(A) Heatmap of normalized RNA-seq glycotransferase and mucin gene counts of HIOs associated with E.coli at 0–96 hr post-microinjection. N = 4 (0 hr), 5 (24 hr), 3 (48 hr), and 4 (96 hr) biological replicates consisting of 5–6 pooled HIOs per replicate. (B) Periodic acid-Schiff and Alcian Blue (PAS-AB) staining of control HIOs or HIOs microinjected with E. coli and cultured for 48 hr at 10X magnification. (C) HIO epithelium from control HIOs or HIOs microinjected with E. coli and cultured for 48 hr stained with H and E, AB, PAS, or PAS-AB and imaged under 100X light microscopy. (D) Confocal micrograph of HIO epithelium from a control HIO or an HIO microinjected with E. coli and cultured for 48 hr. Nuclei are stained blue with DAPI, and fluorescent antibody-labeled proteins E-cadherein and Mucin 5 AC are pseudocolored in white or red, respectively. UEA1 lectin is used to label the carbohydrate moiety Fucα1-2Gal-R, which is pseudo colored in green. 60X optical magnification. (E) Heatmap of normalized RNA-seq glycotransferase and mucin gene counts of HIOs associated with live or heat-inactivated E. coli, E. coli + NF-κB inhibitor (SC-514) or HIOs cultured under hypoxic conditions for 24 hr. Results represent the mean of N = 4–5 biological replicates per treatment condition, with each replicate consisting of 5–6 pooled and identically treated HIOs. (F) PAS-AB staining of HIOs treated as indicated in the figure labels for 24 hr. 10X magnification. Histological and immunofluorescent images in panels B-D and F are representative of three or more independent experiments, each consisting of 5–10 HIOs per treatment group.