Figure 4: Raftlin-mediated endocytosis is essential for PV-RNA-induced IFN-β production by human cells.

(a) Left panels: HEK293 cells were transfected with control, raftlin or TICAM-1 siRNAs (20 pmol), together with the expression vector for human TLR3 and the IFN-β reporter plasmid. Forty-eight hours after transfection, cells were washed and stimulated with 20 μg ml⁻¹ poly(I:C) or PV-RNAs. After 6 h, luciferase reporter activity was measured and expressed as the fold induction relative to the activity of unstimulated cells. Representative data from three independent experiments are shown (mean±s.d.). Student’s t-test was used for statistical analysis. *P<0.05 and **P<0.01. Right panels: Knockdown of raftlin or TICAM-1 was confirmed using western blotting. (b) HeLa cells were transfected with the control or raftlin siRNAs. Forty-eight hours after transfection, cells were washed and stimulated with 20 μg ml⁻¹ PV5 for 3 h. Total RNA was extracted, and quantitative PCR was performed using primers for the IFN-β and raftlin genes. Expression of genes was normalized to glyceraldehyde 3-phosphate dehydrogenase mRNA expression. Data are shown as the mean±s.d. Representative data from three independent experiments are shown. *P<0.05. (c) HeLa cells were incubated with 15 μg ml⁻¹ Cy3-PV5 for 30 min at 4 °C. After washing, cells were incubated for up to 60 min at 37 °C. At timed intervals, cells were fixed and permeabilized. After staining with anti-TLR3 mAb and anti-EEA1 pAb (upper panels) or anti-LAMP1 mAb (lower panels), cells were incubated with an Alexa Fluor-488- or -633-conjugated secondary Ab and then analysed using confocal microscopy. Red, Cy3-PV5; green, TLR3 or LAMP1; blue, EEA1; light blue, nuclei with 4',6-diamidino-2-phenylindole; white, merged PV5 with TLR3 and EEA1; and yellow, merged PV5 with LAMP1. Scale bar, 10 μm. Co-localization between PV5 and TLR3, EEA1 or LAMP1 was analysed by counting the merged PV5 spots with each molecule in 70 PV5-internalizing cells. The data are shown as % of merged PV5 with EEA1, LAMP1 or TLR3 at indicated time points.