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)).
Protocol detail
Cell Culture and Infection Protocols
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Carbenoxolone
Cdna
Cells
Cgamp
Cgas
Cultured media
Embryonic
Epithelial cells
Fibroblasts
Hela cells
Human
Immunostimulatory
Infections
Macrophages
Mouse
Oviduct
Poly da
Poly i c
Trex1
Vector
Corresponding Organization : University of North Carolina at Chapel Hill
Other organizations : University of Arkansas for Medical Sciences, University of Pittsburgh, University of Maryland, Baltimore
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Variable analysis
independent variables
- Treatment with Poly dA:dT/LyoVec (100 µg/ml)
- Treatment with Poly I:C/LyoVec (50 µg/ml)
- Treatment with 2'3' cGAMP (1 mg/ml)
- Treatment with Carbenoxolone (working concentration 0.2 mM)
- Transfection of pBluscript vector (1–2.5 µg) as immunostimulatory DNA (ISD)
- Transfection of cDNA constructs for human cGAS (pCMV-cGAS) and STING (pCMV-STING)
- Infection with C. muridarum, C. trachomatis D and L2 at 1 MOI or as indicated
- Not explicitly mentioned
- Cell lines: HeLa cells, WT (wild-type) and TREX1 KO mouse embryonic fibroblasts (MEFs), HEK293T, mouse J774 cells, mouse BM1.11 cells, and human OE-E6/E7 (oviduct epithelial cells)
- Cell culture conditions: Supplemented DMEM for HeLa cells, WT and TREX1 KO MEFs, and HEK293T; Complete media for macrophages for mouse J774 cells; Supplemented F12-DMEM for mouse BM1.11 cells and human OE-E6/E7 cells
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