Supplemental Figure 2.

(A, B) Effect of culture method and VIP (0 and 330 ng/mL) on the number of goblet cells. (A) Immunofluorescence images of monolayers stained for Muc2 (green). (B) The percentage of the monolayer surface area positive for Muc 2 fluorescence. *P < .05; **P < .005; #not statistically significant. n = 3. (C, D) The hydrated mucus layer generated by VIP-assisted air-liquid interface (ALI) culture. (C) Side-view confocal micrograph showing tissues with bacteria-separating mucus accumulation at 2 and 4 days, respectively. Green: GFP-expressing EC; blue: Hoechst 33342. (D) Scanning electron microscopy of hydrated mucus layer (overlaid with GFP-expressing EC). The mucus layer was partially removed using tweezers to reveal the epithelium (lower arrow) below the mucus. The upper right panel shows bacteria (rod-shaped structures) on the surface of the mucus layer. (E–G) Characterizations of the hydrated mucus layer generated by VIP-assisted ALI culture. (E) Visualization of the mucus layer by overlaying 1-μm red fluorescent beads. Top: adding beads to the mucus layer without rinsing. Bottom: adding beads to the mucus layer after gently rinsing. (F) The mucus layer was overlaid with a mixture of 0.02-μm red fluorescent beads (#F8786; ThermoFisher) and 5_μm green fluorescent beads (#G0500; ThermoFisher) for 4 hours. (G) Immunofluorescence of sectioned monolayers. Red: AGR2 or Meprin β; blue: Hoechst 33342. There was minimal Meprin β expression, as was expected because the tissue is derived from the large and not small intestine and the β subunit is not predominant in the large intestine.⁸