Figure 3

Increased neurite length in NGF-treated PC12-U18666A compared to controls. A. PC12 cells were treated for 24 hrs with 2 μg/ml of U18666A and for another 48 hrs with the same dose of the drug in the presence of NGF (5 ng/ml). After the treatments, PC12 cells were fixed and stained for filipin. B. The number of differentiated cells (cells with a neurite at least twice the diameter of the cell body), the length of the neurites and the number of neurites per cell were quantified in control and PC12-U18666A cells. A total of 100 cells from three different experiments were quantified. The differences between treatments are significant, *p < 0.02. C. PC12 cells were treated for 24 hrs with NGF (5 ng/ml) and for an additional 24 hrs with NGF alone (48 hrs NGF) or in the presence of U18666A (2 μg/ml) (48 hrs NGF + 24 hrs U18). D. The number of differentiated cells, the length of neurites and the number of neurites per cell were quantified in the conditions defined in C. The differences between treatments are significant, *p < 0.02. E. PC12 cells were treated for 72 hrs with NGF (72 hrs NGF) or 24 hrs with NGF followed by 48 hrs with U18666A (72 hrs NGF + 48 hrs U18). Cells treated with the drug are stained, as shown in the representative images. F. PC12 cells were treated for 48 hrs with NGF (48 hrs NGF) or 24 hrs with NGF followed by 24 hrs with U18666A in the absence of NGF (24 hrs NGF + 24 hrs U18). PC12 cells treated with the drug in the absence of NGF retracted neuritis, which is evident compared with cells that were treated with NGF for 48 hrs. G. PC12 cells were treated for 48 hrs with NGF (48 hrs NGF) or for 24 hrs with NGF followed by 24 hrs with wortmannin (100 nM) in the presence of NGF. PC12 cells treated with wortmannin retracted neuritis, indicating that neurite elongation is at least partly regulated by IP3K signaling.