Fig 1.
Effect of missense mutations in STING found in cancer cells on activation of the IFNβ promoter.
(A) Luciferase reporter assay for HEK293T cells cotransfected with pcDNA3, wild-type STING, or STING missense mutants, and an IFNββ promoter reporter construct, pLUC- IFNβ. 5 ng (5) or 25 ng (25) of STING expression construct was transfected as indicated. The normalized relative luciferase activity activity is shown (mean +/- standard deviation) (B) Luciferase reporter assay for HEK293T cells cotransfected with pcDNA3, wild-type STING, or R284M mutant, and the pLUC-PRD(III-I)3 or pLUC-PRD(II)2 reporter construct as indicated.
Fig 2.
Induction of type I IFN production by the R284M mutation.
Human IFNβ enzyme-linked immunosorbent assays at 36 h with tissue culture supernatants of HEK293 cells transfected with pcDNA3, wild-type STING, or STING R284M. Samples were prepared in duplicate. Either 0.5ug or 1.0ug of plasmid was transfected as indicated. The concentration of IFNβ is shown (mean +/- standard deviation). ND, not detectable. Expression of STING and α-tubulin proteins as determined by immunoblot from cell lysates is shown in the insert.
Fig 3.
Protection of cells from VSV infection by the R284M mutant.
Monitoring for VSV-GFP replication by flow cytometry with infected HEK293 cells transiently transfected with pcDNA3, wild-type STING, or R284M mutant. The percentage of cells positive for GFP is indicated (mean +/- standard deviation). Samples were performed in triplicate. *, P value of < 0.005 for comparison with pcDNA3 (n = 3).
Fig 4.
The R284M mutation triggers constitutively active STING-dependent signaling.
(A) Responsiveness of HEK293T cells with stable STING expression to cGAMP. Luciferase reporter assay for HEK293T cells stably expressing empty vector (V), wild-type STING (WT), or STING R284M. All cells were transfected with an IFNβ promoter reporter construct, pLUC- IFNββββ 4 hrs after transfection, new growth media was added and cells were either mock transfected or transfected with 0.5ug/ml cGAMP as indicated. Luciferase activity was measured 16hrs later and analyzed as in Fig. 1. (B) Responsiveness of HEK293T cells with stable STING expression to exogenous cGAS. Luciferase reporter assay for HEK293T cells stably expressing empty vector (V), wild-type STING (WT), or STING R284M. Cells were transfected with pcDNA3 (V) or pCMV-SPORT6-cGAS (cGAS) as indicated. All cells were transfected with an IFNβ promoter reporter construct, pLUC- IFNβ. Luciferase activity was measured 20 hrs following transfection.
Fig 5.
STING with an R284M mutation forms SDS-resistant dimers.
Immunoblot of whole cell lysates from HEK293T cells transfected with wild-type STING or the R284M mutant analyzed by SDS-PAGE using a STING rabbit polyclonal antibody (Cell Signaling). Samples were heated at 95°C for 5 min as indicated (lanes 4 and 5) prior to loading. Positions of protein molecular weight markers are indicated on left-hand side. The arrowhead indicates the 35-kDa STING monomer and the asterisks indicates the 70-kDa presumptive STING dimer. For a loading control, α-tubulin antibody was used.
Fig 6.
Enhanced association of STING with an R284M mutation with TBK1.
Immunoblot of anti-AU1 immunoprecipitates and whole cell lysates from HEK293T cells transfected with AU1-tagged wild-type STING, L374A, or R284M mutant analyzed by SDS-PAGE. Proteins were probed using an AU1 mouse monoclonal or TBK1 rabbit polyclonal antibody as indicated. Cells were lysed 20 hrs following transfection.