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. 2019 Mar 15;20(4):e46293. doi: 10.15252/embr.201846293

Figure 4. STING is important but not sufficient for eCDN‐induced type I IFN response.

Figure 4

  1. qRT–PCR detection of IFNB1 mRNA in scrambled (Scramble) or STING shRNA stably transfected (STING KD) THP‐1 cells stimulated with ec‐di‐AMP (5 μg/ml) and ecGAMP (5 μg/ml) or transfected with ISD or poly(I:C).
  2. ELISA detection of IFNβ protein in supernatants of Scramble or STING KD THP‐1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C).
  3. qRT–PCR detection of Ifnb1 mRNA in WT and Sting −/− (Sting KO) mBMDMs stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD.
  4. ELISA detection of IFNβ protein secretion in supernatants of mBMDMs stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly (I:C).
  5. qRT–PCR detection of Ifnb1 mRNA in WT, Sting KO, or Sting KO complemented with mouse STING (Sting KO+mSTING) RAW264.7 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD.
  6. Western blot detection of indicated proteins in lysates of WT and Sting −/− (KO) mBMDMs stimulated with ecGAMP (5 μg/ml) or transfected with ISD. Data are representative of three independent experiments.
  7. qRT–PCR detection of IFNB1 mRNA levels in STING stable HEK293T cells (HA‐STING‐HEK293T) stimulated with ecGAMP or icGAMP at 5 μg/ml for indicated times.
  8. Western blot detection of indicated proteins in HA‐STING‐HEK293T cells stimulated with increasing amounts of ecGAMP and icGAMP at 5 μg/ml for 24 h. Data are representative of three independent experiments.
  9. qRT–PCR detection of the induction of IFNB1 mRNA in HEK293T cells stably transfected with pcDNA3.1‐HA (HA), HA‐STING, and HA‐STING+HA‐cGAS stimulated with ecGAMP (5 μg/ml) or icGAMP (0.1 μg/ml) or transfected with ISD.
Data information: Data in (A–E, G, I) are means + SD averaged from at least three independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two‐way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.Source data are available online for this figure.