Figure 4. Inhibition of NgR1 enhances neuronal cell production.

Differentiation of NSPs derived from the adult spinal cord was induced by depletion of EGF and FGF-2 in the presence of the indicated reagents. (a and b) Total RNA was prepared from the differentiating NSPs on indicated differentiation day, and its cDNA was used as the template for quantitative RT-PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was employed as an internal standard. Average of three independent experiments was shown and error bars indicate the S.D. (c and d) On the Day 5 (c) and the Day 10 (d) of differentiation, cells were fixed and used for immunofluorescence analysis with primary antibodies against the indicated proteins. The bars indicate 25 µm. In (e), cells expressing the indicated proteins were manually counted. DAPI-positive cells were also counted to obtain the total cell number. Each experiment was repeated 3 times with 3 independent clones of NSPs, and more than 200 cells per sample were examined. (*p < 0.05 vs. vehicle-treated NSPs, Student's t-test). The bar indicates 25 µm. (f) On the Day 0, 5 and 10 of differentiation, cells were harvested and cell number of each samples were counted, as described in the Methods section. The cell numbers were indicated as percentage against the number of cells treated with vehicle at the Day 0. Each experiment was repeated 3 times with 3 independent clones of NSPs.