Mouse primary neuron culture

Embryonic day 15 (E15)–E16 mouse cortical neurons were dissected and plated at a density of 5 × 104 to 20 × 104 cells per well onto coverslips coated with or without poly-d-lysine (0.1 mg/ml; BD Biosciences, San Jose, CA) in plating medium [minimum essential medium (MEM) + 10% fetal bovine serum (FBS); Gibco, Carlsbad, CA]. After 3 to 4 hours, medium was changed to culturing medium (Neurobasal medium + B27 + GlutaMAX; Invitrogen, Carlsbad, CA). Cells were transfected via electroporation with an Amaxa Nucleofector apparatus (Amaxa, Cologne, Germany) at 0 days in vitro (DIV0) according to the manufacturer’s instructions. After DIV5, the cells were fixed for neutric length analyses. For qPCR or Western blot analyses, neurons were infected 2 or 6 days after plating (DIV2 or DIV6) with indicated lentiviruses at a multiplicity of infection of 2, and cells were harvested at DIV6 or DIV15. For dendritic length analyses, neurons were transfected by calcium phosphate transfection at DIV5 and fixed at DIV15 for further analyses. For rescue experiments, we cotransfected each group with three plasmids using a 1:1:0.5 ratio of WT CSDE1 or CTNNB1, shRNAs, and enhanced GFP. For experiments examining the effect of Wnt/β-catenin pathway activation on neurodevelopment in culture, neurons were treated with 5 mM lithium (Sigma, St. Louis, MO) for 7 days. Study protocols complied with all relevant ethical regulations and were approved by the IRB of Central South University.

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