Cd2665

Manufactured by Bio-Techne

Sourced in United Kingdom

The CD2665 is a laboratory equipment product manufactured by Bio-Techne. It is designed to perform specific functions within a laboratory setting. The core function of this product is to facilitate certain procedures or analyses, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

7 protocols using cd2665

Isolation and Characterization of Human Basophils

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Human basophils were isolated from the buffy bags of healthy donors (Centre Trinité, L’Établissement Français du Sang, Paris; EFS-INSERM, 18/EFS/041) as previously described (28 (link)) by using Basophil Isolation Kit (Miltenyi Biotec, Paris, France). Basophils were then cultured in X-Vivo medium, with 100 ng/0.5 M cells/ml of IL-3, or with 10 nM all-trans RA for 6 hr with or without prior treatment with 1 µM each of retinoic acid receptors (RAR) antagonists CD2665 (RARβ/γ antagonist; Tocris, Cat. 3800) and BMS614 (RARα antagonist; Sigma, Cat. SML-1084) for 1 hr or with RAR antagonists for 1h followed by IL-3 for 6h or with RAR antagonists alone for 1h. Untreated basophils (Baso alone) were used as control.
Total RNA from the different experimental conditions was isolated using the RNeasy minikit (Qiagen, Hilden, Germany). cDNAs were synthesized using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Courtaboeuf, France), and quantitative PCR was performed with LightCycler 480 (Roche Diagnostics) and QuantiTect SYBR Green Kit (Qiagen) using the primers as described in Table 3. Relative RNA levels were calculated with human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) as an internal control.

Hachem C.E., Marschall P., Hener P., Karnam A., Bonam S.R., Meyer P., Flatter E., Birling M.C., Bayry J, & Li M. (2023). IL-3 produced by T cells is crucial for basophil extravasation in hapten-induced allergic contact dermatitis. Frontiers in Immunology, 14, 1151468.

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Retinoid Receptor Modulation in Breast Cancer

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The following compounds were used: ATRA (Sigma-Aldrich, https://www.sigmaaldrich.com), AM580 (Tocris, http://www.tocris.com), BMS961 (Tocris), ER50891 (Tocris), CD2665 (Tocris), and UVI2003 (a kind gift of Dr. Angel De Lera, Universidade de Vigo, Spain). Sulforhodamine was from Sigma-Aldrich Co. A list of the cell lines, their characteristics, and origin is available in Supplementary Table S1. The plasmid constructs used for RARα3 over-expression in MDA-MB453 cells and knockdown in SKBR3 cells are described below.

Centritto F., Paroni G., Bolis M., Garattini S.K., Kurosaki M., Barzago M.M., Zanetti A., Fisher J.N., Scott M.F., Pattini L., Lupi M., Ubezio P., Piccotti F., Zambelli A., Rizzo P., Gianni' M., Fratelli M., Terao M, & Garattini E. (2015). Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression. EMBO Molecular Medicine, 7(7), 950-972.

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Retinoid-Mediated Enzyme Activity Assay

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The natural retinoid ATRA, in addition to the synthetic retinoids EC19, EC23, CD437 (RAR-γ selective agonist), AC261066 (RARβ-2 agonist) and CD2665 (Selective RAR-β/γ antagonist), were purchased from Tocris Biosciences (UK; purity ≥ 98% (high-performance liquid chromatography, HPLC)). The stock solutions of retinoids were prepared in DMSO (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 1 mM and stored at −20 °C. The ATRA stock solution and the aliquots of working concentrations were preserved away from laboratory lights during storage and during the experiments. Malachite green, ammonium molybdate, polyvinyl alcohol and ATP were supplied by Piochem (Giza, Egypt) and stored as guided by the manufacturer.

Abdelaal M.R., Ibrahim E., Elnagar M.R., Soror S.H, & Haffez H. (2022). Augmented Therapeutic Potential of EC-Synthetic Retinoids in Caco-2 Cancer Cells Using an In Vitro Approach. International Journal of Molecular Sciences, 23(16), 9442.

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Retinoid Agonists and Antagonists Synthesis

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R667 (palovarotene, CAS410528-02-8)^{(17 )} and NRX 204647^{(13 (link))} are RARγ agonists and were synthesized by Atomax Chemicals (Shenzhen, China). RARγ antagonists CD2665 (CAS 170355-78-9)^{(18 (link))} and MM11253 (CAS345952-44-5)^{(19 (link))} were purchased from Tocris Biosciences (Bristol, UK). RARγ antagonist 7a was synthesized by Atomax Chemicals (Shenzhen, China) according to the world patent application WO 2005/066115 A2. The concentrations of retinoids used for animal experiments were 1 mg/kg for NRX 204647 and 4 mg/kg for all other compounds unless indicated. Stock solutions of retinoids in DMSO (D2650; Sigma-Aldrich, St. Louis, MO) were stored at −30°C under argon. Before administration, 30 μl aliquots of stock solution were mixed with 70 μl of corn oil (C8267; Sigma-Aldrich, St. Louis, MO) for each dose, and administered to mice by oral gavage using a 20-gauge gavage needle (Fine Science Tools, Foster City, CA) at indicated time points. Vehicle control mice received 30 μl DMSO plus 70 μl corn oil in the same manner.

Uchibe K., Son J., Larmour C., Pacifici M., Enomoto-Iwamoto M, & Iwamoto M. (2016). Genetic and Pharmacological Inhibition of Retinoic Acid Receptor γ Function Promotes Endochondral Bone Formation. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 35(5), 1096-1105.

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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).

le Maire A., Teyssier C., Balaguer P., Bourguet W, & Germain P. (2019). Regulation of RXR-RAR Heterodimers by RXR- and RAR-Specific Ligands and Their Combinations. Cells, 8(11), 1392.

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Reprogramming factors and vector systems

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Oct4, c‐Myc, Klf4, Sox2, Rarg, Rara, and Lrh1 cDNAs and the RARA‐DN were cloned into piggyBac (PB) transposons under the control of the CMV early enhancer/chicken β actin (CAG) promoter or the doxycycline (Dox)‐inducible TRE promoter (TRE).
For episomal expression, cDNAs of 4F and 2F were cloned into episomal vectors under the control of CAG promoter to construct two expression vectors: pCEP‐4F and pCEP‐2F.
We also used the PB transposase plasmid, pGL3‐RARE‐Luciferase (Addgene, Cambridge, MA, https://www.addgene.org, plasmid, 13458), pRL‐TK (Renilla luciferase control plasmid) (Promega, Madison, WI, http://www.promega.com), and TOPflash (T‐cell factor [TCF] reporter plasmid) (Merk Millipore, Darmstadt, Germany, http://www.emdmillipore.com, gift from Dr. Jason Wray and Prof. Austin Smith, University of Cambridge, U.K.).
The diagrams of these constructs and plasmids are listed in Supporting Information Figure S1. ATRA, 9cRA, retinol, citral, and IWR‐1 were purchased from Sigma (Gillingham, UK, https://www.sigmaaldrich.com/united‐kingdom.html), and CD437 and CD2665 were obtained from Tocris Biosciences (Abingdon, UK, http://www.tocris.com). PD0325901 (PD) and CHIR99021(CH) were obtained from Axon Medchem, (Groningen, The Netherlands, http://www.axonmedchem.com).

Yang J., Wang W., Ooi J., Campos L.S., Lu L, & Liu P. (2015). Signalling Through Retinoic Acid Receptors is Required for Reprogramming of Both Mouse Embryonic Fibroblast Cells and Epiblast Stem Cells to Induced Pluripotent Stem Cells. Stem Cells (Dayton, Ohio), 33(5), 1390-1404.

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Retinoids for RAR and RXR Activation

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All-trans-RA (ATRA) (Sigma-Aldrich) was dissolved at a concentration of 0.1 M in dimethyl sulfoxide (DMSO) under red light in a dark room, aliquoted into 0.6 ml microtubes stored under nitrogen at − 70 °C, and protected from light. Synthetic retinoids were dissolved in DMSO as 0.01 M stock solutions and stored at − 20 °C, protected from light. A1120 and TTNPB were purchased from Sigma-Aldrich, CD2665 was purchased from Tocris; HX600 and DA124 were obtained from Dr. Kagechika (Tokyo) while fenretinide was obtained from Dr. Nimesh Mody (University of Aberdeen). Other synthetic retinoids and non-retinoid homologues were designed and synthesized as described previously [22 –28 ]. The molecular structures of the RAR and RXR ligands used are shown in Fig. 1. The majority of the compounds are effective activators of RARs, except for A1120 (a RBP4 ligand), HX600 and DA124 (RXR agonists), CD2665 (RAR β/γ antagonist) and DC324, DC329 and DC303, which exhibit extended structures compared to their shorter analogues (DC271, DC375 and EC23, respectively) that significantly reduce their binding affinities for the RARs.Fig. 1

Retinoids used in the study grouped into those that are RAR agonists, RXR agonists, RAR antagonists, poor RAR agonists and antagonists for RBP4

Khatib T., Marini P., Nunna S., Chisholm D.R., Whiting A., Redfern C., Greig I.R, & McCaffery P. (2019). Genomic and non-genomic pathways are both crucial for peak induction of neurite outgrowth by retinoids. Cell Communication and Signaling : CCS, 17, 40.

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