Figure 4.

Large TAg Depletion in HEK293T Cells Does Not Impact cGAS/STING-Driven Activity of the IFN-β Luciferase Reporter or LV Production

(A) SV40 large T-antigen (TAg) mRNA levels in HEK293T cells expressing TAg-specific shRNA or an shRNA control after 5 days of puromycin selection. TAg expression was first normalized to GAPDH and then to expression levels in the unmodified HEK293T cells to generate percentage relative expression. (B) TAg protein expression measured by immunoblot detecting TAg or vinculin as a loading control. Molecular mass markers are indicated. (C) Mean fold change in co-transfected IFN-β-luc reporter activity in HEK293T cells expressing shRNA targeting TAg or an shRNA control (293T shControl) 48 h after co-transfection with the indicated pcDNA-based expression plasmids, assessed using a dual-luciferase reporter assay. Data are presented relative to cells transfected with an equivalent amount of pcDNA (±SD, n = 4). (D) Mean Renilla luciferase activity (mean relative light units ± SD, n = 4) from (C) measured as a control. (E) Culture supernatants from unmodified HEK293T cells or HEK293T cells expressing shTAg or shControl that had been transfected to produce a LV encoding GFP were harvested at 48 h and mean viral titers ± SD of biological replicates (n = 2) were determined in duplicate in HEK293T and THP-1 cells by enumerating GFP-positive cells. TU, transducing units.