TLR3 Regulates Neural Stem Cell Proliferation Figure 2. PIC negatively regulates neurosphere-derived NPC proliferation in a TLR3-dependent manner. Proliferative response of primary cortical neurospheres obtained from WT and TLR32/2 embryos at GD14. Neurospheres were maintained in EGF-containing (20 ng/ml) medium for 7 days and treated with either PBS or PIC for 24 hours. BrdU was added for the last 12 hours of the culture. (a) Dot plots showing BrdU- labeled nestin+ cells in PBS- and PIC-treated neurospheres derived from WT and TLR32/2 mice. Numbers in quadrants represent the percentages of each subpopulation. Three independent cell culture assays were performed with cells isolated from embryos (n = 12); data from one representative assay is shown. (b) Box plots showing percentages of BrdU+nestin+ cells in PBS- and PIC-treated neurospheres derived from WT and TLR32/2embryos. Results are expressed as percentages of total cells (* p,0.05; relative to PBS control group; Mann-Whitney U test). (c) Histograms showing BrdU- negative GFAP+ and Tubulin-b-III+ cells in PBS- and PIC-treated neurospheres derived from WT mice. Numbers in the graph represent the percentages of each subpopulation. (d) Histograms showing the mean fluorescence intensity of GFAP and Tubulin-b-III expression in BrdU-negative cells from PBS- and PIC-treated neurospheres obtained from WT mice. The mean fluorescence intensity is represented as a percentage of the maximum expression: red line: PBS-treated cells; blue line: PIC-treated cells; and gray line: isotype control. (e) Box plots of the percentages of Tubulin-b-III+BrdU2 and GFAP+BrdU2 cells from PBS- and PIC-treated neurospheres derived from WT embryos. Results are expressed as percentages of total cells (*, p,0.05; relative to PBS control group; Mann-Whitney U test). Height of box plots show interquartile range; horizontal line, median. Due to the low numbers involved in the flow cytometry experiments, the box plot upper and lower interquartile ranges also represent the maximum and minimum scores. White bars: PBS-treated mice, hatched bars: PIC-treated mice. doi:10.1371/journal.pone.0026766.g002 expressed Ptch in both PBS- and PIC-treated WT cells (Fig. 3a). WT and TLR32/2 Nestin+ cells expressed the Shh-regulated transcription factor, Gli1 (Fig. 3b, c). To test the effect of PIC on Shh signaling in EGF-responsive NPC, GD14 WT and TLR32/2 cortical neurospheres were grown in EGF-supplemented medium for 7 days, and treated with either PIC or PBS for the last 24 hours of the culture period. FACS analysis of WT neurospheres indicated that PIC treatment decreased the percentage of Gli1+nestin+ cells (n = 12; Mann-Whitney U, p = 0.04; relative to PBS-treated WT control group; Fig. 3b, d) and the mean fluorescent intensity of Gli1 expression (n = 12; Mann-Whitney U, p = 0.04; relative to PBS-treated WT control group; Fig. 3c). In contrast, no differences were observed between TLR32/2 cells treated with PIC or PBS (n = 12; Mann-Whitney U, p = 0.51; relative to PBS-treated TLR32/2 control group; Fig. 3b–d). Together, these results suggest that PIC-mediated inhibition of Shh signaling in nestin+ precursor cells may be occurring downstream to Ptch and that this inhibition requires TLR3 expression. Shh pathway activation reverses PIC-mediated inhibition of neurosphere-derived NPC proliferation Induction of Shh signaling with recombinant Shh protein regulates proliferation of neural stem cells [41]. GD14 WT- derived primary cortical neurospheres grown for 7 days in media containing 5 ng/ml of EGF supplemented with 5 nM recombi- PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26766