HAQ, AQ, and Q293 human cells are resistant to STING agonists-induced death.
(A-B). Total human Lung cells from WT/WT individuals were treated with diABZi (100ng/ml) for 24hrs. Cell death in CD4, CD8 T cells and CD19 B cells were determined by PI staining. (C). Total lung cells from a WT/HAQ (2 individuals) and a WT/WT (3 individuals) were treated with diABZi (25, 100ng/ml) for 24h. Cell death in CD4 T cells was determined by PI staining. (D-E). STING-KO THP-1 cells (Invivogen,, cat no. thpd-kostg) were stably reconstituted with human WT (R232), HAQ, AQ, Q293. Cells were treated with diABZI (200ng/ml) in culture for 24 hrs. Dead cells were determined by Annexin V staining. (F). STING-KO THP-1 cells stably reconstituted with human WT (R232), Q293 were treated with indicated dose of diABZI for 24hs in culture. Dead cells were determined by Annexin V staining. (G). STING-KO THP-1 cells stably reconstituted with human WT (R232), Q293 were treated with indicated dose of diABZI for 2hs in culture. STING activation was detected by anti-STING antibody (Proteintech, #19851-1-AP). (H). STING-KO THP-1 cells stably reconstituted with human WT (R232), HAQ, AQ, Q293 were treated with 50ng/ml diABZI in culture for 24hrs. ISG-54 reporter luciferase activity was determined in cell supernatant and normalized to 10ng/ml IFNβ-stimulated ISG-54 luciferase activity. (I). STING-KO THP-1 cells stably reconstituted with human WT (R232), HAQ, AQ were treated with 50ng/ml diABZI in culture for 2hrs. STING and IRF3 activation were determined by anti-STING antibody and anti-p IRF3 antibody (CST, Ser396, clone 4D4G). Densitometry was determined by ImageLab 5. Data are representative of three independent experiments. Graphs represent the mean with error bars indication s.e.m. p values determined by one-way ANOVA Tukey’s multiple comparison test (B, C, F, H, G) or unpaired student T-test (E, I). * p<0.05, n.s: not significant.