Fig 2: SKI regulated TGF-ß1-mediated activation of the Wnt/ß-catenin signaling pathway in HK-2 cells: (a) immunofluorescence staining demonstrating that ß-catenin protein expression was inhibited by SKI (200×); (b) western blotting images of active ß-catenin, Snail1, TCF4, WNT1, ß-catenin, and PAI-1, which are involved in the Wnt/ß-catenin signaling pathway; (c–h) graphic presentation of active ß-catenin, Snail1, TCF4, WNT1, ß-catenin, and PAI-1 expression in the four groups as indicated; and (i–m) qRT-PCR revealed that SKI reversed the increased level of Snail1, Tcf4, Ctnnb1 (ß-catenin), and PAI-1 mRNA expression.

Fig 3: Cytotrophoblasts (CTBs) of the SC inhibit extravillous trophoblast (EVT) invasion.(a) Immunofluorescence co-localization of np-CTNNB1 and HLA-G in the VC. (b) Immunofluorescence co-localization of KRT6 and HLA-G in the SC. Arrowheads denote CTB and EVT interactions. (c) Schematic of the transwell invasion assay (top). Cells from either chorionic region were plated in the upper chamber of the transwell. After 39 hr of culture, the transwell membrane was fixed and stained with a Pan-cytokeratin antibody. The projections through the membrane are denoted by black arrowheads and quantified below. Results from the VC derived cells are shown in black, and the results from the SC derived cells are shown in blue. (d) Results from three biological replicates from each chorionic region cultured with conditioned medium from either VC or SC cells. The gestational ages of the plated cells are shown adjacent to the y-axis. The gestational ages of the cells from which conditioned medium was collected are noted in the legends at the right. The results for cells cultured with VC cell conditioned medium are denoted by black dots, and the results for those cultured in SC cell conditioned medium are denoted by open squares. p-Values were determined by t-test and are listed above each comparison. (e) Predicted receptor-ligand interactions from CellPhoneDB. The strength of interaction is estimated by mean expression and is plotted in the heatmap. Receptor-ligand interactions and cell pairs are listed such that Molecule 1 is expressed by Cell 1 and Molecule 2 is expressed by Cell 2. For all images, nuclei were visualized by DAPI staining; scale bar = 100 µm. Abbreviations: AV = anchoring villi; FV = floating villi; SC = smooth chorion epithelium; Dec. = decidua.

Fig 4: Silencing of Ctnnb1 gene in mkCF cells. The mkCF cells were transfected with Ctnnb1 siRNA for 72 h and then cultured for 4 further days (7 days in total post-transfection), after which the cells were subjected to molecular and protein analyses. (A) RT-PCR analysis revealed a significant inhibition of Ctnnb1 gene 72 h and 7 days post-transfection compared to control siRNA. (B) Western blot analyses revealed a significant downregulation of ß-catenin 7 days post-transfection. ** p < 0.01, **** p < 0.0001 was considered statistically significant. Data represents mean ± SEM of three independent experiments.

Fig 5: Silencing of ß-catenin prevents the up-regulation of profibrotic genes in fibroblasts in vitro. mTECs pre-treated with 100 µM AA for 4 h were co-cultured with mkCF or mkCF silenced for the Ctnnb1 gene in the presence or absence of HLSC-EVs. Post-experimental analysis revealed a significant upregulation of the profibrotic genes a-Sma (A) and Col1a1 (B) in fibroblasts exposed to AA-treated mTECs, which was significantly reduced in the presence of HLSC-EVs. No significant up-regulation of both profibrotic genes was observed in fibroblast silenced for the Ctnnb1 gene, which codes for ß-catenin in the presence or absence of HLSC-EVs, when compared to control. Data represent the mean ± SD of three independent experiments. A one-way ANOVA with Bonferroni’s comparison test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001 was considered to be statistically significant.