Fig 2: RanBPM stabilizes and deubiquitinates p21 protein. (A) A549 or H1299 cells were transiently transfected with the indicated siRNAs, and the proteins were assessed by western blotting. (B) A549 or H1299 cells were transfected with the indicated plasmids, and the proteins were assessed by western blotting. (C and D) A549 or H1299 cells were infected with the indicated siRNAs (C), or transfected with the indicated plasmids (D), and the mRNA was subjected to qRT-PCR. The error bars represent the SD of triplicate measurements. (E) A549 or H1299 cells were transfected with the indicated siRNAs for 48 h, and then were treated with DMSO or MG132 (20 μM) for additional 6 h. The indicated proteins were analyzed by western blotting. (F) A549 or H1299 cells were transfected with the indicated plasmids for 24 h, and then were treated with DMSO or MG132 (20 μM) for additional 6 h. The indicated proteins were analyzed by western blotting. (G and H) A549 cells were transfected with the siRNAs of scrambled or siRanBPM#1 (G) or siRanBPM#2 (H), and then were treated with 50 μg/mL CHX. The resulting cell extracts were collected at the indicated time points for western blot analysis. The relative values of p21 to GAPDH expression were quantified. (I) A549 cells were transfected with the indicated plasmid constructs, and then were treated with 50 μg/mL CHX. The cells were collected at the indicated times, and proteins were analyzed by western blotting. The relative values of p21 to GAPDH expression were quantified. (J) HEK293T were transfected with plasmids encoding Flag-RanBPM, Myc-p21 and HA-Ubiquitin for 48 h, and then treated with MG132 (20 μM) for 6 h before harvesting. p21 protein was immunoprecipitated with anti-Myc antibody, and the ubiquitination level of p21 protein was analyzed with anti-HA antibody. (K) HEK293T cells were transfected with the indicated siRNA for 24 h, and followed by co-transfection with Myc-p21 and HA-Ubiquitin for another 24 h. Cells were treated with MG132 (20 μM) for 6 h before harvesting. p21 protein was immunoprecipitated with anti-Myc antibody, and the p21 protein ubiquitination were tested with anti-HA antibody

Fig 3: RanBPM regulates DNA damage response in a p21-dependent manner. (A-C) A549 cells were transfected with the indicated siRNAs, and then the cells were treated with the indicated concentrations of Etop (B) or Dox (C). (D-G) A549 or H1299 cells were co-transfected with the indicated siRNAs and plasmid constructs, and the cells were treated with 5 μM Etop or 0.5μM Dox. The same number of cells were seeded for colony formation. Data are representative of three independent experiments and values are expressed in mean ± SEM (***p < 0.001). (H) A549 cells implanted into nude mice were lysed and analyzed using Western blotting. (I-L) The 5 × 106 indicated shRNA-transduced A549 cells were subcutaneously injected into mice. Tumor growth (I), tumor images (J), tumor weight (K) and were shown

Fig 4: DNA damage promotes the translocation of RanBPM and regulates p21 protein stability through ATM-mediated pathways. (A-C) A549 or H1299 cells were treated with 5μM Etop or 0.5μM Dox in the presence or absence of ATM inhibitor KU-55,933 (10μM), and the total proteins were assessed by western blotting. After cell fractionation, the subcellular fractions were blotted with the indicated antibodies(C). (D and E) A549 or H1299 cells were treated with 5μM Etop or 0.5μM Dox in the presence or absence of ATM inhibitor KU-55,933 (10μM), and the cells were stained with the indicated fluorescent antibodies. DAPI was used for nuclei staining. Scale bars, 20 μm

Fig 5: RanBPM is significantly downregulated, and positively correlated with p21 in NSCLC tissues. (A) Representative immunohistochemical (IHC) images of RanBPM in NSCLC tissues or the matched adjacent tissues. (B) Quantitation of RanBPM protein levels from IHC images. (C) Correlation analysis of the mRNA levels of RanBPM and p21 genes in NSCLC samples from TCGA datasets. (D) Representative IHC images of RanBPM and p21 in NSCLC tissues. (E) Regression analysis comparing RanBPM and p21 expression in NSCLC tissues. n = 52