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. 2023 Jan 21;14:349. doi: 10.1038/s41467-023-35965-8

Fig. 6. Disruption of epithelial integrity enables activation of TGF-β signaling.

Fig. 6

a Cultures of WT, CTRLsh, GPC4sh5-c10 and GPC4sh2-c3 hiPSCs treated or not with LPA or Blebbi were stimulated for 1 h with BMP4 (50 ng/mL), and analyzed for pSMAD1,5 (magenta) and ZO1 (green) proteins by immunocytochemistry. Dashed areas highlight zones with disrupted TJs, n = 3. Scale bar: 100 μm. b Percentages of pSMAD1,5 positive cells quantified from staining in a. Box plots represent the median with min and max values, n = 3. c Percentages of pSMAD1,5 positive cells as a function of distance from zones with disrupted TJs quantified from staining in a. 0 μm indicates cells located within zones with disrupted TJs, +30 μm, +60 μm, +90 μm and <+90 μm indicate cells located at 0 to 30 µm, 30–60 µm, 60–90 µm or >90 µm away from zones with disrupted TJs, respectively. Data are represented as mean ± SD, n = 3. d Immunofluorescence analysis of pSMAD1,5 (magenta) and ZO1 (green) in the indicated hiPSC lines grown in transwell plates and stimulated from the basal side with BMP4 (50 ng/mL). Outlined areas highlight zones of disrupted TJs, n = 1. Scale bar: 100 μm. e Immunofluorescence analysis of ZO1 (green) and BMPR1A (magenta), in CTRLsh and GPC4sh5-c10 hiPSCs. Pictures are presented as lateral view of stained cells, n = 3. f Quantification of BMPR1A receptor distance to the plane of ZO1 contacts in CTRLsh and GPC4sh5-c10 hiPSCs. Data are represented as mean +/− SD of n = 10 frames of view. g RT-qPCR analysis of BMPR1A and BMP4 transcripts in CTRLsh and GPC4sh5-c10 hiPSCs. Data are represented as box and whisker plot with a line at the median, n = 8. h Protein extract of the indicated hiPSC lines were analyzed by Western-blot using BMPR1A antibodies. Membrane ponceau staining was used as loading control, n = 2. Statistical analysis for the overall figure: (b) two-way ANOVA, followed by Dunnett’s multiple comparison test, (f) unpaired t-test. P-values: (***) <0.001, (**) <0.01, (*) <0.05, ns not significant. For all panels “n” corresponds to the number of biological replicates. Source data are provided as a Source Data file.