TLR3 Regulates Neural Stem Cell Proliferation Figure 3. PIC decreases neurosphere-derived NPC proliferation by inhibiting Shh signaling. Primary neurospheres were obtained from cerebral cortex cells isolated from WT and TLR32/2 embryos at GD14. Neurospheres were cultured for 7 days in the presence of EGF (20 ng/ml) and treated with either PBS or PIC for the last 24 hours of the culture period. (a) Representative histograms showing Patched expression in nestin-positive cells from PBS- and PIC-treated neurospheres obtained from WT and TLR32/2 mice. Numbers in the graphs represent the percentage of nestin+ cells expressing Patched. Three independent cell culture assays were performed with cells isolated from embryos (n = 12); data from one representative assay is shown. (b) Dot plots showing Gli1+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. (c) Histograms showing the mean fluorescence intensity of Gli1 expression in PBS- and PIC- treated neurospheres obtained from WT and TLR32/2 embryos. 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. (d) Box plot showing percentages of Gli1+nestin+ cells in PBS- and PIC-treated neurospheres derived from WT and TLR32/2 embryos. Results are expressed as percentages of total cells (*, p,0.05 relative to PBS control group; Mann-Whitney U test). Height of box plot shows 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.g003 nant Shh showed increased proliferation as measured by BrdU labeling (Fig. 4a). PIC treatment inhibited BrdU labeling in WT neurospheres cultured in 5 ng/ml of EGF (n = 12; Mann-Whitney U, p = 0.04; relative to PBS-treated WT control group; Fig. 4b, d). Recombinant Shh treatment in the presence of 5 ng/ml of EGF abrogated the PIC-mediated reduction in BrdU labeling (n = 12; Mann-Whitney U, p = 0.52; relative to Shh and PBS- treated WT control group; Fig. 4c, d). Under culture conditions with a higher concentration of EGF (20 ng/ml) in the media, Shh did not have this effect (data not shown), presumably because stimuli for proliferation were already maximal [41]. These results indicate that PIC-mediated inhibition of NPC proliferation can be abrogated by stimulating the Shh pathway under culture conditions wherein EGF concentration is sub maximal. To test for direct evidence that PIC effect is due to Shh signaling inhibition, neurosphere cultures were treated with cyclopamine, a small molecule inhibitor of Shh signaling. Cyclopamine is a steroidal alkaloid that specifically inhibits Shh pathway activation by binding directly to the multipass transmembrane protein, Smo, and regulating its function [55]. WT cortical neurospheres were grown in 5 ng/ml of EGF supplemented with 5 mM of cyclopamine or ethanol (vehicle control group) for 7 days, and treated with PIC or PBS for the last 24 hours of the culture period. FACS analysis of WT neurospheres indicated that PIC treatment in the presence of cyclopamine and EGF decreased the percentage of Gli1+nestin+ cells when compared to PBS/ethanol-treated vehicle control group (Fig. 5a). PIC treatment in the presence of cyclopamine and EGF did not result in any additive effects in the inhibition of Gli1 in comparison to cells that were treated with PIC alone (Fig. 5a). These observations suggest: (i) a lack of synergy between PIC and cyclopamine on Shh signaling, and (ii) in WT neurosphere cells, PIC-mediated effects in the Shh signaling pathway may be on effectors that are downstream to Ptch and Smo. Furthermore, no differences in percentages of Gli1+nestin+ cells were observed in PIC-treated WT cells cultured in the presence of recombinant Shh (5 nM) and EGF (5 ng/ml) in PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26766