Fig 2: Expression analysis of five cellular proteins and comparison of different quantitation methods. Lysates from Ctrl (noninfected cells) and HAdV-B3/C5-infected A549 cells (biological duplicates) were used for Western blot analyses. Alpha-tubulin was used as a loading control for the normalization. Western blot analyses and quantitative comparisons were performed for Gal1 (LEG1_HUMAN) (a), Gal3 (LEG3_HUMAN) (b), AG2 (AGR2_HUAMN) (c), and CD98 (4F2_HUMAN) (d). nq: no quantitation value was obtained.

Fig 3: Up-regulation and altered localization of AGR2 in gastrointestinal tissues of the patients. Confocal images showing AGR2 (red, clone 5G1.1 for gastric and D9V2F for bowel) immunofluorescence staining on small and large intestinal and gastric sections. Intestinal stains were visualized with goat anti-rabbit AF647 secondary while gastric stains were visualized with goat anti-mouse AF546 secondary. Nuclei were stained with Hoechst (blue). Representative images are shown for patients 1 and 2. For small bowel, representative images are shown for 1 of 3 noninflamed controls and 1 of 5 inflamed controls. For large bowel, representative images are shown for 1 of 3 noninflamed controls and 1 of 6 inflamed controls. For gastric sections, representative images are shown for 1 of 3 noninflamed controls and 1 of 4 inflamed controls. Staining was replicated independently at least 3 times. Inflamed controls shown in the figure included ulcerative colitis for gastric, Crohn’s for small bowel, and appendicitis for large bowel. Scale bar: 100 µm. Identical thresholding parameters were applied for each row of images.

Fig 4: AGR2 localization changes in the small bowel of patients to encompass all epithelial surfaces. Fluorescent immunohistochemistry on formalin-fixed paraffin-embedded sections showing AGR2 (red, clone D9V2F) localization and goblet cells, identified by MUC2 (green, clone F-2) and TFF3 (yellow, clone B-1) staining. Nuclei were counterstained with Hoechst 33342 (blue). Images were acquired via confocal microscopy. The 2 large panels are low-magnification images (10× objective) of the small bowel from a noninflamed control and patient 1 and show the change in localization of AGR2 in the patients. In the control, crypts (arrows) show a diffuse staining of AGR2 across all cell types, while villi show near-exclusive staining within goblet cells (arrowheads). The patient on the other hand shows staining across all epithelial surfaces in the section. Smaller panels below show maximum intensity projection of the high-magnification (63× objective) confocal Z-stacks of representative goblet cells from the noninflamed control and patient 1. For each pair of images, top panels show only nuclear (blue) and AGR2 (red) staining, while the bottom panels show the merge for all 4 markers. Patient AGR2 staining is diffuse within the cytoplasm and is present throughout all epithelial cells, in contrast to the goblet cell–specific staining in the control. Accumulation of AGR2 against the mucin granules (arrowhead) observable in the control is lost in the patient. Identical thresholding parameters were applied for all images. Scale bars: 100 µm (large panels); 10 µm (small panels).

Fig 5: Identification of a homozygous missense variant in AGR2. (A) Map of AGR2 protein domains as reviewed by Delom et al24 with the mutation site indicated. (B) Pedigree showing the parents, the patients (P1, P2), and healthy siblings (S1–S5). Only the patients are homozygous for the mutation. (C) Chromatograms from Sanger sequencing of genomic DNA from the family and an unrelated healthy control. (D) Ribbon diagram of the WT AGR2 homodimer (grey/cyan, Protein Data Bank ID: 2LNS) showing the side chain position of H117 (green) at the protein surface in each monomer. (E) Surface location of H117 (green, thin stick representation) and modeling of the H117Y mutant side chain (blue) using the ICM software package (Molsoft). The neighboring residue L118 (grey) packs against V137 (purple, distance 4.3 Å) as shown. (F) H117 residue of AGR2 protein is conserved across species. Multiple sequence alignment of AGR2 orthologues, using Clustal Omega (UCD Conway Institute, Dublin, Ireland), and similarity highlighting were performed through UniProt. The following orthologues are shown with the UniProt identifiers in parentheses in the following order: humans (O95994), mouse (O88312), zebrafish (Q5RZ65), American chameleon (G1KMJ8), Tasmanian devil (G3VGV8), pig (A0A4X1TD21), sperm whale (A0A2Y9FEZ9), Sumatran orangutan (Q5R7P1), Japanese rice fish (A0A3B3IHX1), and Duckbill platypus (F7FAI5).