Human embryonic kidney (HEK) 293T CRL‐3216 cells were purchased from American Type Culture Collection. All cells are mycoplasma free and they are regularly tested. HEK293T was cultured in Dulbecco's modified Eagle's medium with 10% foetal bovine serum (FBS), 2 mM L‐glutamine, penicillin (100 U/ml) and streptomycin (100 mg/ml; Gibco) at 37°C with 5% CO2.
THP‐1 cells were maintained in RPMI (Gibco) supplemented with 10% FBS and Pen/Strep. THP‐1‐IFIT1 cells that had been modified to express Gaussia luciferase under the control of the IFIT1 promoter were described previously (Mankan et al, 2014 (link)). THP‐1 Dual Control and STING KO and MAVS KO cells were previously described (Sumner et al, 2020 (link)). Jurkat and SupT1 T‐cell lines and PBMCs were maintained in RPMI 1640 with L‐glutamine (Corning) and supplemented with 10% FBS (GenClone), penicillin (100 U/ml) and streptomycin (100 mg/ml). STING inhibitor H151 was obtained from Invitrogen.
Mutant construct D185E was generated with the New England Biolabs (NEB) Q5 site‐directed mutagenesis kit (NEB, E0554) against pCRV GagPol WT and mutants (K158A, K158A/T8I, T8I; Mallery et al, 2021 ) using primers designed using the NEBaseChanger online tool.
THP‐1 cells were maintained in RPMI (Gibco) supplemented with 10% FBS and Pen/Strep. THP‐1‐IFIT1 cells that had been modified to express Gaussia luciferase under the control of the IFIT1 promoter were described previously (Mankan et al, 2014 (link)). THP‐1 Dual Control and STING KO and MAVS KO cells were previously described (Sumner et al, 2020 (link)). Jurkat and SupT1 T‐cell lines and PBMCs were maintained in RPMI 1640 with L‐glutamine (Corning) and supplemented with 10% FBS (GenClone), penicillin (100 U/ml) and streptomycin (100 mg/ml). STING inhibitor H151 was obtained from Invitrogen.
Mutant construct D185E was generated with the New England Biolabs (NEB) Q5 site‐directed mutagenesis kit (NEB, E0554) against pCRV GagPol WT and mutants (K158A, K158A/T8I, T8I; Mallery et al, 2021 ) using primers designed using the NEBaseChanger online tool.
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