For early cyclopamine treatment, zebrafish embryos were treated at 1 dpf with 1 μM– 30 μM cyclopamine (Cayman Chemical Company) diluted in embryo medium containing 1% dimethyl sulfoxide (DMSO), and examined for ICH at 2 dpf. For later treatment, zebrafish larvae were treated with 10 μM cyclopamine starting at 5 dpf and assessed for BRB breakdown at 7 dpf.
Cyclopamine
Manufactured by Cayman Chemical
Sourced in United States
Cyclopamine is a small molecule compound. It is a steroidal alkaloid isolated from the plant Veratrum californicum. Cyclopamine functions as an inhibitor of the Hedgehog signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Gdc 0449, by Cayman Chemical (4 mentions)
Bodipy cyclopamine, by Abcam (4 mentions)
Sb431542, by Cayman Chemical (2 mentions)
Xav939, by Cayman Chemical (2 mentions)
Phenoplate, by PerkinElmer (2 mentions)
Y 27632, by Bio-Techne (2 mentions)
Vybrant apoptosis assay kit, by Thermo Fisher Scientific (1 mentions)
Pirfenidone, by Selleck Chemicals (1 mentions)
Sis3 hcl, by Selleck Chemicals (1 mentions)
Pdgf bb, by Cell Signaling Technology (1 mentions)
Vegf receptor 2, by Cell Signaling Technology (1 mentions)
Vegfa, by Cell Signaling Technology (1 mentions)
Hif 1α, by Cell Signaling Technology (1 mentions)
L7 hpsc passaging solution, by Lonza (1 mentions)
Gant61, by Cayman Chemical (1 mentions)
Pyrrolidine dithiocarbamate pdtc, by Merck Group (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
N2 and b27 supplements, by Thermo Fisher Scientific (1 mentions)
Stempro ezpassage tool, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
20 protocols using cyclopamine
1
Cyclopamine Treatment in Zebrafish
2
Hedgehog Pathway Inhibitors in Marine Cell Culture
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GDC-0449 (Vismodegib, Cayman Chemical; Ann Arbor, MI; Cat. No. 13613) and Cyclopamine (Cayman Chemical; Cat. No. 11321) were diluted in DMSO as 50 mM and 10 mM stocks, respectively. These stocks were diluted 1:2000 in 12 ppt filtered artificial sea water (FSW) to generate working solution: 25 µM GDC-0449 and 5 µM Cyclopamine. 1:2000 diluted 100% DMSO (final 0.05%) was applied as a control. All treatments were protected from light and replaced with fresh working solutions every day.
3
Glioblastoma Cell Proliferation Assay
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Cells were plated in 96-well plates at a density of 1 × 103 cells per well and incubated in 10% FBS-supplemented DMEM with 100 μM TMZ (Enzo Life Sciences) and maintained at 37 °C under a humidified atmosphere of 5% CO2 and 95% air for 120 h. Cyclopamine (Cayman chemical) at the concentration of 5 μM was added as indicated in the “Results ” section. Cell proliferation was detected by the cell counting kit-8 (CCK8, Dojindo) and the absorbance was measured by microplate reader at 450 nm.
Cells were cultured in 6-well plates at a density of 2 × 105 cells per well for the apoptosis assay. The apoptotic effects of treatments were determined by using Vybrant Apoptosis Assay Kit (Invitrogen), followed by fluorescence-activated cell sorter analysis using a FACScanto flow cytometry.
Cells were cultured in 6-well plates at a density of 2 × 105 cells per well for the apoptosis assay. The apoptotic effects of treatments were determined by using Vybrant Apoptosis Assay Kit (Invitrogen), followed by fluorescence-activated cell sorter analysis using a FACScanto flow cytometry.
4
Ischemic Stroke Protection Strategies
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Rats were administered intraperitoneally with pirfenidone (250 mg/kg) (Selleck Chemicals, Houston, TX, United States) (Shihab et al., 2002 (link); Burghardt et al., 2007 (link)), SIS3 HCl (2.5 mg/kg) (Selleck Chemicals, Houston, TX, United States) (Li et al., 2010 (link); Liu et al., 2018 (link)) and cyclopamine (10 mg/kg) (Cayman Chemical, Ann Arbor, MI, United States) (Alvarez et al., 2011 (link); Yu et al., 2017 (link)) 30 min before ischemia.
Rats were randomly divided into 9 groups: animals received sham operation and an equal volume of DMSO (Sham); rats received MCAO/R and an equal volume of DMSO (I/R); MCAO/R rats were treated with TGF-β2 inhibitor pirfenidone or Smo inhibitor cyclopamine (I/R + Pir, I/R + CYC); MCAO/R rats were treated with 1.5% ISO post-conditioning for 1 h after immediate reperfusion (ISO); MCAO/R rats were treated with pirfenidone, Smad3 inhibitor SIS3 HCl, cyclopamine, pirfenidone combined with cyclopamine before ISO post-conditioning (ISO + Pir, ISO + SIS3, ISO + CYC, and ISO + Pir + CYC, respectively).
Rats were randomly divided into 9 groups: animals received sham operation and an equal volume of DMSO (Sham); rats received MCAO/R and an equal volume of DMSO (I/R); MCAO/R rats were treated with TGF-β2 inhibitor pirfenidone or Smo inhibitor cyclopamine (I/R + Pir, I/R + CYC); MCAO/R rats were treated with 1.5% ISO post-conditioning for 1 h after immediate reperfusion (ISO); MCAO/R rats were treated with pirfenidone, Smad3 inhibitor SIS3 HCl, cyclopamine, pirfenidone combined with cyclopamine before ISO post-conditioning (ISO + Pir, ISO + SIS3, ISO + CYC, and ISO + Pir + CYC, respectively).
5
Angiogenesis Regulation Pathway Analysis
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Cyclopamine and 1‐stearoyl‐2‐arachidonoyl‐snglycerol (SAG) were supplied by Cayman Chemicals; curcumol was supplied by Selleck Chemicals. They were dissolved in DMSO for experiments. The following primary antibodies were used in this study: PDGF‐BB, VEGF‐A, VEGF receptor 2 (VEGFR2), angiopoietin 2, HIF‐1α, CD31, CD34 and PROX1; and SHH, Smo, Gli1 and Hhip1 (Cell Signaling Tech).
6
Directed Differentiation of iPSCs
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iPSC lines were passaged using Accutase (Innovative cell) and replated onto Geltrex-coated (1:50 dilution in DMEM/F12) 12-well plates at 7–8 × 105 cells/well in mTeSR1 with 10 μM rho-kinase inhibitor (Y-27632; Tocris, 1254). The following day, or when the cells reached 80–100% confluency, the medium was then changed to 3N (50:50 DMEM/F12:neurobasal with N2 and B27 supplements) without vitamin A with 2 μM DMH1 (Tocris, 4126), 2 μM XAV939 (Cayman Chemical, 13596), and 10 μM SB431542 (Cayman Chemical, 13031) (2 mL of medium per well). 75% of this medium was changed daily with 1 μM cyclopamine (Cayman Chemical, 11321) added beginning on day 1. On day 4, the monolayer was cut into squares using the StemPro EZ passage tool (Gibco). The squares were incubated for 2 minutes with L7 hPSC passaging solution (Lonza). After aspirating the L7 solution, the squares were sprayed off the bottom with 1 mL of preconditioned culture media with a P1000 micropipette. An additional 1 mL of fresh culture medium with the 4 inhibitors was added. Approximately 100 μL of resuspended monolayer squares were then transferred into the wells of black-walled thin-bottom 96-well plates (Greiner μClear or PerkinElmer PhenoPlate) preincubated at 37 °C for 30 minutes with 35 μL of 100% Geltrex solution in each well.
7
Glioblastoma Cell Inhibitor Study
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GBM28, SJ-GBM2 and U87MG glioblastoma cells were plated at a density of 0.2–0.3 × 106 cells per well in a six-well plate. After overnight incubation, cells were pretreated for 3h with 25 μM Cyclopamine or 30 μM GANT 61. Cyclopamine and GANT 61 were purchased from Cayman Chemical, Ann Arbor, MI. After pretreatment with inhibitors, cells were treated with 5 μM penfluridol for 48 h and processed for western blotting as explained above.
8
Hedgehog Signaling Modulation in Ox-LDL-Induced Injury
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Ox-LDL was purchased from Yiyuan Biotechnologies (cat. no. YB-002-1). Recombinant human Shh N-terminus protein (rShh-N) was obtained from Sangon Biotech Co., Ltd. (cat. no. C600310) and dissolved in ddH2O. Cyclopamine, a hedgehog signaling inhibitor, was purchased from Cayman Chemical Company (cat. no. 11321). Pyrrolidine dithiocarbamate (PDTC) was purchased from Sigma-Aldrich; Merck KGaA (cat. no. P8765). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (cat. no. CK04). Antibodies specific for Shh (cat. no. 2207; 1:1,000), cleaved caspase 3 (cat. no. 9662S; 1:500), Bcl-2 (cat. no. 4223; 1:1,000), Bax (cat. no. 2773; 1:1,000), tublin (cat. no. 2148; 1:1,000) and β-actin (cat. no. 4970; 1:1,000), a NF-κB pathway sampler kit including antibodies for IκBα, phosphorylated (p)-IκBα, p-IKKα/β, NF-κB p65 and p-NF-κB p65 (cat. no. 9936; all used at 1:1,000) and horseradish peroxidase-conjugated goat anti-rabbit IgG (cat. no. 7074; 1:1,000) and horseradish peroxidase-conjugated goat anti-mouse IgG (cat. no. 7076; 1:1,000) were purchased from Cell Signaling Technology, Inc. Antibodies specific for Smo (cat. no. ab113438; 1:1,000), Ptch (cat. no. ab53715; 1;1,000), Gli1 (cat. no. ab49314; 1:500) and IKKα/β (cat. no. ab178870; 1:1,000) were purchased from Abcam.
9
iPSC Differentiation to Neural Progenitors
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We Accutase passaged 7–8 × 105 iPSC lines onto Geltrex-coated (1:50 dilution in DMEM/F12) 12-well plates in mTeSR1 with 10 µM Y-27632 (Tocris, Bristol, UK). When the cells reached 80–100% confluency, the medium was changed to 3N (50:50 DMEM/F12:neurobasal with N2 and B27 supplements [Thermo]) without vitamin A with 2 µM DMH-1 (Tocris), 2 µM XAV939 (Cayman Chemical, Ann Arbor, MI, USA), and 10 µM SB431542 (Cayman Chemical) with 2 mL of medium per well. Subsequently, 1.5 mL medium were changed daily with 1 µM cyclopamine (Cayman Chemical) added beginning on day 1. On day 4, the monolayer was cut into squares using the StemPro EZ passage tool (Thermo). The squares were incubated for 2 min with the hypertonic sodium citrate solution. After aspiration, the squares were sprayed off the bottom with 1 mL of preconditioned culture media with a P1000 micropipette. An additional 1 mL of fresh culture medium with the 4 inhibitors was added. Approximately 100 µL of resuspended monolayer squares were then transferred into the wells of black-walled thin-bottom 96-well plates (µClear [Greiner Bio-One, Monroe, NC, USA] or Phenoplate [PerkinElmer, Waltham, MA, USA]) preincubated at 37 °C for 30 min with 35 µL of 100% Geltrex solution in each well.
10
Erlotinib and Cyclopamine in Lung Cancer
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Ethical approval was not sought as cell lines were commercially sought. Study protocol followed the principles outlined in the Declaration of Helsinki. Human pulmonary adenocarcinoma PC9 cells (EGFR exon 19 del E-746-A750) and bronchioalveolar carcinoma H358 cells (EGFR wild-type) were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea). Cells were maintained in Roswell Park Memorial Institute (RPMI-1640) medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific Inc, Waltham, MA, USA). Erlotinib and cyclopamine were obtained from Cayman Chemical (Ann Arbor, MI, USA) and Sigma-Aldrich Co. (St Louis, MO, USA), respectively, and were dissolved in dimethyl sulfoxide (DMSO) at 30 and 10 mM, respectively.
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