FIGURE 4 from Identification of Nonfunctional Alternatively Spliced Isoforms of STING in Human Acute Myeloid Leukemia

Novel STING alleles fail to respond to STING agonists. HEK-Blue-ISG-KO-STING cells were reconstituted with indicated novel STING variants (n = 2; A) or STING variants plus WT STING by retroviral transduction (B), then were exposed to 100 µg/mL indicated CDN for 24 hours (n = 2). IRF3/9 activity was measured by SEAP assay. IFNα positive control indicates presence and functionality of the IRF3/9 SEAP reporter in these cells. Statistical significance in B was calculated using repeated measures one-way ANOVA test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. C, STING knockout 293 T cells were transduced to express human WT STING with an HA or 6xHIS tag, the STING-I3R isoform with a 6xHIS tag, or the STING-Ex4D isoform with a 6xHIS tag. Cell lysates were Western blotted with anti-6xHIS antibody (Invitrogen) followed by anti-mouse HRP (Cell Signaling Technology). Anti-human β-actin staining is shown as a loading control (Abcam).