Bms453

Transcriptional activation of RAR‐reporter constructs by tRA was found to be maximal between 0.1 and 10 μM (Allenby et al. 1993), and experiments with glial cultures showed strong effects with 0.01–1 μM (van Neerven, Regen, et al., 2010). To test the molecular regulation of phagocytosis we therefore tested 10 nM, 0.1 μM, and 0.5 μM of the pan‐RAR agonist all‐tRA (R2625; Sigma); 0.1 and 0.5 μM pan‐RXR agonist bexarotene (153559‐49‐0; LC Laboratories); 1 μM pan‐RXR antagonist UVI3003 (3303; Tocris); 1 μM RARβ agonist‐RARα/γ antagonist BMS189453 (SML1149; identical to BMS453; Sigma); 1 and 10 μM pan LXR agonist T0901317 (Cay71810‐10; Cayman); 9 and 18 μM PPARα agonist fenofibrate (Cay10005368‐1; Cayman); 50 nM and 0.1 μM PPARγ antagonist rosiglitazone (LKT‐R5773.100; LKT Laboratories); 0.2 and 0.5 μM PPARβ/δ agonist GW501516 (Cay10004272‐1; Cayman); 1 and 2 μM FXR agonist GW4064 (Cay10006611‐5; Cayman); 0.5 and 1 μM TGM2 inhibitor cystamine dihydrochloride (B22873.14; Alfa Aesar); 15, 25, 50, and 100 μM TGM2 inhibitor ERW1041E (5095220001; Merck); and 1, 5, and 25 μM blocker of scavenger receptor CD36 sulfo‐N‐succinimidyl oleate (SSO; SML2148; Merck).

Fetal bovine serum, Eagle’s minimum essential medium (MEM), and 10X MEM were obtained from Invitrogen-Gibco (Rockville, MD). Reconstitution buffer and acid-solubilized native porcine type I collagen were from Nitta Gelatin (Osaka, Japan). Antibodies to phospho-FAK (phospho-Tyr^576/577) and to FAK were obtained from Cell Signaling (Beverly, MA). The RARα agonist Am580, the RARβ agonist BmS453, collagenase, and antibodies to β-actin and to alpha-smooth muscle actin (α-SMA) were from Sigma-Aldrich (St. Louis, MO), and the RARγ agonist R667 (palovarotene) was from NARD Institute (Hyogo, Japan). Recombinant human TGF-β1 and antibodies to MMPs and to tissue inhibitors of metalloproteinases (TIMPs) were obtained from R&D Systems (Minneapolis, MN). Enzyme immunoassay (EIA) kits for fibronectin and the C peptide of procollagen type I were from Takara Shuzo (Shiga, Japan). Viscoelastic material (Healon) was obtained from Abbott Medical Optics (Santa Ana, CA), 30G needles were from NIPRO (Osaka, Japan), and silicon microtubes with a diameter of 0.3 mm were from ARAM (Osaka, Japan).

5-fluorouracil (5-FU), vincristine sulfate (VCR), cisplatin (CDDP), mitomycin C (MMC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), caspase-3 inhibitor (z-VAD-fmk), G418, BMS453 and CoCl2 were all purchased from Sigma-Aldrich (St. Louis, MO, USA). The RPMI-1640 and fetal bovine serum (FBS) were all purchased from Gibco BRL (Grand Island, NY). The 100 U/ml penicillin and bicinchoninic acid protein assay reagent were purchased from Wuhan Boster (Wuhan, China). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ab9485) and RARβ (ab198557) antibodies were purchased from Abcam (Cambridge, UK). The annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining apoptosis detection kit and TdT-mediated dUTP nick end labeling (TUNEL) kit were obtained from Roche Diagnostics (Basel, Switzerland). Lipofectamine 2000 was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA., USA). The Caspase-3 assay kit was from R&D Systems, Inc. (Minneapolis, MN, USA). All of other chemicals and reagents were the highest quality and purchased from standard commercial sources.