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Nitasha Phatak, Dorota Stankowska, Raghu Krishnamoorthy; Overexpression of the POU Domain Transcription Factor, Brn3b Causes Neurite Outgrowth in Cultured PC 12 Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6364.
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Brn3b is a POU domain transcription factor shown to play key role in regulating retinal ganglion cell axon outgrowth during development of the retina. The purpose of this study was to determine if overexpression of Brn3b could promote neurite outgrowth in cultured PC 12 cells.
Rat Pheochromocytoma cells ( PC 12) were seeded and grown on Poly-D-lysine coated 100 mm dishes and transfected either with pCMV6-Brn3b (an expression vector encoding Brn3b cDNA) or pCMV6-Empty (empty vector). Following transfection for 6 hrs, cells were maintained for 24 hr in complete medium containing NGF (100ng/ml). Protein extracts were isolated from these cells and analyzed for Brn3b and GAP43 protein expression by immunoblot analysis. In another set of experiments, PC 12 cells were seeded on Poly-D-Lysine coated 25mm cover slip and transfected with either pCMV6-Brn3b or pCMV6 -Empty plasmid vectors. Following transfection cells were maintained for 24 hr in complete medium with NGF (100ng/ml). Brn3b, GAP43 and TUBA-1 expression in the transfected cells were analyzed using immunocytochemistry. Morphological changes in PC 12 cells transfected with Brn3b were studied by confocal microscopy.
Immunoblot analysis showed increased levels of GAP-43 in PC12 cells transfected with Brn3b cDNA, compared to those transfected with the empty vector. Overexpression of transcription factor Brn3b in PC12 cells produced morphological changes including increased neurite outgrowth. An increased immunostaining for Brn3b and GAP43, TUBA-1 was also observed in PC12 cells overexpressing Brn3b.
The POU domain transcription factor, Brn3b, could promote neurite outgrowth some of which resembled axon projections in PC12 cells.
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