Journal: Nature Communications
Article Title: Cellular crosstalk mediated by TGF-β drives epithelial-mesenchymal transition in patient-derived multi-compartment biliary organoids
doi: 10.1038/s41467-025-61442-5
Figure Lengend Snippet: a Overview of in vitro A8301-mediated TGF-β signal inhibition in BA-MBOs. Created in BioRender. Ayabe, H. (2025) https://BioRender.com/q1pk62z . b Representative plots and quantification of FACS of day 14 MBOs treated with A8301. Number of hybrid (pan-CK + /VIM + ), cholangiocyte (pan-CK + /VIM - ) and mesenchymal (pan-CK − /VIM + ) cells were quantified (Mean ± SD; n = 12 organoids). c Images of MBOs treated with A8301 captured before collection and after whole-mount immunostaining (CK19: red, CD31: green, VIM: white). Abundance of CK19 + structures (Mean ± SD; n = 9 organoids examined over 3 independent experiments) and size distribution of PBG-like structures were quantified ( n = 39-104 clusters examined over 3 independent experiments). d Immunostaining of ECAD (green), CK19 (red) and Nucleus (blue; DAPI) in MBOs treated with A8301. ECAD + cells in CK19 + cells in the field were quantified (Mean ± SD; n = 12 fields from 3 organoids). e Immunostaining of ZO1 (green), CK19 (red) and Nucleus (blue; DAPI) in MBO treated with A8301. ZO1 + cells in CK19 + cells per field were quantified (Mean ± SD; n = 12 fields from 3 organoids). f Alcian blue staining of MBOs treated with A8301. Mucin production was determined by ratio of Alcian Blue + areas in CK19 + (from separately examined immunofluorescence data) areas (Mean ± SD; n = 12 fields from 3 organoids). g Top enriched terms of MSigDB Hallmark 2020 , and GO , sets in serum proteins (reanalyzed from Lertudomphonwanit et al. ) upregulated in BA subjects ( n = 175 patients) vs age-matched normal controls ( n = 9). MF: Molecular Function, BP: Biological Process. h Heatmap of enriched proteins in the EMT pathway in MSigDB Hallmark 2020 sets. ( n = 9 NC and 175 BA subjects). i Serum TGF-β/Activin ligand levels represented by the relative fluorescent unit (RFU; Mean ± SD). ( n = 9 NC and 175 BA subjects). The p -values were shown in plots and were determined using one-way ANOVA with Tukey’s test ( b – f ) and two-tailed Welch’s t -test ( i ).
Article Snippet: Bright field images were captured using a light microscope (Olympus, BX43) and immunofluorescence images were captured using either fluorescence (Olympus, BX51; Nikon, ECLIPSE Ni-E) or confocal (Nikon, ECLIPSE Ti; Zeiss, LSM880) microscopes.
Techniques: In Vitro, Inhibition, Immunostaining, Staining, Immunofluorescence, Two Tailed Test
![a Phase contrast and H&E staining of NC- and BA-MBOs. Three experiments were repeated with similar results. b Immunostaining of CK19 (green), CD31 (white), VIM (red) and Nucleus (blue; DAPI) in MBOs. Abundance of CK19 + cells ( n = 12 fields for NC or 14 fields for BA examined over 3 independent samples) and average size of gland-like structures ( n = 11 fields for NC or 7 fields for BA examined over 3 independent samples) were quantified (Mean ± SD). c Immunostaining of ZO1 (green), β-catenin (red), CK19 (white) and nucleus (blue; DAPI) in MBOs. Graphs show CK19 + cells exhibiting apical-basal polarity (Mean ± SD; n = 12 fields per 3 organoids). d Alcian Blue staining for MBOs. Mucin production in epithelium was determined by ratio of Alcian Blue + areas in CK19 + (from separately examined <t>immunofluorescence</t> data) areas (Mean ± SD; n = 15 fields [for NC] or 12 fields [for BA] examined over 3 independent samples). e Heatmap (left) and Volcano plot (right) of differentially expressed genes between NC- and BA-MBOs (up in BA: 350; down in BA: 851). f DAB staining of MBOs for MMP-7 and OPN. The staining intensities were quantified by imaging analysis (Mean ± SD; n = 12 fields per 3 organoids). g Representative plots and quantification of FACS for hybrid cells (pan-CK + /VIM + ) and cholangiocytes (pan-CK + /VIM − ) in Mono and MBO. The number and cell ratio were quantified (Mean ± SD; n = 6). For b – d , f , g , the p -values were shown in plots and were determined using two-tailed Welch’s t -test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1497/pmc12271497/pmc12271497__41467_2025_61442_Fig2_HTML.jpg)
