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Nitasha R. Phatak, Alena Z. Minton, Cynthia Elena Mireles, Raghu Krishnamoorthy; Abstract Title: Overexpression of POU Domain Transcription Factor, Brn3b Causes Neurite Outgrowth and Axon Elongation in Cultured Transformed 661W Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2659.
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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 axon outgrowth in cultured transformed 661W cells.
: 661W cells were seeded and grown on 100 mm dish and transfected either with AAV-Brn3b (an expression vector encoding Brn3b cDNA) or AAV-MCS (empty vector). Nuclear extracts were isolated from these cells and analyzed for Brn3b and GAP43 protein expression by immunoblot analysis. In another set of experiments, 661W cells were seeded on 25mm cover slip and transfected with eitherAAV-Brn3b or AAV-MCS. Brn3b and GAP43 expression were analyzed by using immunocytochemistry in the transfected cells. Morphological changes in 661W cells transfected with Brn3b were studied by using confocal microscopy.
Immunoblot analysis showed overexpression of Brn3b in 661W cells transfected with Brn3b cDNA. Overexpression of transcription factor Brn3b in 661W cells produced morphological changes including increased neurite outgrowth and axon elongation. An increased immunostaining for Brn3b and axon-specific GAP43 was also observed in 661W cells overexpressing Brn3b.
The POU domain transcription factor, Brn3b, could promote neurite outgrowth and axon elongation in 661W cells.
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