Figure 1.

CSE exposure decreased TGF-β signaling by disrupting MSC primary cilia. Single-cell-derived human mesenchymal stem cell line (SCP-1 cells (N = 4, n  =  8)) infected with an adenoviral Ad5-CAGA9-MLP-Luciferase reporter constructs (Smad2/3 reporter) were exposed overnight, either with or without Cigarette smoke extract (CSE) (a; 5–10%) or chloral hydrate (CH) (b; 0.5–1 mM). Next, cultures were incubated with recombinant human transforming growth factor beta one (rhTGF-β1) 10 ng/mL for 48 h, and luciferase activity was measured in cell lysates. The results were normalized to total protein content and expressed as relative luminesce units (RLU). Results represent mean ± standard error of the mean (SEM). Statistical significance was determined by the Kruskal–Wallis H test, followed by Dunn’s post-test. Significance was established as *** p < 0.001 compared to TGF-β-treated cells and °°° p < 0.001 compared to untreated cells. (c) Representative immunostaining images of SCP-1 cells stained for acetylated α-tubulin (green), and nuclei (blue), after CH exposure. (d) Primary cilia length quantification of SCP-1 cells treated with and without CH. (e) Percentage of ciliated SCP-1 cells following CH treatment.