OVEREXPRESSION OF POU DOMAIN TRANSCRIPTION FACTOR, BRN3B CAUSES NEURITE OUTGROWTH IN CULTURED PC 12 CELLS

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2013-04-12

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Phatak, Nitasha

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Abstract

Purpose: 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. Methods: Rat Pheochromocytoma cells ( PC 12) were seeded and grown on Poly-D-lysine coated 100 mm dish and transfected either with pCMV6-Brn3b (an expression vector encoding Brn3b cDNA) or pCMV6-MCS (empty vector). Medium changed to complete medium with NGF (100ng/ml) after 6 hours of transfection. Protein extracts were isolated from these cells and analyzed for Brn3b and GAP43, TUBA-1 protein expression in 24 hours 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 -MCS. Medium changed to complete medium with NGF (100ng/ml) after 6 hours of transfection. Brn3b, GAP43 and TUBA-1 expression in 24 hours were analyzed by using immunocytochemistry in the transfected cells. Morphological changes in PC 12 cells transfected with Brn3b were studied by using confocal microscopy. Results: Immunoblot analysis showed overexpression of Brn3b in PC12 cells transfected with Brn3b cDNA. Overexpression of transcription factor Brn3b in PC12 cells produced morphological changes including increased neurite outgrowth. An increased immunostaining for Brn3b and neurite-specific GAP43, TUBA-1 were also observed in PC12 cells overexpressing Brn3b. Conclusions: The POU domain transcription factor, Brn3b, could promote neurite outgrowth in PC12 cells.

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