Verification of morphological changes of co-cultured MSCs and functional category of differentially expressed proteins following iTRAQ.

<p>(A) Microscopic images of C57BL/6-derived pSGCs (upper, first panel), control mMSCs (lower, first panel), and co-cultured MSGs at day 1, 3, 5 and 7 were shown at a 20X magnification. pSGCs showed a typical islet-like cell morphology while control mMSCs exhibited fibroblast-like cell appearance as predicted. Aggregated co-cultured MSCs, which morphologically resembled islet-forming pSGCs, at each culture time point were indicated by black arrowheads in the day 7 panel. Co-culture was carried out for 7 days without replacing media. (B) Total protein lysate isolated from co-cultured mMSCs was subject to western blotting. Control mMSCs were used as a negative control while pSGCs were used as a positive control. Three acinar cell maker proteins (a-amylase, a-Amy; aquaporin-5, AQP5; and muscarinic acetylcholine receptor type-3, M3R) were detected in pSGCs and co-cultured mMSCs. (C) <i>A Venn diagram</i> showed the distribution of number of differentially expressed 280 proteins, selected from statistical analyses of iTRAQ data, at each time point during co-culture. (D) A total of 280 identified proteins were assigned to 45 functional groups using Gene Ontology (GO). All identified unigenes were aggregated into three main categories: Cellular Component, Molecular Function, and Biological Process. Percentages are based on the proportion and number of genes in each set. *WEGO (Web Gene Ontology Annotation Plot).