Fig 2: Comparison of ET-1-dependent Gaq-protein signaling in 2D and 3D VSMC. (A) Representative phase-contrast image (20-fold magnification) of murine aoSMC cultured as a 2D monolayer (scale bar: 100 µm). (B–D) Analyses of the IP-1 level in ET-1-treated (100 nM) murine aortas ((B), n = 3, *** p < 0.001) and in aoSMC cultured as monolayer upon ET-1 treatment ((C), n = 10–12, data were normalized to total protein levels) or treated with the Gaq/11 inhibitor YM-254890 (10 µM) ((D), n = 8, *** p < 0.001). (E) Representative phase-contrast image (20-fold magnification) of aoSMC cultured as 3D spheroids (scale bar: 100 µm). (F,G) IP-1 level measured in 3D aoSMC treated with YM-254890 ((F), n = 4) or ET-1, ((G), n = 4, ** p < 0.01). (H) Ednra expression was determined by qPCR in RNA samples isolated from 2D or 3D cultured murine aoSMC (n = 4–5, ** p < 0.01, RPL32 was used as reference).

Fig 3: ET-1 promotes mesothelial to mesenchymal transition (MMT) through ETAR/ETBR/ß-arr1 axis. The working model describes the crosstalk between MCs and SOC cells. Autocrine and paracrine ET-1 activates ETAR and ETBR in mesothelial cells leading to MMT, migration and invasion and favouring transmesothelial migration of SOC cells. Created with BioRender.com.

Fig 4: ET-1 upregulates Fibronectin and a-SMA expression through ETAR/ETBR. Representative images of IF analysis of MCs stimulated with ET-1 and/or BQ123+BQ788 for 48 h. (A) Cells are stained for F-actin (green), Fibronectin (red), and DAPI (blue) or (B) for F-Actin (green), a-SMA (red) and DAPI (blue). Bars, 20 µm.

Fig 5: Mesothelial cells express ETAR and ETBR along with ß-arr1 and secrete ET-1. (A) Representative Western Blotting (WB) analysis of ETAR and ETBR and ß-arr1 in MCs and MC1s. GAPDH and Tubulin were used as the loading control. (B) Representative RT-PCR analysis showing ETAR, ETBR and ß-arr1 mRNA levels in MCs. CypA was used as a loading control. (C) ET-1 expression evaluated by qPCR in MCs at 24, 48, and 72 h (D) ET-1 secretion evaluated by ELISA assays in MC culture medium at 24, 48, and 72 h. Histograms, the mean ± SD, n = 2. One-way ANOVA.