Cuboidal-like MCs treated with patient rA increase cell-cell interactions and Alcian blue stained extracellular matrix.

(A) Removing EGF from culture media drives mesothelial cells into a cuboidal-like morphology. Filamentous actin visualization with Phalloidin staining (Grayscale) on mesothelial cells in 2% media with EGF and 2% media without EGF. (B) Cartoon depicting cell-cell interaction scores corresponding to changes in MC morphology. Graph of cell-cell interaction scoring results following nested 1-way ANOVA of scoring from 3 independent experiments. Morphological changes were accessed by brightfield, and rA-treated MCs increased cell-to-cell interactions compared to controls (low interaction = 1, processes between cells = 2, dense thread-like fibers = 3, mean score ± SD: controls (2% Control and HS) 1.71 ± 0.46, appy and SBO rA-treated 2.31 ± 0.55, p < 0.05. (C) 48h-rA treated mesothelial cells were stained with Alcian blue at a pH of 2.5 to detect sulfated and carboxylated acid mucopolysaccharides and sulfated and carboxylated sialomucins. Relative Alcian blue staining was solubilized and quantified by microplate optical density (OD) at λ630nm. The two control conditions, 2% Control and HS showed very low levels of Alcian blue staining. Three of the rA showed significantly higher levels of Alcian blue staining after 48h culture compared to HS treatment controls: median optical density at 630nm ± SD: (HS, 0.0602 ± 0.005086; vs APPY 1, 0.1323 ± 0.04955 (ns); APPY 2, 0.2803 ± 0.05526 (* P<0.05); APPY 3, 1.097 ± 0.2398 (***P<0.001); APPY 4, 0.222 ± 0.2033 (ns); SBO 1, 0.8314 ± 0.2951 (**P<0.01); SBO 2, 0.1321 ± 0.04869 (ns); SBO 3, 0.07603 ± 0.01536 (ns). Results are graphed as a boxplot of median scores (interquartile range); *P<0.05; **P<0.01; ***P<0.001.

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