Figure 5.
Effects of increased NG2 expression on neurite growth. A–D, Comparison of the level of NG2 expression in control NG2 cells and in NG2 cells infected with adeno-NG2. A, B, Surface immunofluorescence labeling (live-cell labeling) for NG2 on uninfected (A) and adeno-NG2-infected cells (B; moi 5). Images were acquired under identical exposure conditions below pixel saturation. Scale bar: (in B) A, B, 12 μm. C, Quantification of fluorescence intensity of uninfected NG2 cells and NG2 cells infected with adeno-NG2. Cells transduced with adeno-NG2 expressed fivefold more surface NG2 than uninfected NG2 cells. D, Western blots comparing the amount of NG2 in extracts of uninfected and adeno-NG2-infected NG2 cells. The same blot was probed with antibodies to NG2 (top) and GAPDH (bottom) chABC+: extracts treated with chondroitinase ABC. Smaller amounts of protein extracts from adeno-NG2-infected cells were loaded to allow for densitometric analyses of the samples within the dynamic ranges of the pixels, as seen by the weaker GAPDH bands in lanes 3 and 4. The positions of molecular weight standards are indicated on the left. E–I, Response of P1 hippocampal neurons to NG2 cells expressing elevated levels of NG2. E, F, Double-immunofluorescence labeling for NG2 (red) and βIII tubulin (green) to illustrate the spatial relationship between neurites and NG2 cells expressing normal (E) or increased levels of surface NG2 after adeno-NG2 transduction (F; moi 5). Scale bar: (in F) E, F, 24 μm. G, Percentage of neurite tips contacting uninfected or adeno-NG2-infected NG2 cells. ns, No statistically significant difference between the number of neurite tips contacting uninfected and adeno-NG2-infected NG2 cells (P = 0.239, t test). H, Total neurite lengths of neurons grown on uninfected or adeno-NG2-infected NG2 cells. ns, No significant difference (P > 0.05, one-way ANOVA). They were significantly longer than neurite lengths on PLL (P < 0.001, one-way ANOVA). I, The lengths of the longest neurite of neurons grown on uninfected or adeno-NG2-iinfected NG2 cells. ns, No significant difference (P > 0.05, one-way ANOVA). They were significantly longer than neurite lengths on PLL (P < 0.001, one-way ANOVA). Error bars indicate SE.