Our final results also showed that the anti apoptotic effect of BI was not altered even under the suppression on the MEK ERK pathway whereas, as expected, apoptosis was decreased within the presence of SB . In addition, early neuronal differentiation within the BI overexpressing cells was appreciably decreased inside the presence of MEK ERK inhibitor and was not altered inside the presence of p inhibitor , demonstrating the importance of BI mediated MEK ERK pathway regulation in controlling early neuronal differentiation Proportion of neuronal differentiation in mouse ES cell lines with overexpression of BI As BI altered the expression of early neuronal markers, Neuroectoderm and Nestin, throughout spontaneous differentiation from undifferentiated mES cells,we examined the impact of BI overexpression to the extent of mature neuronal differentiation. To realize differentiation into mature neurons, we developed an optimized stepwise differentiation protocol, based on several different differentiation methods . Immediately after differentiation, we evaluated cell proliferation and mature neuronal differentiation efficiency by double immunostaining and FACS examination for TUJ and the proliferation marker KI .
We observed that the TUJ favourable cells during the BI overexpressing cultures were significantly increased in contrast with that inside the management or BI Coverexpressing cultures. In addition, the proportion of KI TUJ cells in BI overexpressing cultures was markedly greater than that in the management or BI C overexpressing cultures . Subsequent, PF-04691502 in order to investigate neuronal maturity with the gene expression level, we performed real time PCR. Expression levels of doublecortin , Map , and Tuj have been greater in neuronal differentiated BI overexpressing cells, compared to that of handle or BI C overexpressing cells . Even more analysis indicated that choline acetyltransferase , tyrosine hydroxylase , and dopa decarboxylase transcripts, all of that are neurotransmitterrelatedmarkers , were also enhanced in neuronal differentiated BI overexpressing cells . To investigate whether protein expression of mature neuronal differentiationmarkers altered in conjunctionwith BI overexpression,we performed immunocytochemical analysis by using antibodies against TUJ , MAP, HOXB, HB, and CHAT.
Expression of all markers considerably enhanced in neuronal differentiated Voriconazole BI overexpressing cells, compared to that of handle or BI C overexpressing cells , plainly demonstrating that BI overexpression led to induction of neuronal differentiation. Thinking of these findings collectively, our information strongly suggest that BI overexpression significantly increases cellular proliferation and prospects on the resultant grow from the proportion ofmature neurons just after neuronal differentiation Discussion Amajor obtaining of this studywas that BI could possibly regulate each neuronal differentiation and proliferation through differential modulation of MAPK pathways.