Cyclopamine is a Hedgehog (Hh) pathway antagonist with an IC₅₀ of 46 nM in the Hh cell assay. Cyclopamine is also a selective Smo inhibitor.

Treatment with small molecule Hh inhibitors such as HhAntag and the natural product Cyclopamine, both binding to Smo, induces tumor remission in a genetic mouse model of medulloblastoma^[1]. Cyclopamine is a Hedgehog (Hh) pathway antagonist. Cyclopamine suppresses cell growth. Cyclopamine (3 μM) suppression of Hh pathway activity and growth in digestive tract tumour cell lines correlates with expression of PTCHmRNA^[2]. Cyclopamine is a steroidal alkaloid that inhibits Hh signalling through direct interaction with Smo^[3].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Cyclopamine causes durable regression of xenograft tumors. Tumors in Cyclopamine-treated animals, regress completely by 12 days^[2]. Cyclopamine (1.2 mg) treatment blocks tumour formation of human pancreatic adenocarcinoma cells after transplantation into nude mice^[3].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

411.62

C₂₇H₄₁NO₂

4449-51-8

环杷明；环巴胺；11-去氧芥芬胺

Room temperature in continental US; may vary elsewhere.

Powder	-20°C	3 years
	4°C	2 years
In solvent	-80°C	6 months
	-20°C	1 month

In Vitro: 

Ethanol : 33.33 mg/mL (80.97 mM; Need ultrasonic)

DMSO : 10 mg/mL (24.29 mM; ultrasonic and warming and heat to 80°C)

*

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

In Vivo:

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% EtOH    90% corn oil

  Solubility: ≥ 1.67 mg/mL (4.06 mM); Clear solution

  此方案可获得 ≥ 1.67 mg/mL (4.06 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 16.7 mg/mL 的澄清 EtOH 储备液加到 900 μL玉米油中，混合均匀。

• 2.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 1 mg/mL (2.43 mM); Clear solution

  此方案可获得 ≥ 1 mg/mL (2.43 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 10.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• 3.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 0.5 mg/mL (1.21 mM); Clear solution

  此方案可获得 ≥ 0.5 mg/mL (1.21 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 5.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 4.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 0.5 mg/mL (1.21 mM); Clear solution

  此方案可获得 ≥ 0.5 mg/mL (1.21 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 5.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• [1]. Peukert S, et al. Identification and structure-activity relationships of ortho-biphenyl carboxamides as potent Smoothened antagonists inhibiting the Hedgehog signaling pathway. Bioorg Med Chem Lett, 2009, 19(2), 328-331.

  [2]. Berman DM, et al. Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours. Nature, 2003, 425(6960), 846-851.

  [3]. Thayer SP, et al. Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature, 2003, 425(6960), 851-856.

  [4]. Ma W, et al. Reduced Smoothened level rescued Aβ-induced memory deficits and neuronal inflammation in animal models of Alzheimer’s disease. J Genet Genomics. 2018 May 20;45(5):237-246.

  [5]. Qi Wan, et al. Overexpression of Laminin α4 Facilitates Proliferation and Migration of Fibroblasts in Knee Arthrofibrosis by Targeting Canonical Shh/Gli1 Signaling. Connect Tissue Res. 2020 May 24.

Cells are cultured in triplicate in 96-well plates in assay media to which 5E1 monoclonal antibody, ShhNp and/or Cyclopamine (3 μM) are added at 0 h at concentrations indicated in the main text. Viable cell mass is determined by optical density measurements at 490 nm (OD₄₉₀) at 2 and 4 days using the CellTiter96 colorimetric assay. Relative growth is calculated as OD (day 4)-OD (day 2)/OD (day 2)^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[3]
A total of 0.1 mL Hanks’ balanced salt solution and matrigel (1:1) containing 2×10⁶ cells is injected subcutaneously into CD-1 nude mice. Tumors are grown for 4 days to a minimum volume of 125 mm³; treatment is initiated simultaneously for all subjects. Mice are injected subcutaneously with vector alone (triolein:ethanol 4:1 v/v) or a Cyclopamine suspension (1.2 mg per mouse in triolein:ethanol 4:1 v/v) daily for 7 days. At the end of the treatment period, tumours are excised from mice, weighed and then fixed for 3 h at 4°C with 4% paraformaldehyde, embedded in paraffin wax and sectioned (6 µm). Apoptotic cells are identified by TUNEL using recombinant Tdt. Sections are then counterstained with eosin. Eight ×20-magnified fields from regions corresponding to the exterior, middle and interior of two control and two cyclopamine-treated tumours are chosen at random. We counted the number of TUNEL-positive nuclei manually. Haematoxylin/eosin staining is done.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Peukert S, et al. Identification and structure-activity relationships of ortho-biphenyl carboxamides as potent Smoothened antagonists inhibiting the Hedgehog signaling pathway. Bioorg Med Chem Lett, 2009, 19(2), 328-331.

  [2]. Berman DM, et al. Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours. Nature, 2003, 425(6960), 846-851.

  [3]. Thayer SP, et al. Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature, 2003, 425(6960), 851-856.

  [4]. Ma W, et al. Reduced Smoothened level rescued Aβ-induced memory deficits and neuronal inflammation in animal models of Alzheimer’s disease. J Genet Genomics. 2018 May 20;45(5):237-246.

  [5]. Qi Wan, et al. Overexpression of Laminin α4 Facilitates Proliferation and Migration of Fibroblasts in Knee Arthrofibrosis by Targeting Canonical Shh/Gli1 Signaling. Connect Tissue Res. 2020 May 24.