Figure 5.

Large TAg Expression Enhances LV Production but Does Not Impact cGAS/STING-Driven Activity of the IFN-β Luciferase Reporter

(A) TAg protein expression levels measured by immunoblot in unmodified HEK293 cells or in HEK293 or HEK293T after co-transfection of TAg encoding pcDNA. Molecular mass markers are indicated. Vinculin was detected as a loading control. (B) Measurement of LV encoding GFP (SG-PERT RT assay) produced in HEK293 or HEK293T cells co-transfected with empty vector or TAg encoding pcDNA (mean ± SD of biological replicates, n = 2). (C) Infectious titers of LV from (B) were evaluated in HEK293T or THP-1 cells by flow cytometry detecting GFP expression. Data are presented as mean transducing units (TU)/mL ± SD of biological replicates (n = 2) performed in duplicate. (D) Immunoblot detecting TAg protein levels in unmodified HEK293 or HEK293T cells, or HEK293 stably expressing TAg (293-TAg) from gammaretroviral vector pBABE-puro. Molecular mass markers are indicated, and vinculin was detected as a loading control. (E and F) Measurement of LV encoding GFP by (E) SG-PERT or (F) infection measuring GFP by flow cytometry, produced from unmodified HEK293 or 293T or 293 stably expressing TAg (293-TAg) (mean ± SD of biological replicates, n = 2). (G) Activation of IFN-β luciferase reporter in HEK293 (293), HEK293 cells stably expressing TAg-coding vector (293-TAg), or HEK293T cells (293T) after co-transfection with indicated pcDNA-based expression plasmids. Mean fold change in IFN-β activity (±SD, n = 4) was assessed 48 h after transfection and presented relative to cells transfected with an equivalent amount of empty pcDNA. (H) Mean Renilla luciferase activity (mean ± SD, n = 4) from (G) measured as a control.