Fig. 4

RFP-zRICH(H334A) expression promotes branching during neurite regeneration in PC12 cells. A: Summary of the neurite regeneration assay. The diagram summarizes the procedure to analyze the regrowth of neurites. Differentiated PC12 cells were subjected to mechanical damage and then allowed to regenerate neurites for a limited time in medium with NGF. Morphometric analysis was performed by the NeuronJ tracing procedure. B: Example of NeuronJ tracing of a PC12-RFP-zRICH(H334A) cell with an extensive regenerated neurite arbor after 36 h. The top image shows the phase contrast image and the bottom image shows NeuronJ neurite segment tracings (primary in blue and secondary in green). Scale bar represents 50 μm. C: Results of morphometric analysis of neurite regrowth assays with the stable transfectants. From left to right, the graphs represent the number of primary neurite branches, the number of secondary neurite segments, and the total neurite arbor length per cell, respectively. The most pronounced effect of the expression of zRICH(H334A) during neurite regeneration in PC12 cells (by comparison with cells expressing RFP) was again a significant increase of approximately 2.2 fold in secondary neurite segments. A smaller increase of approximately 1.3 fold was observed in the total neurite arbor length, but it was not statistically significant. The bars show the average ± SEM; n = 46 PC12-RFP and 39 PC12-zRICH(H334A) cells. Statistics: ** t-test, p < 0.01. The experiment presented is representative of 3 independent assays