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

Fig. 5. MES fate acquisition relies on disruption of epithelial integrity.

Fig. 5

a CTRLsh and GPC4sh5-c10 029 hiPSCs were treated or not with LPA or Blebbi and subjected to immunofluorescence analysis of ZO1 (green), n = 3. Scale bar: 100 μm. b Immunofluorescence analysis of ZO1 (green) and Na+/K+ ATPase (magenta), in CTRLsh and GPC4sh5-c10 029 hiPSCs treated or not with LPA or Blebbi. Pictures are presented as lateral view of stained cells, n = 3. c, d CTRLsh and GPC4sh5-c10 029 hiPSCs treated or not with LPA or Blebbi were differentiated into MES and the expression levels of MES specific markers were analyzed by (c) immunofluorescence analysis of EOMES (red; n = 3; Scale bar: 100 μm.) and (d) RT-qPCR analyses of MIXL1 and GSC transcript levels. Transcript levels were normalized to the ΔCt of reference WT control sample. Box plots represent the median with min and max values, n = 3. e GPC4sh5-c10 and GPC4sh2-c3 029 hiPSCs were differentiated into MES using ACTIVIN A and subjected to immunofluorescence analysis of ZO1 (green) and BRACH (magenta). Outlined areas highlight the extent of zones with disrupted TJs, n = 3. Scale bar: 100 μm. f Percentage of BRACH positive cells as a function of distance from the zone with disrupted TJs were quantified from staining shown in e. 0 μm indicates cells located within the zones of disrupted TJs, +30 μm, +60 μm, +90 μm and >+90 μm indicate cells located at 0–30 µm, 30–60 µm, 60–90 µm or >90 µm away from zones of disrupted TJs, respectively. Slope of the curves are represented as m. Data are represented as mean ± SD. n = 3. Statistical analysis for the overall figure: (d) two-way ANOVA, followed by Dunnett’s multiple comparison test, (f) linear regression analysis. P-values: (***) <0.001, (**) <0.01, (*) <0.05, ns not significant. For all panels “n” corresponds to the number of biological replicates unless stated otherwise. Source data are provided as a Source Data file.