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. 2023 Feb 23;220(5):e20220666. doi: 10.1084/jem.20220666

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© 2023 Jeremiah et al.

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Figure S2. — Impact of RELA on IFN response to cGAMP in CD4⁺ T cells MDDCs, MDMs, and THP-1 cells. (A) RT-qPCR of IFNL1 and IFNB expression 5 h following stimulation with cGAMP (6 µg/ml) in CD4⁺ T cells and MDDCs transduced with either control or RELA (n = 3 donors combined from two independent experiments). RQ normalized to average of controls, each symbol represents one donor, bars represent geometric mean, paired one-way ANOVA with Tukey’s multiple comparison test. (B) Activity of IFNB promoter and NF-κB promoter luciferase reporter in 293FT following transfection with BFP, human STING or zebrafish STING and stimulation with cGAMP (4 µg/ml). Each symbol represents one experiment (n = 3 independent experiments). (C) IFN-I/III concentration following cGAMP (6 µg/ml) stimulation of CD4⁺ T cells, transduced with control (BFP), human STING, or zebrafish STING (n = 4 donors combined from two independent experiments). Each symbol represents one donor, bars represent mean, paired one-way ANOVA with Tukey’s multiple comparison test. (D) Proliferation measured using a cell proliferation dye (CPD) of CD4⁺ T cells transduced with either control (GFP) RELA or RELA K5R lentivector on day 4 and 6 post TCR stimulation (n = 4 donors combined from two independent experiments). Each symbol represents one donor, bars represent mean + SEM, paired one-way ANOVA with Tukey’s multiple comparison test. (E) Western blot of RELA and actin in CD4⁺ T cells, MDMs, and THP-1 transduced with either control, RELA, or RELA K5R (representative of two individual experiments). (F) IFN-I/III concentration following cGAMP (6 µg/ml) stimulation of (top) CD4⁺ T cells, MDMs, and (bottom) THP-1 transduced with control (GFP), RELA, or RELA K5R (n = 4 donors combined from two independent experiments, for THP-1 combined from three independent experiments). Each symbol represents one donor, bars represent geometric mean, paired one-way ANOVA with Tukey’s multiple comparison test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are available for this figure: SourceData FS2.