Fig 2: Model for the involvement of RECQL4 in OGG1-mediated removal of 8-oxoG and its regulation by SIRT1 deacetylase. In response to oxidative stress, RECQL4 becomes hyperacetylated, which enhances its interaction with OGG1 to promote 8-oxoG repair. After repair, SIRT1 outcompetes OGG1 from interaction with RECQL4 to return it to a hypoacetylated state. However, 8-oxoG repair is impaired in RECQL4- deficient cells, because of the loss of stimulatory effect of RECQL4 on OGG1, which leads to increased genomic 8-oxoG lesions.

Fig 3: SIRT1 controls the interaction between OGG1 and RECQL4 following oxidative stress and maintains RECQL4 in a hypoacetylated state. (A) Western blot analysis of acetylation of RECQL4 in shCtrl or shSIRT1 cells. Knockdown of SIRT1 significantly increased RECQL4 acetylation. shCtrl and shSIRT1 cells were transfected with FLAG-RECQL4. 24 hours after transfection, whole-cell lysates were subjected to immunoprecipitation with FLAG-M2 magnetic beads followed by Western blot analysis of the immunoprecipitated material with anti-acetylated lysine and anti-RECQL4 antibodies. A representative gel is shown on the left. The graph on the right shows quantification of relative acetylated RECQL4 levels normalized to total RECQL4. Data are shown as mean ± SD from two independent experiments. ***P < 0.001, using unpaired two-tailed Student's t test. (B) Immunoprecipitation (IP) was performed in shCtrl and shSIRT1 cells expressing FLAG-tagged RECQL4 with FLAG-M2 magnetic beads and analyzed by western blot with the indicated antibodies. shCtrl and shSIRT1 cells expressing FLAG-tagged RECQL4 were treated with 50µM menadione for 1 h, then the media were replaced with fresh media and the cells were collected at indicated time points. Interaction between RECQL4 and SIRT1 decreased immediately after menadione treatment (0 h recovery) both in shCtrl and shSIRT1 cells. This interaction increased at 6 h recovery, compared with 0 h recovery. Lamin B1 was used as a loading control. A representative gel is shown on the left. The graph on the right shows the relative ratio of immunoprecipitated SIRT1 to FLAG-RECQL4 from shCtrl cells. Data are shown as mean ± SD from two independent experiments. *P < 0.05, **P < 0.01, using unpaired two-tailed Student's t test. (C) Time-course dependent interaction between RECQL4 and SIRT1 following 1 h 50 µM menadione treatment. Cells were transfected with FLAG-RECQL4 and cell lysates were collected at 0, 1, 2, 4, 6, and 12 h after menadione treatment. Western blot analysis of the IP complexes was carried out by anti-FLAG and anti-SIRT1 antibodies. A representative gel is shown on the left. The graph on the right shows the relative ratio of immunoprecipitated SIRT1 to FLAG-RECQL4 from shCtrl cells. Data are shown as mean ± SD from three independent experiments. *P < 0.05, ***P < 0.001, using unpaired two-tailed Student's t test. (D) Immunoprecipitation (IP) was performed in shCtrl and shSIRT1 cells expressing FLAG-tagged RECQL4 with FLAG-M2 magnetic beads and analyzed by Western blot using the indicated antibodies. SIRT1 knockdown significantly affects the interaction between RECQL4 and OGG1. A representative gel is shown on the left. The graph on the right shows the relative ratio of immunoprecipitated OGG1 to FLAG-RECQL4 from shCtrl and shSIRT1 cells. Data are shown as mean ± SD from three independent experiments. ***P < 0.001, using unpaired two-tailed Student's t test. (E) Immunoprecipitation (IP) was performed in cells expressing FLAG-tagged RECQL4 (WT), RECQL4 (KQ) mutant, and RECQL4 (KR) mutant with FLAG-M2 magnetic beads treated with or without menadione, and analyzed by Western blot using the indicated antibodies. Five lysine mutants impaired the interaction between RECQL4 and OGG1. A representative gel is shown on the left. The graph on the right shows relative ratio of immunoprecipitated OGG1 to FLAG-RECQL4. Data are shown as mean ± SD from two independent experiments. *P < 0.05, using unpaired two-tailed Student's t test.

Fig 4: SYT7 is a potential target gene of OGG1. (A, B) After A549 cells were transfected with scrambled or OGG1 siRNA for 48 h, the mRNA levels of OGG1 (A) and SYT7 (B) were examined. (C) Cells were treated as panel (A). Western blotting was used to determine the protein levels of OGG1 and SYT7. Right, quantification of OGG1 and SYT7. (D, E) The mRNA (D) and protein (E) levels of SYT7 in the WT and OGG1‐KO cells were examined by RT‐qPCR and Western blotting. (F) Analysis of CpG islands 2 kb upstream of the TSS region of SYT7. Upper graph: A plot of the ratio of observed GC content to expected GC content. Middle graph: GC content percentage. Lower graph: The CpG island was predicted to exist at −844 to −55 nucleotides (nt) upstream of the TSS region of SYT7. (G, H) The mRNA (G) and protein (H) levels of SYT7 in A549 cells treated with or without 20 µM H2O2 were analysed. Right, quantification of SYT7 level. (I, J) After cells were treated with 20 µM H2O2 and 10 mM NAC for 24 h, the mRNA (I) and protein (J) levels of SYT7 were analysed. Right, quantification of SYT7. (K, L) After cells were treated with or without 10 µM Th5487 for 24 h, the mRNA (K) and protein (L) levels of SYT7 were analysed. Right, quantification of SYT7 level. (M, N) After cells were treated with or without 10 µM OGG1‐IN‐08 for 24 h, the mRNA (M) and protein (N) levels of SYT7 were analysed. Right, quantification of SYT7 level. All of the data are expressed as mean values ± SEM (n = 3); **p < 0.01, ***p < 0.001. (Student's t‐test).

Fig 5: Schematic diagram of OGG1 affecting SYT7 expression to mediate EVs release. This model shows that under oxidative stress, OGG1 increases the accessibility of NF‐κB in the SYT7 promoter region, upregulates its expression, further promotes the secretion of EVs and participates in the EMT process.