Fig 2: K46 of Mxi1 is the major ubiquitination target residue of UBE2O.a Mass spectrometry analysis was used for identifying the ubiquitination sites in Mxi1. The di-glycine-modified K46 in Mxi1 was detected, and the corresponding peptide was LQHSKPPR. b Alignment of the K46 corresponding amino acids sequences in Mxi1 from different species. c HeLa (upper panel) or H1299 (lower panel) cells were co-transfected with vector expressing HA-UBE2O together with indicated plasmids. After 24 h, cells were lysed for immunoblot analysis (n = 3). d HeLa cells were co-transfected with the indicated plasmids for 24 h, followed by treatment with MG132 (10 µM) for 4 h prior to collection. Cells lysates were subjected to co-IP with S beads followed by western blotting (n = 3). e Left panel: HeLa cells were transfected with SFB-Mxi1-WT or SFB-Mxi1-K46R plasmid for 24 h, and then treated with cycloheximide (CHX, 20 µg/mL) for the indicated times before harvesting. Cells lysates were analyzed by immunoblotting. Right panel: Mxi1 band intensity was measured by ImageJ software (n = 3). ***P < 0.001.

Fig 3: UBE2O interacts with Mxi1 in vitro and in vivo.a HEK293T cells transfected with indicated plasmids for 24 h were treated with MG132 (10 µM) for 4 h. Cells were lysed with NETN buffer and then analyzed by co-IP with S beads or Myc agarose followed by western blotting (n = 3). b HeLa cells were transfected with SFB-Mxi1 or SFB-UBE2O for 24 h and treated with MG132 (10 µM) for 4 h. Cells were collected for co-IP with S beads followed by western blotting (n = 3). c HeLa, H1299, and A549 cell were treated with MG132 (10 µM) for 4 h and then harvested. Cell lysates were analyzed by co-IP with anti-IgG, Mxi1, or UBE2O antibodies followed by western blotting (n = 3). d Recombinant GST-only and GST-Mxi1 protein were purified from BL21 bacteria. Cell lysates from HEK293T cells transfected with SFB-UBE2O were incubated with GST or GST-Mxi1 proteins coupled to GST beads overnight and then detected by immunoblotting (n = 3).

Fig 4: Loss of UBE2O reduces tumor growth and enhances radiosensitivity in lung cancer in vitro and in vivo.a Upper panel: cell lysates were analyzed by immunoblotting with anti-UBE2O antibody. Lower panel: quantification of UBE2O band intensity was shown (n = 3). b H1299 cells stably expressing sh-Control or sh-UBE2O were collected and examined the knockdown efficiency of UBE2O by western blotting (n = 3). c Nude mice were subcutaneously injected with H1299 cells stably expressing sh-Control or sh-UBE2O. The tumor volumes were measured every 3 days (n = 4 mice/group). Data were shown as mean ± SEM. **P < 0.01. d Left panel: representative images of the comet tail. Scale bar, 50 µm. Right panel: quantification of the olive tail moment in each group were shown (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. e Left panel: representative immunostaining images of Rad51 foci. A cell containing 10 or more foci was considered as a foci-positive cell. Scale bar, 10 µm. Right panel: the percentage of Rad51 foci-positive cells in each group were shown (n = 3). **P < 0.01, ***P < 0.001. f H1299 and A549 cells were transfected with indicated siRNAs and irradiated at different doses as indicated respectively. After 14 days, cell colonies were fixed and counted, and the survival fraction was calculated (n = 3). **P < 0.01, ***P < 0.001. g The growth curves of the xenograft tumors for each group are represented. H1299 cells stably expressing sh-Control or sh-UBE2O were subcutaneously injected in the right hind limb of nude mice, respectively. The mice were irradiated with 10 Gy when the tumor volume reached 100 mm3. Data were shown as mean ± SEM (n = 5 mice/group). *P < 0.05, **P < 0.01.

Fig 5: UBE2O promotes lung cancer cell proliferation and radioresistance through negative regulation of Mxi1.a H1299 cells transfected with indicated siRNAs were lysed and analyzed by immunoblotting (n = 3). b H1299 cells transfected with indicated siRNAs were seeded in six-well plates with 1 × 104 cells and counted every other day (n = 3). **P < 0.01, ***P < 0.001. c H1299 cells transfected with the indicated siRNAs were seeded in six-well plates with 200 cells/well. Two weeks later, cell colonies were fixed and counted (up to 50 cells) (n = 3). ***P < 0.001. d Representative images of EdU labeled cells in each group. H1299 cells were transfected with the indicated siRNAs for 48 h, and then incubated with EdU for 2 h (n = 3). Scale bar, 50 µm. e Left panel: representative comet tail images in each group. Scale bar, 50 µm. Right panel: quantification of the olive tail moment was shown (n = 3). **P < 0.01. f Left panel: representative images of Rad51 foci in each group. Scale bar, 10 µm. Right panel: the percentage of Rad51 foci-positive cells in each group were shown (n = 3). ***P < 0.001. g H1299 cells were transfected with the indicated siRNAs and then irradiated at various doses. Two weeks later, the colonies were calculated (up to 50 cells). The survival fraction was shown as indicated (n = 3). *P < 0.05.