tion of the transcriptional repressor HES1. Notch regulation of NGN3 was investigated to determine if it plays a similar role in the adult pancreas. Notch signaling results in proteolytic cleavage and nuclear translocation of the Notch intercellular domain. Both NICD and HES1 were detected in whole cell and nuclear extracts of CD133+ but not CD133D cells. Furthermore, mRNA from CD133+ and CD133D cell populations were screened with a panel of genes involved in Notch signaling, Notch downstream targets and pathways that crosstalk with Notch. Of 59 genes with >2-fold changes in mean expression, 54 are upregulated in the CD133+ cell population compared to CD133D. Expression of all four Notch genes as well as ligands jagged and delta-like also were detected in CD133+ and CD133D PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19752540 transcriptomes. To determine if Notch signaling is actively regulating expression of downstream genes in exocrine tissue, the mean mRNA expression PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19752537 levels of HES1, NGN3 and pancreas-specific transcription factor 1A were measured following four days of Notch inhibition with DAPT, a gamma-secretase inhibitor, which prevents Notch cleavage. HES1 mRNA decreased significantly in the presence of DAPT, consistent with transcriptional activation by Notch. NGN3 and PTF1A mRNAs, which are regulated negatively by Notch signaling, increased significantly as did the mean percentage of cells expressing NGN3 protein. Conversely, the mean percentage of cells expressing NGN3 protein decreased significantly in the presence of Notch agonist, JAG-1 peptide compared to cells treated with an equal concentration of a negative control peptide comprised of scrambled JAG1 amino acids. The presence of HES1 in CD133+ protein extracts was unexpected given the repressive role of HES1 on NGN3 transcription and their segregation during murine fetal development. This observation was supported by NGN3 and HES1 colocalization in the nuclei of 10.2 1.8% 8 / 26 Endocrine Transdifferentiation by NGN3 Expressing Exocrine Cells 9 / 26 Endocrine Transdifferentiation by NGN3 Expressing 
Exocrine Cells Fig 3. Expression of Notch pathways genes. A, Western blot analyses of Notch intercellular domain, hairy enhancer of split 1 and endogenous control gene glyceraldehyde-3-phosphate dehydrogenase in cells isolated from human exocrine tissue. Whole cell lysates from CD133+ and CD133-depleted cells. Nuclear and cytoplasmic extracts from CD133+ cells. B, Volcano plot of Notch pathway gene mean SEM mRNA level differences in expression level from CD133+ cells compared to CD133D shown on X-axis as Log2 of fold difference. Significance determined by Student’s t-test shown on Y-axis as p value. Magenta vertical lines mark a 2-fold difference in expression. Blue horizontal line marks the significance cutoff. Selected gene names shown. Genes are: receptor tyrosine-protein kinase erbB-2, frizzled class receptor 7, E1A binding protein p300, MFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase, H19, imprinted maternally expressed transcript, LIM domain only 2 , JW 55 web inhibitor of DNA binding 1, hairy enhancer of split 4, cyclin D1, matrix metallopeptidase 7, mastermind-like 2, jagged 1, Notch 2, hes-related family bHLH transcription factor with YRPW motif-like, snail family zinc finger 2, recombination signal binding protein for immunoglobulin kappa J region-like. C, Normalized mRNA expression level of neurogenin 3, HES1 and pancreas transcription factor 1 subunit alpha in exocrine tissue after 4 days of culture in the presenc
