Fig. 5: ARIH1-mediated degradation of DNA-PKcs promotes STING pathway activation.

a HEK293T cells were transfected with ARIH1^-WT-HA or ARIH1^-C357S-HA for 36 h. Proteins that co-immunoprecipitated (Co-IP) with ARIH1 were analyzed by mass spectrometry. DNA repair-related proteins are shown in heatmap. b Co-IP of ARIH1 with DNA-PKcs in HEK293T cells. Endogenous DNA-PKcs was immunoprecipitated using anti-DNA-PKcs, and the immunoprecipitates were analyzed with anti-ARIH1. lgG, immunoglobulin G. c Immunoblot (IB) analysis for ubiquitination of DNA-PKcs from HEK293T cells co-transfected with the indicated constructs. d In vitro ubiquitination assay of purified DNA-PKcs. The reactions were performed with purified His-ubiquitin, GST-UBA1 (E1), His-UBCH7 (E2), and GST-ARIH1^-WT or its ligase-dead mutant (GST-ARIH1^-C357S) or in the absence of UBA1, UBCH7, ubiquitin, ARIH1 or DNA-PKcs. e, f. IB analysis of DNA-PKcs levels in U2OS cells. The cells were incubated with small interfering RNAs (siRNAs) against ARIH1 (f), or transfected with ARIH1-HA (e). g IB analysis of DNA-PKcs in U2OS cells transfected with the indicated constructs. Cells were treated by MG132, Bafilomycin or NH₄Cl+Leup for 6 h. h Immunofluorescent staining of DNA-PKcs and Lamp2 in CTRL and Arih1^-WT-OE 4T1 cells and their quantifications. The nuclei were stained with DAPI. Scale bar, 10 μm; insets: Scale bar, 2 μm. Data represent means ± SEM, ^****P < 0.0001. Each dot in the graph represents the percentage of counted cells with co-localization in each sample, and the total number of counted cells in each group is as follows: CTRL group (n = 381 cells), Arih1^-WT-OE group (n = 321 cells). i IB analysis of pSTING-S366, pTBK1-S172 and DNA-PKcs in HEK293T-STING cells co-transfected the indicated constructs. j cGAS-KD MCF-7 cells were transfected with cGAS WT or the phosphorylation-mimic mutants and ARIH1-HA. WCLs were analyzed by immunoblotting. k qRT-PCR measurement of ISGs expression in cGAS-KD HeLa cells transfecting with cGAS WT or the phosphorylation-mimic mutants and ARIH1-HA. IFNB1 (n = 3/group), IFNA2 (n = 3/group), IL6 (n = 4/group). Data represent means ± SEM, ^***P < 0.001 (P = 0.0002), ^****P < 0.0001. In e–g, the numbers under the blots represent the gray scale quantification (DNA-PKcs/Actin). For k data, One-way ANOVA test. For h data, Two-tailed t-test. Data shown in b, c, e–h, and j, k are representative of two independent experiments. Data shown in a, d and i are representative of three independent experiments. Source data are provided as a Source Data file.