*2.6. Primary Neuronal Cultures*

Primary cortical culture was prepared from fetal cortices of *NestinCre*/+; *TCTPf*/+ intercrossed with *TCTPf*/*f* mice at embryonic day 16.5 (E16.5) or postnatal day 0.5 as previously described [25]. Briefly, the fetal cortices were removed and dissected, followed by mechanical trituration in Hanks' balanced salt solution (GIBCO #14185, Thermo Fisher, Waltham, MA, USA) containing 2.5 U/mL dispase and 2 U/mL DNase. The supernatant that contained cortical neurons was filtrated through a 70-μm filter (BD Falcon #REF352350, New York, NY, USA) and transferred into a 15-mL autoclaved tube, and then immediately centrifuged at 1500 × *g* for 10 min. The pellet containing neurons was resuspended in minimum essential medium (MEM) (GIBCO #12561) containing 10% heat-inactivated fetal bovine serum (FBS), 10 g/<sup>L</sup> glucose (Sigma #G7021, St. Louis, MI, USA), 0.176 g/<sup>L</sup> L-glutamine (GIBCO

#25030), 0.12 g/<sup>L</sup> sodium pyruvate (Sigma #p2256), 2.2 g/<sup>L</sup> sodium bicarbonate, 0.238 g/<sup>L</sup> HEPES (Sigma #H4034), and 10 mL/L 100 × penicillin–streptomycin (BioWest #L0022, Les Ulis, France). Cells were seeded at a density of 2.5 × 10<sup>5</sup>/well in 0.5 mL medium in a 24-well culture plate. The culture dishes were precoated with poly-d-lysine hydrobromide (50 μg/mL) (BD Bioscience #354210) for 2 h. The dishes were then washed twice with autoclaved deionized water. After 4 h, the MEM was replaced by Neurobasal medium (GIBCO #21103-049) supplemented with B27 (GIBCO #17504-044). Cells were incubated at 37 ◦C in a humidified atmosphere of 5% CO2 and 95% air.
