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. 2022 Jan 12;13(5):1530–1553.e4. doi: 10.1016/j.jcmgh.2022.01.008

Figure 9.

Figure 9

Characterizing adult human gallbladder epithelial cells in vitro. (A) One site (-275) in the insulin promoter region and another (+1318) in the exon 3 region were assessed for the presence of H3K9 Me3 (inactive mark) as well as H3H4 Ac and H3K9 Ac (active transcription marks) using ChIP assay in freshly isolated human islets (n = 6) and gallbladder epithelial cells (n = 2, pooled from 3 to 5). (B) Promoter regions of insulin, PDX1, NEUROG3, and HES1 were assessed for the presence of H3K9 Me3 and H3K9 Me2 (inactive marks) as well as H3K4 Me2 (active transcription mark) using ChIP assay in passages 5–10 of gallbladder-derived mesenchymal cells (n = 3–6 different biological preparations). Significance was calculated using 2-way analysis of variance with the Tukey multiple comparisons test. ∗∗P < .01. (A and B) Data are presented relative to input and normalized to IgG. The dotted line represents expression in isotype control (IgG) samples. Data in bar graphs present means ± SD and individual points present a different sample/pool. (C) Representative phase-contrast images of the differentiation process involving trypsinization (day 0), reaggregation (day 1), and cluster formation (days 7–14). (D) Real-time qPCR data for insulin genes in differentiated gallbladder cells using 5 different DNMT (DNA methyltransferase) and HDAC (Histone deacetylases) inhibitors/hypomethylation agents. Data are presented as fold-over vehicle control from 9 to 10 different biological samples/experiments. (E) Phase-contrast images of transduction efficiency using GFP-expressing adenoviral vectors. Real-time qPCR data for insulin genes in differentiated gallbladder cells using a vehicle (GFP alone) or a dominant-negative HES1 or a combination of PDX1, NEUROG3, and MAFA. Data are presented as fold-over vehicle control from 4 to 15 different biological samples and analyzed using the Kruskal–Wallis test. The exact P values after adjusting for multiple comparisons are presented. (D and E) Each dot in the violin plots represents a different sample. The horizontal solid line within each polygon represents the median, the horizontal black dotted line represents quartiles, and the polygons represent the density of individual data points and extend to minimum/maximum values. Scale bars: 20 μm. 5-Aza, 5-aza-2′-deoxycytidine; Dex, dexamethasone; GB, gallbladder; SB, sodium butyrate; TSA, trichostatin A; VPA, valproic acid.