Quantification of DENV C protein-mediated translocation of ssDNA, GFP-encoding plasmid and TLR3 siRNA into cells.

<p><b>A)</b> Flow cytometry fluorescence distribution histograms of trypsinized BHK-21 cells incubated with ssDNA-Alexa488 in the presence or in the absence (control) of DENV C protein. The percentage of BHK-21 cells positive for DENV C protein-mediated ssDNA internalization is indicated. <b>B)</b> Real time-flow cytometry of DENV C-mediated ssDNA intracellular delivery in BHK-21 cells at 4°C (black) or 37°C (green). The average fluorescence intensity signal of ssDNA is plotted over time. <b>C)</b> DENV C-mediated ssDNA delivery was also carried out on astrocytes at 37°C and 4°C, and imaged by confocal microscopy. Nuclear staining with Hoechst 33342 at 1 mg.mL⁻¹– blue. <b>D)</b> Confocal imaging of BHK-21 cells cultured for one day and then transfected with pEGFP-C3 using DENV C protein or Lipofectamine (control – no transfecting agent). Positive GFP expression (green) was imaged 48 h after transfection. Nuclear staining with Hoechst 33342 at 1 mg.mL⁻¹– blue, as in C). The right panel shows GFP fluorescence excitation (dashed lines) and emission (solid lines) spectra in BHK-21cells suspensions after Lipofectamine- (red) and DENV C-mediated (green) transfections (control – black). <b>E)</b> Percentage of GFP-positive cells, quantified by flow cytometry after transfection with Lipofectamine (red), Tat (black and white) or DENV C protein (green). Flow cytometry histograms of BHK-21 cells after transfection with Lipofectamine (red) or DENV C protein (green) are shown on the right. The fraction of GFP-positive cells is indicated. Statistical analysis was carried out using the two-tailed <i>t-</i>test (n.s. – non-significant; *** – p<0.0003)<b>. F)</b> TLR3 gene silencing assay using siRNA sc-36685 in BMEC. Transfection was performed using Lipofectamine (red), DENV C protein (green), pepM (blue) or pepR (orange). TLR3 gene silencing was analyzed by real-time PCR 48 h after transfection. Results reflect TLR3 expression changes in the presence and absence of the TLR3 siRNA for each transfection agent tested. Relative expression values <i>vs.</i> control (absence of siRNA) were analyzed by the two-tailed <i>t</i>-test (***– p<0.0007; * – p<0.025). Relative expression values <i>vs</i>. Lipofectamine were also analyzed (n.s. – non-significant; # – p<0.04). The resulting data are the mean of three experiments with standard deviation.