TLR3 Regulates Neural Stem Cell Proliferation Figure 9. PIC treatment inhibits the EGF-Shh-mediated Ras-ERK signaling pathway in primary cortical neurospheres. Shh binds to the cell surface receptor Ptch, relieves the inhibition of Smo and triggers Shh signaling. Activation of the Shh pathway results in increased levels of Gli1 expression and activation of the downstream Ras-MEK-ERK pathway, causing cell proliferation. In WT neurospheres cultured in the presence of EGF, PIC signals via TLR3 to inhibit Gli1 expression and interfere with MEK and ERK phosphorylation. Inhibition of MEK-ERK activation results in the decreased proliferation and induced Fas expression and apoptosis observed after PIC treatment of primary cortical neurospheres. doi:10.1371/journal.pone.0026766.g009 ACTCCTTTGGGGGACTTTTG and TLR3_R CAGG TTCGTGCAGAAGACAA. After 10 generations, heterozygous mice were inter-crossed, and congenic homozygous TLR32/2 mice on a C57BL/6 background were selected to maintain the colony. Time-pregnant TLR32/2 mice were obtained by selecting vaginal plug positive (GD 0) dams from breeding pairs of homozygous TLR32/2 mice. Primary Neurosphere Cultures Embryonic cortical neurosphere cultures were obtained using the method of Reynolds and colleagues [8,9] with minor modifications. Embryos were obtained from cesarean sections of GD14 pregnant dams sacrificed under anesthesia. The embryos were sacrificed by decapitation and the dorsal telencephalon was dissected, trimmed and stripped of meninges in cold Hanks solution containing 5 mM HEPES at pH.8.0, penicillin/streptomycin (Invitrogen, Carlsbad, CA) and 0.8% D-glucose (Sigma-Aldrich, Saint Louis, MO, USA). The tissue was then mechanically dissociated by gentle trituration with fire-polished, glass Pasteur pipettes to yield single cell suspensions. The cells were incubated to generate primary neuro- spheres in NeuroCult NSC proliferation medium supplemented with 20 ng/ml of human epidermal growth factor (EGF) and Neurocult NSC proliferation supplements (StemCell Technologies, Vancouver, BC, Canada). The cell suspensions were incubated at 37uC, 5% CO2 atmosphere in 10 ml at a concentration of 16105 cells/ml in T-25 tissue culture flasks and left undisturbed for 7 days to generate neurospheres. For experiments with recombinant Shh protein or cyclopamine, cells were cultured in NeuroCult NSC proliferation media containing 5 ng/ml of EGF and supplemented with either 56nM of recombinant human Shh (Invitrogen, San Diego, CA) or 5 mM of cyclopamine (Toronto Research Chemicals Inc., Ontario, Canada). PIC treatment Polyinosinic-polycytidylic potassium salt (Sigma Aldrich; St. Louis, MO, USA) dissolved in PBS was heated to 55uC for 56min PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e26766