Additional file 1 of ACOX2 destabilizes the MRE11-RAD50-NBS1 complex and boosts anticancer immunity via the cGAS-STING pathway in clear cell renal cell carcinoma

Supplementary Material 1. Figure S1. ACOX2 is downregulated in KIRP and KICH and associated with a poorer prognosis in ccRCC. A, B Relative mRNA expression of ACOX2 in tumor and normal tissues of KIRP (A) and KICH (B) from TCGA and GTEx databases. C, D Kaplan–Meier survival curves for progression-free survival (PFS) (C) and disease specific survival (DSS) (D) of ccRCC patients with low or high expression of ACOX2 from TCGA-KIRC cohort. Statistical significance was determined by two-tailed unpaired t-test (A, B) and the two-sided log-rank (Mantel–Cox) test (C, D). * P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, and ns P ≥ 0.05. Figure S2. ACOX2 inhibits the tumor biological characteristics of ccRCC. A, B Immunoblotting of ACOX2 in the indicated 769-P (A) and A-498 (B) cells. C, D Colony formation assay of indicated 769-P (C) and A-498 (D) cells. E, F Growth curves of indicated 769-P (E) and A-498 (F) cells using CCK-8. G, H Wound healing assay of indicated 769-P (G) and A-498 (H) cells. Scale bar: 200μm. I, J Transwell invasive assay of indicated 769-P (I) and A-498 (J) cells. Scale bar: 200 μm. K, L Percentage of apoptosis cell of indicated 769-P (K) and A-498 (L) cells with flow cytometry analysis. M, N The tumor figure of the indicated 786-O (M) and Caki-1 (N) CDX. Statistical significance was determined by two-tailed unpaired t-test (C-L). * P< 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, and ns P ≥ 0.05. Experiments were independently repeated three times with similar results; data of one representative experiment are shown (A-D, G-L). Figure S3. ACOX2 neither interacts with RAD50 or NBS1 nor affects MRN complex component expression. A Co-IP between Flag-ACOX2 and RAD50 or NBS1, with or without ACOX2 overexpression. B Immunoblotting of RAD50, NBS1, MRE11, and ACOX2 in Caki-1 cells infected with indicated lentiviruses. Experiments were independently repeated three times with similar results; data of one representative experiment are shown (A and B). Experiments were independently repeated three times with similar results; data of one representative experiment are shown (A, B). Figure S4. ACOX2 inhibits homologous recombination repair in ccRCC cell. A The indicated 769-P cells were treated with or without CPT (2 μM) at the indicated time, and cell lysates were immunoblotted with the indicated antibodies. B The indicated 769-P cells were treated with or without CPT (4 h) at the indicated concentrations, and cell lysates were immunoblotted with the indicated antibodies. C The indicated 769-P cells were treated with or without CPT (2 μM, 4 h), and cell lysates were immunoblotted with the indicated antibodies. D, E Representative micrographs (D) and quantification data (E) for γ-H2AX foci formation in the indicated 769-P cells treated with or without CPT (2 μM, 4 h). Scale bar: 10 μm. F, G Representative micrographs (F) and quantification data (G) of comet assay in the indicated 769-P cells treated with or without CPT (2 μM, 4 h). Scale bar: 20μm. Statistical significance was determined by two-tailed unpaired t-test (E, G). * P < 0.05, **P < 0.01,*** P < 0.001, **** P < 0.0001, and ns P ≥ 0.05. Experiments were independently repeated three times with similar results; data of one representative experiment are shown (A-C, D, F). Figure S5. ACOX2 increases double-strand breaks in ccRCC cell. A The indicated HEK293T-DR-GFP cells transfected with I-Scel plasmids for HRR detection by flow cytometry analysis. B The indicated HEK293T-EJ5-GFP cells transfected with I-Scel plasmids for NHEJ detection by flow cytometry analysis. C The indicated 769-P cells were treated with or without CPT (2 μM, 4 h), and cell lysates were immunoblotted with the indicated antibodies. D-G Representative micrographs (D) and quantification data (E-G) for CtIP, RAD51, and RPA32 foci formation in the indicated 769-P cells treated with or without CPT (2 μM, 4 h). Scale bar: 10 μm. H The indicated 769-P cells were treated with or without CPT (2 μM, 4 h), and cell lysates were immunoblotted with the indicated antibodies. I-K Representative micrographs (I) and quantification data (J, K) for 53BP1 and RIF1 foci formation in the indicated 769-P cells treated with or without CPT (2 μM, 4 h). Scale bar: 10μm. Statistical significance was determined by two-tailed unpaired t-test (E-G, J, K). * P< 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, and ns P ≥ 0.05. Experiments were independently repeated three times with similar results; data of one representative experiment are shown (A-D, H, I). Figure S6. ACOX2 activates cGAS-STING pathway in ccRCC A The dynamics of protein abundance identified. Proteins are quantified as normalized iBAQ value and transformed to log10 Intensity. B The cumulative number of identified proteins in 10 cell samples. C, D Representative micrographs (C) and quantification data (D) for cytosolic dsDNA detected with the PicoGreen staining in the indicated 769-P cells. Scale bar: 10 μm. E Immunoblotting of cGAS-STING pathway markers in the indicated 769-P cells. F ELISA of IFN-α, IFN-β, CCL5, and CXCLl0 in the indicated 769-P cells. G Gating strategy for flow cytometry analysis. H Representative hematoxylin-eosin (H&E) staining for intratumoral TLS in postoperative ccRCC specimen. Scale bar: 100 μm. Statistical significance was determined by two-tailed unpaired t-test (D, F). * P< 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, and ns P ≥ 0.05. Experiments were repeated three times independently with similar results; data of one representative experiment are shown (C, E). Figure S7. ACOX2 sensitizes ccRCC to olaparib in vitro and in vivo. A Immunoblotting of ACOX2 in the indicated 769-P cells. B IC50 of olaparib in the indicated 769-P cells. C Colony formation assay in the indicated 769-P cells in response to olaparib D Immunoblotting of ACOX2 in the indicated A-498 cells. E IC50 of olaparib in the indicated A-498 cells. F Colony formation assay in the indicated A-498 cells in response to olaparib. G Tumor figures of Caki-1 CDX. H Tumor figures of ccRCC PDX. Statistical significance was determined by two-tailed unpaired t-test (C, F). * P < 0.05, **P < 0.01,*** P < 0.001, **** P < 0.0001, and ns P ≥ 0.05. Experiments were independently repeated three times with similar results; data of one representative experiment are shown (A, C, D, F). Figure S8. PARPi plus anti-PD-1 shows greater efficacy when ACOX2 elevated without obvious hepatotoxicity and nephrotoxicity. A Tumor figures of immunocompetent mouse model. B Effects of saline, olaparib, anti-PD-1, and olaparib plus anti-PD-1 on mice body weight. C, D Effects of saline, olaparib, anti-PD-1, and olaparib plus anti-PD-1 on ALT (C) and AST (D) of mice. E, F Effects of saline, olaparib, anti-PD-1, and olaparib plus anti-PD-1 on CREA (E) and UREA (F) of mice. G, H Representative HE staining for liver (G) and kidney (H) of mice treated with saline, olaparib, anti-PD-1, and olaparib plus anti-PD-1. Scale bar: 100 μm. I Gating strategy for flow cytometry analysis. Statistical significance was determined by two-tailed unpaired t-test (B-F). * P < 0.05, **P < 0.01, *** P < 0.001, **** P< 0.0001, and ns P ≥ 0.05. Experiments were independently repeated three times with similar results; data of one representative experiment are shown (G, H).