Cell viability was determined using an MTT assay. Theca cells were seeded into 96-well plates (10,000 cells/well) and cultured overnight in basic medium. After cell attachment, cells were treated with a range of TBT (Cat# T50202, >96%, Sigma, St. Louis, MO, USA) concentrations (0, 1, 10, 20, 50, 100, 200, 500, 1,000, and 2,000 ng/ml) and/or an RXR antagonist (UVI3003;Cat# 847239-17-2, >98%, Tocris Bioscience, Bristol, UK; concentrations: 0, 0.1, 0.2, 0.5, 1, 2, 4, 8, 20 and 50 μM) in basic medium for 72 h. TBT and UVI3003 were dissolved in DMSO at 0.1% (v/v). All control groups were exposed to DMSO at 0.1% (v/v). Thereafter, medium was replaced with 100 μl of phenol red-free MTT working solution (50 μg/ml) and incubated for 4 h. MTT working solution was then discarded and 100 μl of DMSO added into each well. Plates were vortexed for 10 min at room temperature and cell viability determined by absorbance quantification at 570 nm using a microplate reader (SpectraMax M5e, Molecular Devices LLC, Sunnyvale, CA, USA).
Uvi3003
Manufactured by Bio-Techne
Sourced in United Kingdom
The UVI3003 is a UV/Visible Spectrophotometer designed for accurate and reliable absorbance measurements. It features a wavelength range of 200-1100 nm and a photometric range of -0.3 to 3.0 Abs. The instrument provides consistent and reproducible results, making it suitable for a variety of laboratory applications.
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Lab products found in correlation
Bexarotene, by LC Laboratories (3 mentions)
Cd3254, by Bio-Techne (3 mentions)
Bms614, by Bio-Techne (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Bms493, by Merck Group (2 mentions)
Lg100268, by Bio-Techne (2 mentions)
Am580, by Bio-Techne (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Puromycin, by Merck Group (1 mentions)
Anti pd l1, by Cell Signaling Technology (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Agn193109, by Bio-Techne (1 mentions)
Anti sox2, by Cell Signaling Technology (1 mentions)
Anti oct4a, by Cell Signaling Technology (1 mentions)
Cellytic mt, by Merck Group (1 mentions)
Anti nanog, by Cell Signaling Technology (1 mentions)
Anti klf4, by Cell Signaling Technology (1 mentions)
Anti mpges 1, by Cayman Chemical (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Bms493, by Bio-Techne (1 mentions)
12 protocols using uvi3003
1
Theca Cell Viability Assay with TBT and RXR Antagonist
2
Murine Myoblast Isolation and Differentiation
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Mouse primary myoblasts were isolated and differentiated as previously described (AlSudais et al., 2016 (link)). C2C12 myoblasts acquired from the American Type Culture Collection (ATCC) were maintained in growth medium (GM), Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (P/S), at 37°C with 5% CO2. For differentiation, GM of 80% confluent C2C12 culture was replaced with differentiation medium (DM), DMEM supplemented with 2% horse serum, for the indicated duration and treatment. Bexarotene (Gniadecki et al., 2007 (link)) was purchased from the LC Laboratories, UVI3003 (Nahoum et al., 2007 (link)) from the Tocris and puromycin from the Sigma.
3
Stemness and Immune Markers Assay
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Where indicated, cells were treated with RAR antagonist AGN193109 (5758, Tocris Bioscience, Bristol, UK) and RXR antagonist UVI3003 (3303, Tocris Bioscience) for 48 h (1 µM).
Total protein lysates were obtained using celLytic MT (C2978-50 mL, Sigma Aldrich), a cell lysis reagent, as described [32 (link)]. Antibodies used are as follows: anti-Oct-4A (2840 Cell Signaling, Milan, Italy), anti-Sox2 (3579 Cell Signaling), anti-KLF4 (4038 Cell Signaling), anti-Nanog (4903 Cell Signaling), anti-c-MYC (5605 Cell Signaling), anti-PD-L1 (13684; Cell Signaling); anti-ALDH3A1 (TA332730, Origene); anti-mPGES1 (160140) and anti-COX-2 (160112, Cayman Chemical, Arcore, Italy); anti-CD133 (PA1217, Boster), anti-NFkB (sc-372, Santa Cruz, CA, USA), and anti-β-actin (Sigma Aldrich). Images were digitalized with CHEMI DOC Quantity One program (Biorad, Milan, Italy
Total protein lysates were obtained using celLytic MT (C2978-50 mL, Sigma Aldrich), a cell lysis reagent, as described [32 (link)]. Antibodies used are as follows: anti-Oct-4A (2840 Cell Signaling, Milan, Italy), anti-Sox2 (3579 Cell Signaling), anti-KLF4 (4038 Cell Signaling), anti-Nanog (4903 Cell Signaling), anti-c-MYC (5605 Cell Signaling), anti-PD-L1 (13684; Cell Signaling); anti-ALDH3A1 (TA332730, Origene); anti-mPGES1 (160140) and anti-COX-2 (160112, Cayman Chemical, Arcore, Italy); anti-CD133 (PA1217, Boster), anti-NFkB (sc-372, Santa Cruz, CA, USA), and anti-β-actin (Sigma Aldrich). Images were digitalized with CHEMI DOC Quantity One program (Biorad, Milan, Italy
4
Receptor-mediated Transcriptional Regulation
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RXR is referred to RXRα hereafter. The pSG5-based Gal-RARα LBD (Gal-RARα), Gal-RXR LBD (Gal-RXR), Gal-TIF2 NRID (Gal-TIF2), Gal-SMRT NRID (Gal-SMRT), Gal-NCoR NRID (Gal-NCoR), RARα LBD-VP16 (RARα-VP16), RXRΔAB-VP16 (RXR-VP16), RXRΔAB, RARαΔAB, RXR, and RARγ expression vectors, and the (17m)5x-βGlob-Luc and the (RARE)3x-tk-Luc reporter genes have been described [52 (link),53 (link),54 (link)]. CD3254, UVI3003, LG100754 (LG754), LE135, CD2665, BMS961, BMS614, and BMS493 were from Tocris. BMS948 was provided by Bristol-Myers Squibb (New York, NY, USA) and AGN192870 (AGN870) by Galderma (Lausanne, Switzerland). UVI3002 was a gift of Angel de Lera (University of Vigo, Vigo, Spain). Am580 and TTNPB were from Sigma France (Saint Quentin Fallavier, France).
5
Retinoic acid and fusion assay
6
Retinoic Acid Modulation of EECD34 Cell Proliferation
7
Neuronal Oxidative Stress Analysis
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Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Sigma Chemical (St. Louis, MO, USA). Penicillin–streptomycin solution was purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan). Fetal bovine serum (FBS), neurobasal medium (phenol red and glutamine minus), and B27 supplement were obtained from Thermo Fischer Scientific (Waltham, MA, USA). Bexarotene and triciribine were obtained from Cayman Chemicals (Ann Arbor, MI, USA). UVI3003 was purchased from Tocris Bioscience (Bristol, UK). Methylmercury chloride was obtained from Tokyo Chemical Industry (Tokyo, Japan). Carboxy-H2DCFDA was obtained from Molecular Probes (Eugene, OR, USA).
8
Molecular Regulation of Phagocytosis
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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).
9
C2C12 Myoblast Differentiation with Bexarotene
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Cells of the murine myoblasts cell line C2C12 (ATCC) were maintained in growth medium (GM), Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), at 37°C with 5% CO2. To induce differentiation, the medium of 80% confluent cell cultures was changed to differentiation medium (DM), DMEM supplemented with 2% HS (29 (link)) and 50 nM of Bexarotene was used for treatment conditions. Bexarotene was purchased from the LC Laboratories and UVI 3003 was from Tocris. The RXRα shRNA knockdown cells have been described previously (28 (link)).
10
Synthesis and Procurement of PPARγ Ligands
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