Cyclopamine hydrate

Manufactured by Merck Group

Sourced in United States

Cyclopamine hydrate is a chemical compound that functions as a selective inhibitor of the Hedgehog signaling pathway. It is commonly used as a research tool in the study of cellular development and signaling mechanisms.

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Lab products found in correlation

Lipopolysaccharide from, by Merck Group (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) U0126, by Cell Signaling Technology (1 mentions) Hydroxyproline detection kit, by Nanjing Jiancheng (1 mentions) Pentobarbital sodium salt, by Merck Group (1 mentions) Foetal bovine serum (fbs), by GE Healthcare (1 mentions) Dulbecco s modified eagle medium dmem, by GE Healthcare (1 mentions) Smoothened agonist sag, by Merck Group (1 mentions) Sorafenib tosylate, by Selleck Chemicals (1 mentions) Primescript rt reagent kit, by Takara Bio (1 mentions) Sybr premix ex taq, by Takara Bio (1 mentions) Tgf β1, by Thermo Fisher Scientific (1 mentions) Fitc annexin 5 apoptosis detection kit, by BD (1 mentions) Growth factor reduced matrigel, by BD (1 mentions) Alanine aminotransferase, by Nanjing Jiancheng (1 mentions) Aspartate aminotransferase, by Nanjing Jiancheng (1 mentions) Cy3 conjugated anti mouse secondary antibody, by Merck Group (1 mentions) Mouse anti 5 bromo 2 deoxyuridine brdu monoclonal antibody, by Roche (1 mentions) Eclipse te2000 s microscope, by Nikon (1 mentions) Nis elements ar software, by Nikon (1 mentions)

5 protocols using cyclopamine hydrate

Shh Regulation of NPC Responses to Inflammation

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To assess the effects of Shh on the NPCs, they were cultured on 96-well plates and we either added Shh as a treatment directly (20 nM Shh; R&D Systems, Minnesota, MN, USA) or blocked the Shh pathway with Cyclopamine (20 µM Cyclopamine hydrate; Sigma-Aldrich, Burlington, MA, USA). Furthermore, the cells were either cultured under normal conditions in the above-mentioned growth medium or with the addition of Lipopolysaccharide from E. coli (20 µg/mL LPS; Sigma-Aldrich, Burlington, MA, USA) for the first 8 h to simulate an inflammatory environment upon binding to Toll-Like-Receptor 4 (TLR4) like the postinjury environment in the CNS. The medium was changed after 8 h and every day after that until the end of the experiment (Figure 1).
These treatment regimens resulted in the following six treatment groups: Shh only (20 nM Shh), Cyclopamine only (20 µM Cyclopamine hydrate), LPS only (20 µg/mL Lipopolysaccharide from E. coli), Shh+LPS, Cyclopamine+LPS and untreated control (growth medium) (Table 1).

Tail M., Zhang H., Zheng G., Hatami M., Skutella T., Unterberg A., Zweckberger K, & Younsi A. (2022). The Sonic Hedgehog Pathway Modulates Survival, Proliferation, and Differentiation of Neural Progenitor Cells under Inflammatory Stress In Vitro. Cells, 11(4), 736.

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Cyclopamine and inhibitor effects

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For in vitro experiments, cyclopamine hydrate was purchased by Sigma-Aldrich (St. Louis, MO, USA). Cyclopamine was dissolved in dimethyl sulfoxide (DMSO) at concentration of 3 mM and stored at –80°C. U0126 was provided by Cell Signaling, (Danvers, MA, USA) and used at a 10 μM concentration. 5E1 blocking antibody was purchased from The Developmental Studies Hybridoma Bank, University of Iowa, USA, and used at the concentration of 2.5 mg/ml.

Parascandolo A., Laukkanen M.O., De Rosa N., Ugolini C., Cantisani M.C., Cirafici A.M., Basolo F., Santoro M, & Castellone M.D. (2017). A dual mechanism of activation of the Sonic Hedgehog pathway in anaplastic thyroid cancer: crosstalk with RAS-BRAF-MEK pathway and ligand secretion by tumor stroma. Oncotarget, 9(4), 4496-4510.

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Liver Fibrosis Inhibition Protocol

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PB2, pentobarbital sodium salt, cyclopamine hydrate and Smoothened agonist (SAG) were purchased from Sigma‐Aldrich (Merck KGaA). Sorafenib tosylate was purchased from Selleck Chemicals. CCl4 was purchased from China Sinopharm International Corporation. TGF‐β1 was obtained from PeproTech. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hydroxyproline detection kits were purchased from Nanjing Jiancheng Bioengineering Institute. Oligonucleotide primers were synthesized by Generay Biotech Co., Ltd. The PrimeScript RT Reagent kit and SYBR Premix Ex Taq were purchased from TaKaRa Biotechnology. The primary antibodies used in the study are listed in Table 1. Anti‐goat or antimouse secondary antibodies were obtained from Dako. Dulbecco's Modified Eagle Medium (DMEM) and foetal bovine serum (FBS) were from HyClone (GE Healthcare). The Annexin V‐FITC apoptosis detection kit was purchased from BD Biosciences. Growth factor‐reduced Matrigel was purchased from BD Biosciences. PB2, sorafenib and cyclopamine were dissolved in DMSO (<0.1% [v/v]) for in vitro treatment.

Feng J., Wang C., Liu T., Li J., Wu L., Yu Q., Li S., Zhou Y., Zhang J., Chen J., Ji J., Chen K., Mao Y., Wang F., Dai W., Fan X., Wu J, & Guo C. (2019). Procyanidin B2 inhibits the activation of hepatic stellate cells and angiogenesis via the Hedgehog pathway during liver fibrosis. Journal of Cellular and Molecular Medicine, 23(9), 6479-6493.

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Neurosphere Proliferation Assay

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For proliferation analysis, neurospheres were dissociated in a single cell suspension and plated onto poly-l-ornithine-coated 24-well chamber slides at a density of 3 × 10⁴ cells per well in a DMEM/F-12 medium containing EGF (20 ng/ml), FGF (20 ng/ml) and 2% FBS. Cells were treated with ELN 1 nM and, in some experiments, with 10 μg/ml cyclopamine hydrate (Sigma) for 24 h. Cells were then treated with BrdU for additional 6 h, paraformaldehyde fixed and stained with a mouse anti-5-bromo-2-deoxyuridine (BrdU) monoclonal antibody (1:100; Roche Applied Science) and a Cy3-conjugated anti-mouse secondary antibody (1:200; Sigma). Samples were counterstained with Hoechst-33258. Digital images were captured using an Eclipse TE 2000-S microscope and the NIS-Elements AR software (Nikon).

Giacomini A., Stagni F., Trazzi S., Guidi S., Emili M., Brigham E., Ciani E, & Bartesaghi R. (2015). Inhibition of APP gamma-secretase restores Sonic Hedgehog signaling and neurogenesis in the Ts65Dn mouse model of Down syndrome. Neurobiology of Disease, 82, 385-396.

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Evaluating Compound Effects on Neural Differentiation

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The cells were exposed to the test compounds during neural commitment until the 8th day of differentiation, thus encompassing the acquisition of polarized contractile neuro-epithelial properties and the generation of neural precursors. Different concentrations of each chemical: theophylline (Sigma T1633) at 2, 6, 20, 60, 200, 600, 2000 and 6000 µM, saccharin sodium salt hydrate (Sigma S1002) at 0.002, 0.02, 0.2, 2, 20 and 50 mM, cyclopamine hydrate (CPA; Sigma C4116) at 0.016, 0.05, 0.16, 0.5, 1.6, 5 and 16 µM, valproic acid sodium salt (VPA; Sigma P4543) at 0.08, 0.25, 0.8, 2.5 and 8 mM, ochratoxin A (OTA; Sigma 32937) at 0.5, 1, 2, 4, 10, 25, 62,5 and 125 nM and mycophenolic acid (MMF; Sigma M3536) at 11.5, 23, 46, 92, 185 and 470 nM were added at day 2 and day 5 after plating. At day 8, the rosettes were counted into each well under a Leica microscope at 10x magnification and the cells then harvested. For chemicals prepared into DMSO, the final DMSO concentration was 0.1% for all conditions including untreated samples. Experiments with CPA and VPA were performed on all 4 hESC lines, except cytotoxicity testing that was done with CHES6. Experiments with OTA and MMF were performed only on CHES6. All data presented are from three independent experiments, each performed in duplicate.

Feutz A.C, & De Geyter C. (2019). Accuracy, discriminative properties and reliability of a human ESC-based in vitro toxicity assay to distinguish teratogens responsible for neural tube defects. Archives of toxicology, 93(8).

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