Investigating STING and IRF3 Signaling

STING (Cell Signaling Technology Cat# 13647, RRID:AB_2732796), IRF3 (Cell Signaling Technology Cat# 11904, RRID:AB_2722521) and pIRF3(S396) (cat#29047) antibodies were purchased from Cell Signaling (Boston, MA, USA) and procedures for immunoblotting and immunofluorescence were performed as in our previous publication [3 (link)]. For R-loop immunofluorescence, the S9.6 antibody was purchased from Kerafast (Cat# ENH001, RRID:AB_2687463, Boston, MA, USA). The immunofluorescence protocol was modified, and the cells were fixed with 4% paraformaldehyde in PBS for 5 min and permeabilized with 0.01% saponin in PBS. Live staining with Sir-Hoechst (Spirochrome Inc, cat#SC007, Stein am Rhein, Switzerland) was performed following manufacturer instructions at 1 µM concentration in media. Picogreen staining was performed according to manufacturer instructions using Quant-iT^TM Picogreen^TM dsDNA Assay Kit (Cat#P7589. DAPI was purchased from Thermofisher Scientific (cat#62247, Waltham, MA, USA). For quantification, images were taken from 5 random positions across 2 slides of each treatment, positive cytoplasmic DNA staining was counted and representative images were shown. Three independent experiments were performed.