Fig 2: NEDD4 attenuates cellular damage and release of inflammatory factors in ox-LDL-induced HUVECs via regulating APEX1 expression. (A) RT-qPCR and (B) western blotting were employed to detect the interference level of APEX1 following the transfection of siRNA-APEX1-1 and siRNA-APEX1-2. (C) CCK-8 was used to test cellular activity damage. (D) An LDH kit was used to measure cytotoxic damage under the treatment of ox-LDL, overexpression of NEDD4 and interference with APEX1. (E) Detection of TNF-a, IL-1ß and IL-6 expression was conducted by RT-qPCR. **P<0.01 and ***P<0.001. NEDD4, neuronally expressed developmentally downregulated 4; ox-LDL, oxidized low density lipoprotein; HUVECs, human umbilical vein endothelial cells; APEX1, apurinic/apyrimidinic endodeoxyribonuclease 1; RT-qPCR, reverse transcription-quantitative PCR; si, short interfering; LDH, lactate dehydrogenase.

Fig 3: NEDD4 binds to APEX1 and its overexpression promotes the expression of APEX1. Expression of APEX1 in ox-LDL-induced HUVECs was detected by (A) RT-qPCR and (B) western blotting. (C and D) That NEDD4 bound to APEX1 in HUVEC cells was demonstrated by co-immunoprecipitation assay. Expression of APEX1 was determined after overexpression of NEDD4 by (E) RT-qPCR and (F) western blotting. ***P<0.001. NEDD4, neuronally expressed developmentally downregulated 4; APEX1, apurinic/apyrimidinic endodeoxyribonuclease 1; ox-LDL, oxidized low density lipoprotein; HUVECs, human umbilical vein endothelial cells; RT-qPCR, reverse transcription-quantitative PCR.

Fig 4: The endonuclease activity of APE1 is required for cell survival after IR treatment. a–b HeLa and SiHa cell lines were plated and treated with IR at the indicated doses. Colony formation assays were performed to compare sensitivities of the NC and shAPE1 cells. c–f HeLa and SiHa cells were pre-incubated with Inhibitor III c, e or E3330 d, f at the indicated doses for 6 h prior to 3 Gy IR treatment. Colony formation assays were then performed to analysis of survival ability. (g-i) NC and shAPE1 cells of HeLa and SiHa cell line were treated with IR, and allowed to recover for 96 h, followed by DAPI staining. The representative graph (HeLa) treated by IR was shown in g. h–i Quantitative analysis of proportion of the number of cells with MNs. j The APE1 expression was evaluated by IHC in tumor tissues and paired peri-tumor tissues of 16 cervix cancer samples. The representative images were shown (left) and the bar graph showing the percentage of each score level of APE1 in peri-tumor and tumor tissue, respectively (right). k–l The tissue expression of APE1 were evaluated by IHC in 46 cervix cancer patients receiving radical chemo-radiotherapy. Protein levels were scored for four categories: score-, score + , score + + , and score + + + . The representative images were shown k. Kaplan–Meier plot showing different overall survival of cervical cancer patients according to APE1 expression l. The data were presented as the mean ± SEM from three independent experiments. The p-values were determined using an unpaired Student’s t-test (**p < 0.01, *p < 0.05)

Fig 5: APE1 initiates DSBs generation at an early phase following IR exposure. a–b APE1 affected temporal formation of DSBs following IR Exposure. NC and shAPE1 cells were treated with 10 Gy IR (treatment for IR was used throughout the study unless stated otherwise) and allowed to recover for various time from 1 to 64 h in HeLa a and SiHa b cell line. The γ-H2AX and cleaved-PARP level were assayed by immunoblotting. c–d The endonuclease capacity of APE1 involved in DSBs formation at early phase following IR exposure. HeLa c or SiHa d WT cells were pre-incubated for 6 h with E3330 (10 μM) or Inhibitor III (2.5 μM) prior to IR treatment and allowed to recover for 1 h. e Inducible HeLa APE1shRNA cells were stably transfected with the expression vector encoding wildtype APE1 (WT) or redox activity mutant of APE1 (C65S), respectively. APE1 levels in both cells pre- and post 14-day induction by doxycycline (Dox) were measured by immunoblotting. f The γ-H2AX was independent on the redox activity of APE1 at early phase post-damage. HeLa WT-APE1 and HeLa C65S-APE1cells were treated with IR then allowed to recover for 0.5 h or 1 h. g–h SSB and DSB level of HeLa NC and shAPE1 cell line was evaluated using alkaline and neutral Comet assay, respectively. Cells were treated with IR and allowed to recover for 1 h, followed by comet assays. Tail moment values were calculated for > 100 cells and plotted via a distribution dot plot. The data were presented as the mean ± SEM from three independent experiments. The p-values were determined using an unpaired Student’s t-test (****p < 0.0001)