Fig. 3

RFP-zRICH(H334A) expression increases branching during PC12 differentiation. A: Summary of the computer-assisted random field analysis procedure. The diagram summarizes the morphometric procedure to analyze differentiated PC12 cells in a sample of random field images. The phase contrast images are processed to quantitate various parameters with the help of the “Cell Counter” and “Grid” plugins of ImageJ software. B: Example of ImageJ processing of a random field image captured from cultured PC12-RFP-zRICH(H334A) cells treated with NGF for 7 days. The top-left panel shows the original phase contrast microscopy image (scale bar represents 50 μm), and the bottom-left panel shows the image processed with ImageJ to count various parameters. The 6 small images on the right side represent magnified views to illustrate an example of each of the parameters quantitated on the processed image (marked with arrowheads). C: Results of morphometric analysis of sets of random field images with the stable transfectants. From left to right, the graphs represent the number of primary neurite roots, the number of branching points, and the number of horizontal grid crosses, normalized by the Neuronal Cell Count parameter for each image, respectively. The most pronounced effect in differentiated PC12 cells expressing zRICH(H334A), when compared with neurons expressing RFP, was a 2.2 fold increase in the number of branching points per neuronal cell when compared with cells expressing RFP. The number of horizontal grid crosses increased by 1.3 fold, but it was not statistically significant. A modest, not statistically significant, 1.1 fold increase in neurite roots was observed. The bars show the average ± SEM; n = 20 random field images for both PC12-RFP and PC12-zRICH(H334A) differentiation assays. Statistics: * t-test, p < 0.05. The experiment presented is representative of 3 independent assays