Fig 2: AGR2 up-regulation in the patients is similar to other inflamed conditions. Although AGR2 up-regulation occurs in multiple inflammatory conditions, patients 1 and 2 showed a strikingly greater up-regulation of BiP than the other conditions. Large-bowel sections from patients 1 and 2 and patients suffering from appendicitis, polyp, and ulcerative colitis were co-stained for AGR2 (green, clone 5G1.1) and BiP (red, C50B12). Nuclei were stained with Hoechst (blue). Confocal Z stacks were acquired with a 10× objective and visualized using maximum intensity projection. Scale bars: 100 µm. When comparing other conditions with the AGR2 mutant patients, we found increased AGR2 to be common in inflammatory conditions of the gut, but the BiP increase was greatest in AGR2 mutant patients. These observations support the impaired capacity of AGR2 H117Y to regulate ER stress.

Fig 3: 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 4: 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 5: Expression of canonical breast cancer subtype-markers in breast cancer cells and clinical samples. a Expression of canonical breast cancer subtype-markers in two normal breast cell lines, eight luminal, and seven BLBC cell lines by western blot (S: Short exposure; L: Long exposure). b Expression levels of selected canonical breast cancer subtype-marker mRNAs with Her-2 positive, luminal or basal-like specific based on bc-GenExMiner v4.1 according to the Sørlie’s subtypes (*p < 0.05; ****p < 0.0001; Red star: the former > the latter; Green Star: the former < the latter). Expression of selected luminal breast cancer markers (c) and basal-like specific marker Caveolin-1 (d) in representative Her-2 positive, luminal A, luminal B and basal-like breast cancer tissues by IHC (Red scale bar = 200 µm; Purple scale bar = 40 µm). e The association of selected luminal or basal-like specific markers expression between basal-like and non-BLBC tissue subtypes were assessed using Pearson’s ?2 test (for CK18, p = 0.0076; for FoxA1, p < 0.0001; for AGR2, p = 0.0002; for Caveolin-1, p < 0.0001; Red star, basal-like > non-basal-like; Green star, basal-like < non-basal-like)