gy) and cultured for indicated time. Cells were fixed and subjected to immunofluorescence analysis. n = 120, 85, 59, 96, 73, 58, 62, 75, 96, 62, 68, 93, 71, 81, 64, and 68 (from left to right column). Scale bars, 10 µm. C 4-OHT induces the nuclear import of ER-RPLP0. Cells were treated with 300 nM 4-OHT for 4 h before IF assay. Scale bars, 10 µm. D Nuclear RPLP0 enhances DNA repair. Thirty minutes before irradiation (2 Gy), 300 nM 4-OHT was added to medium. Cells were cultured for 20 h after irradiation. n = 203 (DMSO), n = 170 (4-OHT). Scale bars, 10 µm. E Depletion of RPLP0 suppresses the ligation of linearized DNA in vitro. pCSCMV-tdTomato plasmid was linearized with BamHI and incubated with nuclear protein of U2OS cells. The ligation product was separated using 0.8% agarose gels. F RPLP0 silencing suppresses translation. Forty-eight hours after siRNA transfection, cells were subjected to polysome profile assay. The impact of translation inhibitor cycloheximide (CHX) on DNA repair capacity was examined using western blotting (G) or IF assay (H). HCT116 cells, treated with 100 µg/ml CHX for 12 h, were irradiated and cultured for indicated time before western blotting (G) or IF assay (H). n = 148 (Ctrl), n = 183 (CHX). ***, P < 0.001; ns no significance.

Fig 3: NONO and RPLP0 bind to damaged DNA and increase the phosphorylation of DNA-PK at pT2609.A RPLP0 colocalizes with ?-H2A.X after irradiation. U2OS cells were irradiated (2 Gy) and cultured for 30 min before analysis. Scale bars, 10 µm. B NONO and RPLP0 load into chromosome upon DNA damage. Twelve hours after seeding, cells were treated with radiation (10 Gy) and cultured for indicated time before chromosome fractionation. C 4-OHT induces DNA damage by facilitating the nuclear import of endonuclease ER-AsiSI. Cells were treated with 300 nM 4-OHT for 4 h before IF assay. Scale bars, 10 µm. D The binding of NONO and RPLP0 to damaged DNA was examined with ChIP assay. Four hours after 4-OHT or DMSO treatment, ER-AsiSI-expressing U2OS cells were analyzed with ChIP assay. E, F NONO depletion or RPLP0 silencing inhibits the phosphorylation of DNA-PK at T2609. U2OS cells were irradiated (10 Gy) and cultured for indicated time before nuclei isolation, which were further analyzed by western blotting. *, unspecific band. G The construction of NONO-RPLP0 fusion protein. (GGGGS)3, triplication of GGGGS; P2A, GSGATNFSLLKQAGDVEENPGP. H NONO-RPLP0 fusion protein promotes DNA repair. n (Vector) = 178 nuclei, n (NO-P0) = 199 nuclei, n (P2A) = 179 nuclei. Scale bars, 10 µm. ***; P < 0.001; ns no significance.

Fig 4: Irradiation increases the association between NONO and ribosomal protein RPLP0.A Screening of IR-induced NONO-binding protein. HEK 293T cells were transfected with indicated plasmid and treated with IR (5 Gy). After recovery (30 min), the protein interacting with NONO was assessed by CoIP, separated with SDS-PAGE, and identified with LC/MS assay. GFP was used as a negative control. B The differentially expressed band at 36 kDa in Fig. 1A was analyzed by LC/MS. C The sample in Fig. 1A was analyzed with western blotting. D Flag-RPLP0 and Myc-NONO were interacted with each other in HEK 293T cells. Twenty-four hours after transfection with Flag-RPLP0 and Myc-NONO plasmid, cells were subjected to CoIP assay with anti-Myc or anti-Flag antibodies. E endogenous RPLP0 and NONO are associated in HCT116 cells. NONO or RPLP0-binding protein was assessed by CoIP assay and analyzed using western blotting. F IR enhances the interaction of NONO and RPLP0. HCT116 or U2OS cells were treated with IR (10 Gy) and recovered for indicated time before CoIP assay. G Duolink PLA assay was performed to analyze radiation-induced interaction between NONO and RPLP0. Cells were irradiated with 10 Gy X-ray and cultured for 1 h before PLA assay. n = 130 (0 Gy), n = 90 (10 Gy, antibodies), n = 109 (10 Gy, IgG). ***, P < 0.001.

Fig 5: IR-induced paraspeckles recruit RPLP0 and enhances the interaction of NONO and RPLP0.A Purified NONO-GFP protein formed condensates in vitro. Ten µM NONO protein and 100 ng/ul total RNA extracted from HEK 293T cells were used. Scale bars, 5 µm. B NONO-GFP form condensates with high rate of FRAP in vivo. n = 3 biological replicates. Scale bars, 5 µm. C NONO condensates recruit RPLP0 protein in vitro. Purified NONO-GFP protein formed condensates in vitro, which were then incubated with nuclear proteins of U2OS cells for 10 min. NONO condensates were separated by centrifugation and subjected to western blotting analysis. D 1,6-Hexanediol disrupts the association between NONO and RPLP0. Cells were irradiated (10 Gy) and cultured with complete medium containing 1.5% 1,6-Hexanediol for 30 min before Duolink PLA assay. n = 74 (Ctrl), n = 78 (1,6-Hex). Scale bars, 5 µm. E IR induces paraspeckle formation. U2OS cells were treated with radiation (5 Gy) and allowed to recover for indicated time before IF assay. n = 3 biological replicates. Scale bars, 10 µm. F RNA-immunoprecipitation assay showed that RPLP0 was associated with NEAT1. 28s rRNA and U6 were used as the positive and negative control, respectively. G RNase A disrupts the association between NONO and RPLP0. RNase A (0.1 µg/µl) was added to U2OS cell lysate before CoIP assay. To verify the RNase A-mediated removing of RNA, total RNA was extracted from 10% of flow through lysate and analyzed with gel electrophoresis. H siNEAT1 reduced the interaction between NONO and RPLP0. HCT116 cells were transfected with siNEAT1 (pool of siNEAT1-1 and siNEAT1-2) for 48 h before CoIP (right panel) or RT-PCR (left panel) analysis. ***, P < 0.001.