Additional file 1 of APE1 promotes non-homologous end joining by initiating DNA double-strand break formation and decreasing ubiquitination of artemis following oxidative genotoxic stress
Creators: name: Zhang, Qin
name: Yang, Lujie
name: Gao, Han
name: Kuang, Xunjie
name: Xiao, He
name: Yang, Chen
name: Cheng, Yi
name: Zhang, Lei
name: Guo, Xin
name: Zhong, Yong
name: Li, Mengxia
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Science Policy
subject: Cancer

description: Additional file 1. Figure S1. APE1 involves in DSBs formation at early phase following IR stress. (a–b) HeLa or SiHa NC and shAPE1 cells were mock-treated or treated with dose-depended IR as indicated in figures and allowed to recover for 1 h. Whole cell lysates were obtained and immunoblotting was performed to assess the γ-H2AX. H2AX and tubulin or actin was used as a loading control. (c) Representative images of Figure 1H-I were shown. Figure S2. APE1 involves in DSBs formation at early phase following TBHP exposure. HeLa scramble and shAPE1(7958) cell lines were treated with 100 μM TBHP for 1h and allowed to recover for 1, 8, 16 and 36h, the distribution of γ-H2AX foci were assessed by IF. Figure S3. APE1 involves in DNA damage response following genotoxic stress. (a–b) IR induced DDR are attenuated in the APE1 deficient cells. HeLa or SiHa NC and shAPE1 cells were mock-treated or treated with dose-depended IR as indicated in figures and allowed to recover for 1 h. Whole cell lysates were obtained and immunoblotting was performed to assess the DNA-PKcs pS2056, ATM pS1981, KAP1 pS824. Actin was used as a loading control. (c) Cell cycle distribution after TBHP exposure. HeLa NC and shAPE1 cells were mock-treated or treated with TBHP for 1 h and allowed to recover for 1 h. Flow cytometry were performed to analysis of the distribution of cell cycle, representative images of figures were shown. (d) The data from (c) is presented as mean ± SD from three independent experiments. (e) HeLa scramble and shAPE1(7958) cells were treated with TBHP and allowed to recover for various time from 0 h to 48 h. The γ-H2AX, DNA-PKcs, and cleaved-PARP level were assayed by immunoblotting. (f) The interaction between APE1 and DNA-PKcs in HeLa WT cells, assayed by APE1 immunoprecipitation, was significantly increased post- IR treatment and allowed to recover 1 h. Figure S4. APE1 deficiency leads to increased Artemis protein degradation. (a) The top 20 significantly enriched KEGG pathways of the differentially expressed genes (DEG) between HeLa NC cells and shAPE1 cells. (b) The quantification of Artemis/actin ratio Figure 4A normalized to the value of NC cell line, respectively. (c) Protein expression levels of Artemis in scramble and shAPE1(7658) of HeLa and SiHa cells. (d) HeLa WT cell line was transfected with NC, shAPE1, siArtemis, or both, then cells were treated with TBHP for 1h, and proliferation were analyzed by EdU assay 24 h later. (e) quantitative analysis (d) of proportion of the EdU positive cells. Figure S5. APE1 deficiency have a synergistic lethal effect with ATM inhibitor in vivo. (a-b) Resected tumors after completion of treatment (Figure 6e and 6g) are shown.