TLR3 Regulates Neural Stem Cell Proliferation Figure 7. PIC prevents EGF/Shh–mediated ERK activation and induces Fas expression in primary cortical neurospheres. Primary neurospheres were obtained from cerebral cortex cells isolated from WT embryos at GD14. Neurospheres were cultured for 7 days in the presence of EGF (20 ng/ml) and treated with PBS or PIC for 24 hours. (a, b) Representative histograms showing phospho-ERK and Fas-positive cells in PBS- or PIC- treated WT neurospheres. Numbers in gates represent the percentages of phospho-ERK+ and Fas+ cells. Three independent cell culture assays were performed with cells isolated from embryos (n = 12); data from one representative assay is shown. (c, d) Representative histogram showing the mean fluorescence intensity of phospho-ERK and Fas expression in PBS- and PIC-treated WT neurospheres. The mean fluorescence intensity is represented as a percentage of the maximum expression, ——, PBS-treated cells; ——, PIC-treated cells; and ——, isotype control. (e, f) Box plots showing the percentages of phospho-ERK+ and Fas+ cells in PBS- and PIC-treated neurospheres. 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.g007 Figure 8. Shh pathway activation reverses PIC-induced ERK activation. Primary neurospheres were obtained from cerebral cortex cells isolated from WT embryos at GD14. Neurospheres were cultured for 7 days in the presence of EGF (5 ng/ml) and in the presence or absence of recombinant Shh protein (5 nM), and treated with PBS or PIC for the last 24 hours of the culture. (a) Representative histograms showing percentage of cells expressing phospho-ERK in WT neurospheres. Numbers in gates represent the percentages of phospho-ERK+ cells. Three independent cell culture assays were performed with cells isolated from embryos (n = 12); data from one representative assay is shown. (b) Histogram showing the mean fluorescence intensity of phospho-ERK expression in PBS- and PIC-treated WT neurospheres cultured in the presence or absence of recombinant Shh. The mean fluorescence intensity is represented as a percentage of the maximum expression: red line: EGF and PBS-treated cells, blue line: EGF and PIC-treated cells, green line: EGF + Shh and PIC-treated cells supplemented with Shh, and gray line: isotype control. doi:10.1371/journal.pone.0026766.g008 PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e26766