Journal: PLoS ONE
Article Title: Synergy between Sphingosine 1-Phosphate and Lipopolysaccharide Signaling Promotes an Inflammatory, Angiogenic and Osteogenic Response in Human Aortic Valve Interstitial Cells
Figure Lengend Snippet: Receptors involved in the cooperative effect. AVICs were pre-treated with the indicated drugs, activated for 12 h, and cell lysates and supernatants were analyzed by Western blot and ELISA, respectively. A) Representative immunoblots with densitometry data demonstrate inhibition of the cooperative effect on COX-2 and ICAM-1 expression (100% value) using S1P 1/3 antagonists (n = 6–12). B) Silencing S1P 1/3 attenuated the cooperative effect (n = 3 control AVIC). Scramble, siRNA control; vehicle, 0.1% DEPC. C) Immunoblots showed cooperation between FTY720 and LPS (n = 3). D) ELISA quantification of sICAM-1 levels show inhibition by S1P 1/3 antagonists (n = 6–10). E) Immunoblots demonstrate inhibition of the cooperative effect by TLR4 antagonists (n = 3). Cay indicates 5 µM CAY10614; CLI, 3 µM CLI-095; FTY, 1 µM FTY720; JTE, 10 µM JTE-013; S+L, S1P+LPS; PTX, 100 ng/ml pertussis toxin; R, resting; Sur, 10 µM suramin; W146, 10 µM W146. * p
Article Snippet: In pharmacological studies, cells were pre-treated for 30 min with either S1P receptors antagonists W146 (Cayman Chem., Ann Arbor, MI); VPC 23019 (Avanti Polar Lipids, Alabaster, AL); JTE-013 (Tocris, Bristol, UK); suramin (Biomol, Santa Fe, NM), or TLR4 signaling antagonists CLI-095 (InvivoGen, San Diego, CA); CAY10614 (Cayman Chem., Ann Arbor, MI) or signaling cascades inhibitors NF-κB SN50, ALLN, SB203580, and GF109203X (Calbiochem, Darmstadt, Germany); PD98059 (Tocris, Bristol, UK); pertussis toxin (PTX) and SP600125 (Sigma, St. Louis, MO).
Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Inhibition, Expressing