Fig 1: Neuronal differentiation of N2A cells and mESCs.(A) Cellular morphology of N2A cells cultured in low-serum medium with RA (10 µM) for 3 and 6 d. Scale bars, 50 µm. (B) Immunoblots of Ngn2 and actin in N2A cells untreated and cultured in RA (10 µM) for 3 and 6 d. (C) Cellular morphology of undifferentiated pluripotent ESCs (ES-D0), EBs of ES differentiated 8 d in KSR medium (ES-D8), and EBs trypsinized in N2 medium for an additional 4 d (ES-D12). Immunostaining of ES-D12 neuronal cells with Tuj1 (red, Alexa Fluor 568) and DAPI (blue). Scale bars, 200 µm. (D) The expression of pluripotent markers Oct4 and Nanog, neural progenitor markers Nestin and Sox1, and neuronal markers Tuj1 and Map2 was determined by RT-PCR. Samples were collected as described in C. Data plotted are from two independent experiments, each with triplicate qPCR reactions; error bars represent SD from independent samples.