MATERIALS AND METHODS Reagents and cells LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate) was purchased from Avanti Polar Lipids (Alabaster, such information AL, USA) and diluted in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Basel, Switzerland) including 0.1% fatty acid-free bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA). AM095 was the kind gift of Amira Pharmaceuticals (San Diego, CA, USA). Sodium pyruvate, NEAA mixture, and penicillin/streptomycin were obtained from Lonza. l-glutamine was from Cellgro (Manassas, VA, USA). Y27632, pertussis toxin, cycloheximide, and DMSO were from Sigma-Aldrich. Latrunculin B was from Invitrogen (Grand Island, NY, USA). CCG-1423 was from Cayman Chemical (Ann Arbor, MI, USA). Permeable C3 toxin was from Cytoskeleton (Denver, CO, USA).

Recombinant TGF-��1, monoclonal anti-TGF-��1, -��2, and -��3 neutralizing antibody (1D11), and normal rabbit IgG were from R&D Systems (Minneapolis, MN, USA). NIH3T3 fibroblasts were purchased from American Type Culture Collection (Manassas, VA, USA). Mice We purchased C57Bl/6 mice from the National Cancer Institute (NCI)-Frederick Mouse Repository (Frederick, MD, USA). Experiments comparing LPA1-knockout (KO) and wild-type (WT) mice used offspring of mice heterozygous for the LPA1 mutant allele, which were hybrids of the C57Bl/6 and 129Sv/J genetic backgrounds (kindly provided by Dr. Jerold Chun, Scripps Research Institute, La Jolla, CA, USA; ref. 26). Experiments to identify fibroblasts used type I collagen (COLI)�Cgreen fluorescent protein (GFP) transgenic mice generated on the C57Bl/6 background (kindly provided by Dr.

Jeremy Duffield, University of Washington, Seattle, WA, USA; ref. 27). All experiments used sex- and weight-matched mice at 6�C10 wk of age maintained in specific pathogen-free environments and were performed in accordance with U.S. National Institutes of Health (NIH) guidelines and protocols approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee. Peritoneal fibrosis model Peritoneal fibrosis was induced by intraperitoneal injection of 0.1% CG (Wako Pure Chemical Industries, Osaka, Japan) dissolved in 15% ethanol/phosphate buffered saline (PBS). CG was injected every other day over a period of 21 d. Histology and peritoneal thickness measurement A sample of peritoneal tissue from each mouse was fixed in 10% buffered formalin (pH 7.

2) and embedded in paraffin. We then stained 5-��m sections with Masson’s trichrome according to our standard protocol (4). Peritoneal thickness was measured from the junction of the parietal peritoneum with the musculature of the abdominal wall to the serosal surface of the parietal peritoneum, as described Entinostat previously (9), on photomicrographs (��200) of Masson’s trichrome-stained sections at 5 randomly selected sites per high-power field (HPF) per section.