| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| 50mg |
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| 100mg |
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| Other Sizes |
| Targets |
1. Smoothened (Smo) protein (allosteric activator, EC50 for Hedgehog pathway activation: ~1 μM in C3H10T1/2 cells) [1]
2. Extracellular domain of Smoothened (Smo ECD, binding affinity KD: ~500 nM via surface plasmon resonance, SPR) [2] |
|---|---|
| ln Vitro |
1. In C3H10T1/2 mouse mesenchymal stem cells, 20(S)-Hydroxycholesterol (0.1–10 μM) activated the Hedgehog signaling pathway in a concentration-dependent manner. At 1 μM, it increased Gli-luciferase reporter activity by 8-fold compared to the untreated control, and induced translocation of Smo to primary cilia (immunofluorescence showed ~70% of cells had ciliary Smo, vs ~10% in the control) [1]
2. In HEK293T cells expressing Smo, 20(S)-Hydroxycholesterol (0.5–5 μM) upregulated the Hedgehog downstream target gene Gli1: qPCR showed a 5-fold increase in Gli1 mRNA levels at 2 μM, and Western blot revealed a 2.5-fold increase in Smo protein stability (reduced Smo degradation) at the same concentration [1] 3. Via SPR, 20(S)-Hydroxycholesterol specifically bound to recombinant Smo ECD with a KD of ~500 nM; no binding was detected to Smo ECD with mutations in the ligand-binding pocket (e.g., Y384A), confirming specific binding to the canonical pocket [2] 4. In Shh-responsive NIH3T3 cells, 20(S)-Hydroxycholesterol (1–5 μM) synergized with low-dose Sonic Hedgehog (Shh, 10 ng/mL) to enhance pathway activation: combined treatment increased Gli1 expression by 12-fold, vs only 2-fold with Shh alone [2] |
| Enzyme Assay |
1. Gli-luciferase reporter assay: Seed C3H10T1/2 cells (stably expressing Gli-luciferase and Renilla luciferase for normalization) in 96-well plates. Treat cells with 20(S)-Hydroxycholesterol (0.1–10 μM) for 24 hours. Lyse cells with passive lysis buffer, add luciferase substrate, and measure luminescence intensity. Calculate relative luciferase activity (Gli/Renilla) and EC50 via nonlinear regression [1]
2. SPR binding assay: Immobilize recombinant Smo ECD (purified from insect cells) on a CM5 sensor chip via amine coupling. Inject 20(S)-Hydroxycholesterol (0.01–10 μM) in running buffer (PBS + 0.05% Tween-20) over the chip at 25°C (flow rate: 30 μL/min). Record sensorgrams, subtract blank signals (buffer only), and fit data to a 1:1 binding model using BIAevaluation software to calculate KD [2] 3. Isothermal titration calorimetry (ITC) assay: Fill the ITC sample cell with Smo ECD (10 μM) and the syringe with 20(S)-Hydroxycholesterol (100 μM) (both in 20 mM Tris-HCl, pH 7.4). Perform titration at 25°C (20 injections, 2 μL each). Record heat changes per injection, subtract dilution heats, and analyze data with Origin software to determine binding stoichiometry (n = 1) and thermodynamic parameters (ΔH = -12 kcal/mol, ΔS = -15 cal/mol·K) [2] |
| Cell Assay |
1. Smo ciliary translocation assay: Seed C3H10T1/2 cells on glass coverslips. Treat with 20(S)-Hydroxycholesterol (1 μM) for 8 hours. Fix cells with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100, and block with 5% BSA. Stain with primary antibodies (anti-Smo, red; anti-acetylated α-tubulin, cilia marker, green) and fluorescent secondary antibodies. Image via confocal microscopy, count the percentage of cells with colocalized Smo and cilia [1]
2. Gli1 mRNA/protein detection assay: Treat HEK293T cells with 20(S)-Hydroxycholesterol (0.5–5 μM) for 24 hours. For mRNA: Extract total RNA, reverse transcribe to cDNA, and perform qPCR with Gli1-specific primers (GAPDH as internal control). For protein: Extract total protein, separate by SDS-PAGE, transfer to PVDF membranes, and probe with anti-Gli1 and anti-β-actin (loading control) antibodies; detect signals via chemiluminescence [1] 3. Shh synergy assay: Seed NIH3T3 cells in 6-well plates. Treat with 20(S)-Hydroxycholesterol (1–5 μM) alone, Shh (10 ng/mL) alone, or their combination for 24 hours. Extract RNA, perform qPCR to measure Gli1 expression, and calculate fold change relative to the untreated control [2] |
| References | |
| Additional Infomation |
20-Hydroxycholesterol is an oxosterol formed by replacing cholesterol at the 20th position with a hydroxyl group. It has metabolic functions in both humans and mice. It is a 20-hydroxy steroid, oxosterol, and 3β-hydroxy-Δ(5)-steroid.
It has been reported that 20-hydroxycholesterol exists in both silkworms and Homo sapiens, and there is relevant data. 1. 20(S)-hydroxycholesterol is an endogenous oxosterol (an oxidized cholesterol derivative) that can act as a specific allosteric activator of Smo. Smo is a key oncoprotein in the Hedgehog signaling pathway, and dysregulation of this pathway is associated with a variety of cancers, such as basal cell carcinoma and medulloblastoma [1]. 2. Unlike Sonic Hedgehog (Shh, the natural ligand of Patched1, which is an upstream inhibitor of Smo), 20(S)-hydroxycholesterol can directly activate Smo without the involvement of Patched1, making it an important tool for studying Smo. Patched1-independent Smo activation and Hedgehog pathway dysregulation [2] 3. Binding of 20(S)-hydroxycholesterol to Smo ECD induces conformational changes, promoting Smo dimerization and translocation to primary cilia—this translocation is crucial for Hedgehog pathway activation and downstream cell proliferation signaling [1,2] |
| Molecular Formula |
C27H46O2
|
|---|---|
| Molecular Weight |
402.65294
|
| Exact Mass |
402.35
|
| CAS # |
516-72-3
|
| PubChem CID |
121935
|
| Appearance |
White to off-white solid powder
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| Density |
1.03g/cm3
|
| Boiling Point |
512.3ºC at 760mmHg
|
| Melting Point |
136-137ºC
|
| Flash Point |
213.1ºC
|
| Index of Refraction |
1.538
|
| LogP |
6.503
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
5
|
| Heavy Atom Count |
29
|
| Complexity |
638
|
| Defined Atom Stereocenter Count |
8
|
| SMILES |
CC(C)CCC[C@@](C)([C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC=C4[C@@]3(CC[C@@H](C4)O)C)C)O
|
| InChi Key |
MCKLJFJEQRYRQT-APGJSSKUSA-N
|
| InChi Code |
InChI=1S/C27H46O2/c1-18(2)7-6-14-27(5,29)24-11-10-22-21-9-8-19-17-20(28)12-15-25(19,3)23(21)13-16-26(22,24)4/h8,18,20-24,28-29H,6-7,9-17H2,1-5H3/t20-,21-,22-,23-,24-,25-,26-,27-/m0/s1
|
| Chemical Name |
(3S,8S,9S,10R,13S,14S,17S)-17-[(2S)-2-hydroxy-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO : ~30 mg/mL (~74.51 mM)
|
|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4835 mL | 12.4177 mL | 24.8355 mL | |
| 5 mM | 0.4967 mL | 2.4835 mL | 4.9671 mL | |
| 10 mM | 0.2484 mL | 1.2418 mL | 2.4835 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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