Pcmv sting

HEK293T cells were transfected with pCMV-cGAS (Origene), pCMV-STING (Origene) or pcDNA3.1 vector (60 ng DNA). Cells were trypsinized 24 post transfection and re-plated individually or mixed at 1:1 ratio, such that each well contained a total of 4 × 10⁵ cells/well in a 24 well dish. Cells were infected, or permeabilized with positive control ligands cGAMP (1µg/ml) or transfected with ISD (1µg), 6 hours after plating and harvested at indicated times and processed for RNA and qRT-PCR. Immunoblots for cGAS or STING protein in HEK293, HEK293T and HeLa cells were carried out using cell lysate prepared using RIPA buffer (Pierce-Thermo Scientific) and protease inhibitor cocktails (Sigma). Anti cGAS Ab (Cat# AP10510c, Abgent), anti-STING poly clonal Ab (Cat# PA5-26751, Thermo Scientific) and anti-actin Ab (Cat# A2228, Sigma) were used at the 1:500, 1:500 and 1:1000 concentration respectively.

HeLa cells, WT (wild-type) and TREX1 KO mouse embryonic fibroblasts (MEFs), and HEK293T were cultured in supplemented DMEM and mouse J774 cells cultured in complete media for macrophages, as described earlier (25 (link)). Mouse BM1.11 cells (26 (link)) and human OE-E6/E7 (oviduct epithelial cells) (27 (link)) were cultured in supplemented F12-DMEM as described (25 (link)). Poly dA:dT/LyoVec (100 µg/ml), Poly I:C/LyoVec (50 µg/ml) and 2’3’ cGAMP (1 mg/ml) were purchased from Invivogen. Carbenoxolone (working concentration 0.2 mM) was purchased from SIGMA. pBluscript vector (1–2.5 µg) was used as immunostimulatory DNA (ISD). cDNA constructs for human cGAS (pCMV-cGAS) and STING (pCMV-STING) were purchased from Origene. C. muridarum, C. trachomatis D and L2 were propagated in McCoy cells and infections performed at 1 MOI or as indicated, as previously described (25 (link)).