Taurine Effect on Stem Cells and Synaptogenesis Figure 6. ERK1/2 is involved in taurine induced NPC proliferation. The cells were incubated with or without 100 mM taurine for 48 hrs. ERK1/ 2 inhibitor, PD98059 was added to the media 30 min before taurine treatment. Representative immunoblots showing protein levels of phosphorylated ERK1/2 (A) and total ERK1/2 (C). Graphs showing the ratio of densitometric measurements of phosphorylated ERK1/2 and total ERK1/ 2, normalized with total protein levels of a-tubulin. (B and D). Graph showing fold change in phosphorylated ERK1/2 upon treatments as indicated, normalized with total ERK1/2 (E) * P,0.0 5 and ** P,0.01 as compared with the control. The western blots are representative blots from at least three individual experiments. doi:10.1371/journal.pone.0042935.g006 For differentiation experiments, cells were seeded onto poly- L-ornithin and laminin coated coverslips at a density of 25,000/ cm2 per well in 24 well plates in defined medium composed of DMEM/F-12, N2 supplement and B27 supplement (Invitrogen), 2 mM glutamine and 2 mg/ml pen/streptomycin, supplemented with fibroblast growth factor (5 ng/ml). Taurine was added to the media and the medium was changed every alternate day for 5 days. The cells were then fixed with PFA for 30 min, washed with PBS and processed for immunocytochemistry. Data were analyzed by t-test. P,0.05 is considered as significant. For biochemical analysis, NPCs were cultured in 6 cm plates, treated with taurine for 2 days, and then processed for western blot analysis. Primary Neuronal Cultures Ethics statement: All procedures involving mice and rats were in accordance with IACUC guidelines. Hippocampal neurons were isolated from the hippocampus of embryonic day 18 Sprague-dawley rat embryos as previously described [47]. Briefly, hippocampus were dissected from E18 rat brains under sterile conditions in EBSS (Gibco) containing 10 mM HEPES and the hippocampal tissue was dissociated in papain and passed through a fire polished Pasteur pipette and resuspended in MEM (GIBCO supplemented with 0.2 mM fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin and N2 supplement (GIBCO). Dissociated neurons were cultured on poly-l-lysine coated plates or coverslips at a density of 2.5 6 104 cells per well. Cultures were maintained in a humidified incubator at 37uC with PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e42935