Figure 2.

Activation of DNA Sensing in HEK293T Elicits a Transcriptional Pro-inflammatory Response but Does Not Result in the Secretion of Measureable Type 1 IFN or CXCL10

(A) qRT-PCR for the indicated genes was performed on RNA extracted from HEK293T 48 h after transfection with the indicated pcDNA-based expression plasmids. Bars represent means ± SD (n = 2) of biological replicates relative to cells transfected with an equivalent amount of pcDNA. (B) Supernatants from HEK293T cells transfected with the indicated plasmids were harvested after 48 h, filtered, and incubated with THP-1 reporter cells bearing Gaussia Luciferase under the control of the endogenous IFIT1 promoter. Luciferase was measured after 24 h as a measure of IFN-β. Exogenous IFN-β (0.01–10 ng/mL) was used as a control. (C) CXCL10 in the supernatant of HEK293T 48 h post transfection with the indicated plasmids was measured by ELISA. Supernatant from THP-1 cells stimulated for 24 h with cGAMP (1 μg/mL) was used as a positive control. * indicates below the limit of detection. (D) HEK293T cells were co-transfected with the indicated pcDNA-based expression plasmids, an IFIT1 firefly luciferase reporter and pRL-TK Renilla luciferase. 24 h later, cells were incubated with DMSO vehicle or 2 μM ruxolitinib. At 48 h post transfection, luciferase activity was measured and firefly luciferase values were normalized to Renilla luciferase values. Bars represent mean fold induction of IFIT1 luc activity (±SD, n = 3) presented relative to cells transfected with an equivalent amount of pcDNA. Cells were treated with 10 ng/mL exogenous IFN-β as a control.