NE102 Final Lab Write-Up

Taken at 40x c micrographs of differentiated pc12

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Taken at 40x. c) Micrographs of differentiated PC12 cells transfected with a stable vector and treated with NGF. Taken at 20x. d) Micrographs of differentiated PC12 cells transfected with a stable vector and treated with NGF. Taken at 40x. e) Light micrographs of undifferentiated PC12 cells transfected with ZnErg1 without treatment of NGF. Taken at 20x. f) Light micrographs of undifferentiated PC12 cells transfected with ZnErg1 without treatment of NGF. Taken at 40x. g) Micrographs of undifferentiated PC12 cells transfected with ZnEgr1 with treatment of NGF. Taken at 20x. h) Micrographs of undifferentiated PC12 cells transfected with ZnEgr1 with treatment of NGF. Taken at 40x. Figure 9. Fluorimager immunoblot of stably transfected PC12 cells with a vector or ZnEgr1 with and without NGF treatment. a) Results for presence of β-actin in transfected cells. b) Results for presence of Erg1 in transfected PC12 cells.
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Results In the experiments performed, we explored neuronal differentiation in PC12 cells with treatment of NGF. We focused on the role of Ras in neuronal differentiation, the localization of NFT L and Egr1 in differentiated cells, and the necessity of Egr1 for this process. Figure one depicts a simple image of PC12 cells, differentiated or undifferentiated with or without treatment of NGF. We can see that in image (a) the cells weren’t treated with NGF and did not differentiate. Image (b), however, is an image of PC12 cells that were treated with NGF and it is clear that the cells differentiated. This shows that NGF causes neurites to grow from undifferentiated PC12 cells. Figure two and three are results of the second part of the experiment. We tested the localization of NFT L and Egr1 in the differentiated PC12 cells. Figure two shows the ICC and fluorescence images for Egr1. More specifically, image (a) illustrates the differentiated cells, which fluoresce green. The dark circles that appear are the nuclei, which we know by comparing to the DAPI stain in image (b). It makes sense that the nuclei don’t fluoresce green as much as the rest of the cell because it shows that Egr1 is not present in undifferentiated cells. Image (c), however, shows the nuclei being much more brighter and greener in color. This represents the abundant presence of Egr1 in the differentiated PC12 cells. A similar analysis can be to figure three, which are the ICC results for NFT L. Our main focus was on the neurites of the differentiated cells because that is where NFT L is likely to be found. In the cells that were not treated with NGF, we saw very little green fluorescing, as shown in image (a). But in the cells treated with NGF, image (c), we can see there is a significant amount of green color. We know that the green color is not coming from the nuclei because they were compared to the DAPI stains in images (b) and (d).
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The next portion of our study focused on the role Ras plays in neuronal differentiation. We hypothesized that Ras does induce differentiation in PC12 cells and that it would be overexpressed in cells transfected with pRas-G12V. The first step we took was to create restriction digests of the pGFP and pRas-G12V plus pGFP plasmids. This was to ensure the plasmids were made correctly with the right components. Figure
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Taken at 40x c Micrographs of differentiated PC12 cells...

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