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. 2015 Aug 19;10(8):e0133862. doi: 10.1371/journal.pone.0133862

Fig 3. Expression of Notch pathways genes.

Fig 3

A, Western blot analyses of Notch intercellular domain (NICD), hairy enhancer of split 1 (HES1) and endogenous control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cells isolated from human exocrine tissue. Whole cell lysates from CD133+ (+) and CD133-depleted (D) cells. Nuclear (N) and cytoplasmic (C) extracts from CD133+ cells. B, Volcano plot of Notch pathway gene mean ± SEM mRNA level (n = 3 exocrine cultures) 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 (p<0.05). Selected gene names shown. Genes are: receptor tyrosine-protein kinase erbB-2 (ERBB2), frizzled class receptor 7 (FZD7), E1A binding protein p300 (EP300), MFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase (MFNG), H19, imprinted maternally expressed transcript (H19), LIM domain only 2 (rhombotin-like 1) (LMO2), inhibitor of DNA binding 1 (ID1), hairy enhancer of split 4 (HES4), cyclin D1 (CCND1), matrix metallopeptidase 7 (MMP7), mastermind-like 2 (MAML2), jagged 1 (JAG1), Notch 2 (NOTCH2), hes-related family bHLH transcription factor with YRPW motif-like (HEYL), snail family zinc finger 2 (SNAI2), recombination signal binding protein for immunoglobulin kappa J region-like (RBPJL). C, Normalized mRNA expression level of neurogenin 3 (NGN3), HES1 and pancreas transcription factor 1 subunit alpha (PTF1A) in exocrine tissue after 4 days of culture in the presence of 20 μM Notch inhibitor DAPT. Results reported as mean ± SEM percent of levels in DMSO carrier control. mRNA levels normalized to the level of cyclophillin A. Significance determined by Student’s t-test, ***, p<0.001 (n = 3 exocrine cultures). D, Expression of NGN3 protein following treatment with 20 ∞M DAPT and 47 ∞M Notch agonist JAG-1 peptide (JAG-1). Mean ± SEM percent of DMSO carrier only control or 47 ∞M scrambled JAG-1 peptide, respectively indicated on Y-Axis. Significance determined by Student’s t-test, ***, p<0.001 (n = 3 exocrine cultures). E-H, Orthogonal analysis of colocalized HES1 and NGN3 in nuclei of exocrine tissue after 4 days of culture. Nuclei counterstained with Hoechst 33342 (H). E, Overlay of 3 channels. 0.5 ∞m confocal section. Scale bar is 50 ∞m. F-H, Higher magnification of crosshair region in all three channels shown at right. Scale bars are 20 ∞m. I, Coprecipitation of ID proteins with HES1. Whole cell lysate from exocrine tissue after 4 days of culture immunoprecipitated with antibody to HES1. ID1, 2 and 4 detected following SDS PAGE and western blotting. Predicted molecular weights of ID proteins (ID) and immunoglobulin heavy chain used for precipitation (HC) shown at right. Molecular weight marker positions shown at left in kDa.