Fig 2: RPRM binds to ATM and down-regulates ATM levels(A–C) Co-IP was performed 30 min after X-irradiation to demonstrate the interaction between ATM and RPRM in H460 (A), H1299 (B) and H460-RPRM (C) cells.(D) Representative immunofluorescence images of co-localization of RPRM and ATM in RPRM-/- MEFs re-expressing RPRM at different times after 20 Gy X-irradiation.(E and F) Change in the levels of total ATM and phospho-ATM in H460-NC (N)/RPRM (R) cells at different times after 2 Gy X-irradiation. Quantification of ATM levels (F), which was the average of three independent experiments.(G and H) The ATM levels of WT MEFs were lower than those of RPRM-/- MEFs at different times after 20 Gy X-irradiation. Bar graph (H) shows quantification of ATM levels.(I and J) RPRM re-expression reduced the ATM levels in RPRM-/- MEFs after 20 Gy X-irradiation. Bar graph (J) shows quantification of ATM levels.Data shown represent the means (±SEM) of three biological replicates, *p < 0.05, **p < 0.01; significance was determined by unpaired two-sample t test.

Fig 3: RPRM inhibits DNA repair signaling pathway and enhances cellular radiosensitivity(A) Representative immunofluorescence images of γ-H2AX in H460-NC/RPRM cells after 2 Gy X-irradiation and quantification of the number of γ-H2AX foci per cell. At least 200 nuclei were scored. Scale bars, 20 μm.(B) Immunofluorescence images of 53BP1 foci after X-irradiated with 2 Gy and quantification. At least 200 nuclei were scored. Scale bar, 20 μm.(C) Immunofluorescence images of RAD51 foci after IR and quantification. Scale bars, 10 μm. At least 500 nuclei for each sample were scored in each experiment.(D) Comet assay on H460-NC/RPRM cells at different times after 2 Gy X-irradiation. The tail moments were quantified using CometScore. At least 500 cells were scored per sample. Scale bars, 50 μm.(E) RPRM overexpression decreased the colony formation ability of cancer cells. Sensitization enhancement ratio (SER) was calculated according to the following formula: The radiation dose required for the control group to obtain 30% cell survival/the radiation dose required to obtain the same cell survival rate after overexpression of RPRM.(F) RPRM overexpression increased micronucleus formation in cancer cells upon 2 Gy X-irradiation.(G and H) RPRM knockdown significantly increased cellular radio-resistance manifesting as increased plating efficiency (G, 4 Gy) and decreased micronucleus formation (H, 2 Gy) in irradiated H460 cells.(I) RPRM knockout significantly reduced the micronucleus formation in MEFs upon 5 Gy X-irradiation.(J) Micronucleus formation of RPRM−/− MEFs transfected with NC and WT RPRM plasmids after exposed to 5 Gy X-irradiation.Data shown represent the means (±SEM) of three biological replicates, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; significance was determined by unpaired two-sample t test.

Fig 4: RPRM promotes the nuclear export and degradation of ATM(A and B) Kinetics of the change in the ATM levels of H460-NC/RPRM cells with and without irradiation in the presence of cycloheximide (CHX, 0.1 mg/mL) examined by immunoblotting. (*, RPRM vs NC; #, RPRM + IR vs NC + IR; &, RPRM + IR vs RPRM).(C and D) Change in the ATM levels of H460-NC (N)/RPRM (R) cells at the indicated time points after 2 Gy X-irradiation with and without MG132 (10 µM) pre-treatment.(E) Change in the subcellular localization of ATM in RPRM-/- MEFs transfected with NC and RPRM vectors after 20 Gy X-irradiation and quantification of the ratios of cytoplasmic ATM fluorescence intensity to nuclear ATM fluorescence intensity. Scale bars, 10 µm.(F) Representative immunofluorescence images of ATM in H460-RPRM cells pre-treated with leptomycin B (LMB, 10 nM) for 1 h at different times after 2 Gy X-irradiation and quantification of the cytoplasmic/nuclear ATM fluorescence intensity ratios. Scale bar, 20 µm.(G) The effect of LMB pre-treatment on the total ATM levels of H460-NC/RPRM cells at different times after 2 Gy X-irradiation.(H) Effect of LMB on the micronucleus formation of H460-NC/RPRM cells irradiated with 2 Gy X-rays.Data shown represent the means (±SEM) of three biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ###p < 0.001, &p < 0.05; significance was determined by unpaired two-sample t test.

Fig 5: CDK4/6 phosphorylate RPRM at serine 98, but the unphosphorylation status of RPRM is critical for its stabilization and nuclear translocation(A and B) Mass spectrometry analysis of the phosphorylation site of RPRM corresponding to CDK4 (A) and CDK6 (B).(C) Time-course analysis of the changes in the RPRM levels of H460-RPRM cells after treated with different inhibitors.(D) Diagrams of full-length RPRM protein and different mutants.(E) Representative immunofluorescence images of the RPRM-/- MEFs exogenously expressing full-length RPRM, RPRM-S98A and ?RPRM (79–109) at 30 min after 20 Gy X-irradiation. Scale bars, 10 µm.(F) Co-IP assay confirmed an interaction between ATM and both RPRM-S98A and ?RPRM (79–109) in H460 cells.(G and H) Change in the ATM levels of H460-NC/RPRM/S98A cells at different times after 2 Gy X-irradiation.(I) Change in the ATM expression of H460-NC/RPRM/S998A cells after cisplatin (CDDP, 15 µM) treatment for 24 h.(J) Relative plating efficiency of H460-NC/RPRM/S98A cells irradiated with 4 Gy X-rays.(K) Exogenous expression of both RPRM and RPRM-S98A increased micronucleus formation in H460 cells upon 2 Gy X-rays.(L) Relative plating efficiency of H460-NC/RPRM/S98A cells treated with 15 µM CDDP.(M) Micronucleus formation of RPRM-/- MEFs expressing full-length RPRM, RPRM-S98A and ?RPRM (79–109) after exposed to 5 Gy X-irradiation.Data shown represent the means (±SEM) of three biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001; Significance was determined by unpaired two-sample t test.