Figure 3. PIC decreases neurosphere-derived NPC proliferation by inhibiting Shh signaling.

Primary neurospheres were obtained from cerebral cortex cells isolated from WT and TLR3^−/− 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 TLR3^−/− 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 TLR3^−/− 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 TLR3^−/− 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 TLR3^−/− 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.