Supplementary Figure 1: TLR3 responses in primary neurons and fibroblasts.

(a) WT mice (n = 8 per group) were intracranially infected with indicated doses of HSV-1 and mice survival was evaluated. (b) Primary neurons, astrocytes, and microglia were stained with antibodies to a neuron marker NeuN, an astrocyte marker GFAP, and a microglia marker CD11b (red histograms). Black histograms show staining with the second reagent alone. (c) Primary neurons were left unstimulated or stimulated with poly(I:C) (PIC) at 25 μg/ml, cGAMP at 7 μM with Lipofectoamine or ISD 1 μg/ml with Lipofectoamine for 24 h. Real-time PCR was conducted to measure Ccl5 mRNA. These results are normalized by HPRT mRNA and represented by the mean value ± s. d. of triplicate wells. Statistical analysis was performed by the two-tailed Student’s t test. ***P < 0.001. (d) WT and Tlr3^–/– primary neurons were left uninfected (U) or infected (HSV-1) with HSV-1 at an MOI of 10. Real-time PCR was conducted to measure Ifnb1 mRNA. The results were analyzed as in (c). (e) Membrane permeabilized staining of TLR3 and Unc93B1-Flag in NIH3T3 cells or those expressing the indicated molecules (red histograms). Black histograms indicate labeling with the secondary antibody alone. (f) NIH3T3 cells or those expressing the indicated molecules were left unstimulated (U) or stimulated with poly(I:C) at 5 (PIC 5) or 25 (PIC 25) μg/ml for 24 h. The concentration of CCL5 in the supernatant was determined by ELISA. The results are represented by the mean value ± s. d. of triplicate wells. (g, h) NIH3T3-TLR3-Unc93B1 cells were treated with vehicle, Rapamycin at 50 or 250 nM or Torin 1 at 50 or 250 nM for 2 h. The treated cells were left unstimulated (U) or stimulated with poly(I:C) at 5 (P5) or 25 μg/ml (P25) for 24 h (g) or at 25 μg/ml for the indicated periods of time (h). Immunoblotting of the indicated molecules was conducted (h). Grb2 is shown as a loading control. The concentration of CCL5 in the supernatant was determined by ELISA. The results are represented by the mean value ± s. d. of triplicate wells. Statistical analysis was performed by the two-tailed Student’s t test. ***P < 0.001. These experiments were repeated more than three times. (i) The deletion of RICTOR is shown by immunoblot of the whole cell lysate. Grb2, a loading control. Shown on the right is membrane permeabilized staining of TLR3 in WT and Rictor^Δ/Δ fibroblasts (red histograms). Black histograms indicate labeling with the secondary antibody alone