Fig 2: Abnormal ASB3 expression levels in IBD patients and mice with DSS-induced colitis. (a) The mRNA expression of ASB3, IL-1β, IL-6, and TNF-α was measured in both UC groups (n = 7) relative to controls. (b) Representative immunoblotting of p-IκBα, IκBα, ASB3, villin, EpCAM, and β-actin protein expression in UC. (c) The mRNA expression of ASB3, IL-1β, IL-6, and TNF-α was measured in both CD groups (n = 5) relative to controls. (d) Representative immunoblotting of p-IκBα, IκBα, ASB3, villin, EpCAM, and β-actin protein expression in CD. (e) The expression of p-IκBα, IκBα, ASB3, and β-actin proteins in the colons of DSS-treated or untreated mice was detected by Western blotting. (f) The mRNA expression levels of ASB3 in the colons of DSS-treated or untreated mice were determined by quantitative real-time RT-PCR (qPCR) assay. (g) The expression of p-IκBα, IκBα, ASB3, and β-actin proteins in TNF-α-treated or untreated organoids was detected by Western blotting. (h) The mRNA expression levels of ASB3 in the organoids of TNF-α-treated or untreated mice were determined by qPCR assay. (i) HT-29 cells were treated with TNF-α (150 ng/mL) for 12 h and then stained with indicated antibody and secondary antibody. The nuclei were stained by DAPI. Scale bars, 50 µm. (j) Representative IHC staining and quantification of ASB3 in colon tissues collected from IBD patients. Scale bars, 50 µm. P values less than 0.05 were considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) in panels a and c by paired Student’s t test and in panels c, f, h, and j by unpaired Student’s t test.

Fig 3: SARS-CoV-2 putative target cells are enriched in the human duodenum.(A) Schematic of protocol for isolation of different tissues for scRNA-Seq using Seq-Well S3, to identify cell types. (B) Uniform manifold approximation and projection (UMAP) of 32,381 cells colored by tissue source. (C) Left: UMAP of epithelial cells showing expression of ACE2 (top), TMPRSS2 (middle), and TMPRSS4 (bottom) among all tissue sources from human donors. Color coding is as follows: purple, RNA positive; gray, RNA negative. Right: Corresponding violin plots of expression values for ACE2 (top), TMPRSS2 (middle), and TMPRSS4 (bottom). (D) Representative fluorescence immunohistochemistry image of gut tissue showing ACE2 (red), TMPRSS2 (orange), EpCAM (green), and DAPI (blue) of duodenum and colon. Bars: 20 μm for all images. (E) Quantification of ACE2 and TMPRSS2 proportion of total cells stained with EpCAM. Data shown as median ± SD.

Fig 4: Light microscopy images acquired during EpCAM detection; dashed red lines indicate the mean levitation height of the related PSMs. (a) bare PSMs, (b) PSMs with anti-EpCAM, (c) PSMs with anti-EpCAM + 5 nM EpCAM, and (d) PSMs with anti-EpCAM + 100 nM EPCAM.

Fig 5: Scheme depicting the proposed model of active RhoA dynamics in control and EPCAM-KD cells during spreading.a In control cells, active RhoA (RhoA-GTP) dynamics are promoted by EpCAM, to and from the Rab35+/EHD1+ endosomal platform. The resulting transient signal induces normal myosin-II-dependent contractility at the level of the transverse arcs during spreading. At the cellular level, dynamic RhoA-GTP can be remodeled in a front–rear gradient as the cell spreads, participating to the acquisition of front–rear polarity. Correct contractility at the levels of the transverse arcs allows the formation of ventral stress fibers and proper actomyosin cytoskeleton reorganization to promote epithelial cell migration. b In EPCAM-KD cells, active RhoA is blocked in the endosomal platform preventing the remodeling necessary for correct spreading, symmetry breaking, and polarity establishment. RhoA-sustained activity increases myosin-II contractility at the transverse arcs level, which hinders the formation of ventral stress fibers. Active RhoA also increases formin activity, producing longer dorsal fibers in EPCAM-KD cells. The absence of active RhoA and actomyosin cytoskeleton remodeling impedes symmetry breaking, giving EPCAM-KD cells a characteristic unpolarized fried-egg shape and preventing efficient cell migration.