Figure 4.

Differentiation of neuroblasts into neurons and siRNA-based downregulation of CSE in neuroblasts. A representative phase contrast microscopy 40× images of ED18 neuroblasts grown with and without serum and the yellow arrows indicate lengthy neurite outgrowth in serum free-exposed neuroblasts (n = 4) (A); The cells grown as in panel A were immunostained with antibody against neuronal marker, neurofilament-200 (NF-200) after 48 h and counter-stained with nuclear dye, DAPI. The cells positive for NF-200 (Green stain) indicate neuritogenesis (yellow arrow) and the DAPI (blue) indicate nuclei (n = 4) (B); Illustrative 40× immunofluorescent images of the cells maintained as in panel A stained with an antibody to the widely used neuronal nuclei marker, Neuronal Nuclei antigen (NeuN). Nuclear DNA was labeled with DAPI (n = 10). The yellow arrowheads in the merged panel (BF/DAPI/NeuN) indicate the neuronal nuclei that are double-positive for fluorescent staining of DAPI (blue) and NeuN (red) (C); Fluorescent images of immunofluorescent staining showing cortical neuroblasts under serum conditions show ready expression of the proliferation marker, proliferating cell nuclear antigen (PCNA) (red stain in the top panel), which was lost when differentiated (the bottom panel). The yellow arrowheads in the PCNA/DAPI merged panel indicate the proliferating/undifferentiated neuroblasts nuclei that is double-positive for fluorescent staining of DAPI and PCNA (n = 4) (D); A representative immunoblot image of CSE and GAPDH protein at the end of 48 h of transfection with the indicated concentrations of either non-targeting scramble siRNA or a SMARTpool mix of four siCse in serum free media (E) and the densitometric quantification of CSE band relative to GAPDH (n = 3) (F). Values represent the mean ± SEM. * p < 0.05 was considered significant. In panels A–D, the scale bar indicates 100 μm.