Figure 2.

Cytosolic delivery of Ld-DNA induces TREX-1-dependent IFN-β. (A–D) In two parallel sets of experiment, either mouse RAW264.7 (A,C) or BALB/c mouse BMDM (B,D) cells were pre-transfected with targeted TREX-1 siRNA or non-targeted (NT) control siRNA. These cells were either mock transfected or Ld-DNA transfected [2.5 ug/ml with DOTAP or Lipofectamine 2000] or left untreated. Poly I:C (1 µg/ml for 18 hrs) was used as control (C,D). Total RNA were extracted from all sets of cells and relative transcript levels of IFN receptors [IFNGR1, IFNGR2, IFNAR1, IFNAR2] were analyzed by qRT-PCR. In another set of experiment, TREX-1 siRNA modified mouse RAW264.7 cells were infected with L.donovani parasites (parasite: macrophage = 10: 1) and relative transcript levels of IFNGR1 was evaluated. Mean of relative expression to GAPDH expression (fold change) from three sets of experiments were determined (A,B). The cells from both set of experiments were harvested, 8 hrs post-transfection of mock or Ld-DNA [2.5 ug/ml] and the culture supernatant was analyzed for released IFN-β by ELISA. (E,F) Mouse RAW264.7 reporter derived TREX-1 ablated [TREX-1 KO] cells and control cells [ISG-WT] were mock transfected or Ld-DNA transfected [2.5 ug/ml with DOTAP or Lipofectamine 2000] or transfected with dsDNA control Poly(dA-dT). Production of IFN-β was estimated at the relative transcript level (to GAPDH expression levels) by qRT-PCR (E) and at the release level in the culture supernatant by ELISA. All the experiments were repeated at least three times (n = 4 for A,B; and n = 3 for C–F) and the mean values are presented. Statistically significant results are marked as **p < 0.01 and ***p < 0.0001. [Legend for A and B: 1 = Ctrl siRNA+untreated; 2 = Ctrl siRNA +Ld-DNA; 3 = TREX-1 siRNA +untreated; 4 = TREX-1 siRNA ++Ld-DNA]