Figure 4.

ST6Gal-I overexpression in gastric epithelial organoids is maintained in organoid-derived monolayers and inhibits TNF-mediated apoptosis. A, epithelial stem cell organoids derived from normal gastric antrum (Normal) with ST6Gal-I knockdown (KD) or ST6Gal-I overexpressed (OE) or empty vehicle control (EV) (see “Experimental procedures”) were analyzed on days 3–4 for ST6GAL1 gene expression (n = 4). B, organoids were stained with an ST6Gal-I antibody (FITC), phalloidin (Alexa Fluor 594) and DAPI (representative images; n = 3; scale bar, 50 μm). C, normal, KD, and OE gastric organoids (day 0) and their derived epithelial monolayers (days 1–4) were analyzed for ST6GAL1 gene expression. D, normal, KD and OE epithelial monolayers (day 2) were stained with an ST6Gal-I antibody (FITC) as well as phalloidin (Alexa Fluor 594) and DAPI (monolayers from a representative donor (n = 3; 20×; scale bar, 50 μm). E, ST6Gal-I normal, KD or OE organoid-derived monolayers were analyzed for surface TNFR1 (FITC) by flow cytometry (representative data shown, n = 4). F, mRNA from day 2 monolayers was analyzed for TNFRSF1 expression by qPCR (n = 7). G, ST6Gal-I normal, OE, and KD organoid-derived monolayers (n = 5–10 in four independent experiments) were treated with TNF 50 ng/ml for 24 h or media and evaluated for caspase 3/7 activity by CaspaseGlo 3/7 luminescence assay. A–C significance: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.