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Kaad cyclopamine

Manufactured by Merck Group
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KAAD-cyclopamine is a synthetic compound developed by Merck Group. It is a small molecule that acts as a modulator of the Hedgehog signaling pathway. The core function of KAAD-cyclopamine is to inhibit the activity of the Smoothened (SMO) protein, which is a key component of the Hedgehog signaling cascade. This compound is primarily used as a research tool to study the Hedgehog signaling pathway and its role in various biological processes and disease states.

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Lab products found in correlation

Dual luciferase reporter assay system, by Promega (2 mentions) Fluostar optima, by BMG Labtech (2 mentions) Bovine serum albumin (bsa), by GenScript (2 mentions) Recombinant shh c25ii, by R&D Systems (2 mentions) Ihh c28ii, by GenScript (2 mentions) N acetyl l cysteine, by Merck Group (2 mentions) Vorinostat, by Merck Group (1 mentions) 96 well plate, by Greiner (1 mentions) Dulbecco modified eagle medium (dmem), by Lonza (1 mentions) Dmem high glucose, by Lonza (1 mentions) Fetal bovine serum (fbs), by Lonza (1 mentions) Doxycycline, by Merck Group (1 mentions) Chloroquine, by Merck Group (1 mentions) Anti β actin antibody ac 74, by Merck Group (1 mentions) Bafilomycin a1, by Merck Group (1 mentions)

7 protocols using kaad cyclopamine

1

Hedgehog Signaling Pathway Assay

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Shh-Light2 cells [38 (link)], a clonal NIH-3T3 cell line that stably expresses 8xGLI-binding site-firefly and TK-Renilla luciferase reporters, were co-cultured with LAD cell lines in 24-well plates until confluent and then treated with KAAD-cyclopamine (Millipore) 200 nM, 5E1 antibody 10 µg/ml or recombinant SHHN protein 1 µg/ml in DMEM containing 0.5% (vol/vol) bovine calf serum. Luciferase activity was measured by Fluostar Optima (BMG Labtech) using Dual Luciferase Assay Reporter System (Promega).
Kasiri S., Chen B., Wilson A.N., Reczek A., Mazambani S., Gadhvi J., Noel E., Marriam U., Mino B., Lu W., Girard L., Solis L.M., Luby-Phelps K., Bishop J., Kim J.W, & Kim J. (2020). Stromal Hedgehog pathway activation by IHH suppresses lung adenocarcinoma growth and metastasis by limiting reactive oxygen species. Oncogene, 39(16), 3258-3275.
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2

MEF Cell Hedgehog Pathway Activation

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SAG was purchased from Adipogen, Life Sciences (Liestal, Switzerland). For Hh pathway activation, MEF cells were incubated overnight in serum-free medium, containing 1% BSA, and then treated with 200 nM SAG for the indicated time points. Kaad-cyclopamine was purchased from Millipore (cat. #239804), ATO and vorinostat were purchased from SIGMA (cat. #1010 and cat. #SML0061 respectively), mocetinostat (MGCD0103) was synthetized in house as a dihydrobromide salt as described35 (link).
Coni S., Mancuso A.B., Di Magno L., Sdruscia G., Manni S., Serrao S.M., Rotili D., Spiombi E., Bufalieri F., Petroni M., Kusio-Kobialka M., De Smaele E., Ferretti E., Capalbo C., Mai A., Niewiadomski P., Screpanti I., Di Marcotullio L, & Canettieri G. (2017). Selective targeting of HDAC1/2 elicits anticancer effects through Gli1 acetylation in preclinical models of SHH Medulloblastoma. Scientific Reports, 7, 44079.
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3

Expansion and Preparation of Antibodies

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5E1 antibody was expanded in our laboratory (see supplemental material and methods) and prepared in PBS. IgG1 (InVivoMab, BE0083) was diluted in PBS. KAAD-cyclopamine (Millipore) was prepared in DMSO. Recombinant SHH (C25II) (R&D Systems) and IHH (C28II) (Genscript) were prepared in PBS containing 0.1% bovine serum albumin (BSA). N-Acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich and prepared in PBS for i.p. injection or sterile tap water for supplemented drinking water. For NAC solution, pH was adjusted to 7.4.
Kasiri S., Chen B., Wilson A.N., Reczek A., Mazambani S., Gadhvi J., Noel E., Marriam U., Mino B., Lu W., Girard L., Solis L.M., Luby-Phelps K., Bishop J., Kim J.W, & Kim J. (2020). Stromal Hedgehog pathway activation by IHH suppresses lung adenocarcinoma growth and metastasis by limiting reactive oxygen species. Oncogene, 39(16), 3258-3275.
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4

Shh-Light2 Cell Assay for Hedgehog Signaling

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Shh-Light2 cells [39 (link)], a clonal NIH-3T3 cell line that stably expresses 8xGLI-binding site-firefly and TK-Renilla luciferase reporters, were co-cultured with LAD cell lines in 24-well plates until confluent and then treated with KAAD-cyclopamine (Millipore) 200nM, 5E1 antibody 10 μg/ml or recombinant SHHN protein 1μg/ml in DMEM containing 0.5% (vol/vol) bovine calf serum. Luciferase activity was measured by Fluostar Optima (BMG Labtech) using Dual Luciferase Assay Reporter System (Promega).
Kasiri S., Chen B., Wilson A.N., Reczek A., Mazambani S., Gadhvi J., Noel E., Marriam U., Mino B., Lu W., Girard L., Solis L.M., Luby-Phelps K., Bishop J., Kim J.W, & Kim J. (2020). Stromal Hedgehog pathway activation by IHH suppresses lung adenocarcinoma growth and metastasis by limiting reactive oxygen species. Oncogene, 39(16), 3258-3275.
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5

Preparation of Reagents for SHH/IHH Study

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5E1 antibody was expanded in our laboratory (see Supplementary material and methods) and prepared in PBS. IgG1 (InVivoMab, BE0083) was diluted in PBS. KAAD-cyclopamine (Millipore) was prepared in DMSO. Recombinant SHH (C25II) (R&D Systems) and IHH (C28II) (Genscript) were prepared in PBS containing 0.1% bovine serum albumin (BSA). N-Acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich and prepared in PBS for i.p. injection or sterile tap water for supplemented drinking water. For NAC solution, pH was adjusted to 7.4.
Kasiri S., Chen B., Wilson A.N., Reczek A., Mazambani S., Gadhvi J., Noel E., Marriam U., Mino B., Lu W., Girard L., Solis L.M., Luby-Phelps K., Bishop J., Kim J.W, & Kim J. (2020). Stromal Hedgehog pathway activation by IHH suppresses lung adenocarcinoma growth and metastasis by limiting reactive oxygen species. Oncogene, 39(16), 3258-3275.
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6

Coculture of GBS-GFP Reporter Cells with Primary Cancer Cells

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Mouse embryonic fibroblasts stably transduced with the GBS-GFP reporter construct (GGM cells, [21 (link)]) were grown under standard cell culture conditions in high glucose DMEM (Lonza) containing 8% FBS (Lonza) and 0.5% Penicillin/Streptamycin (Lonza). For signaling assay GGM cells were seeded in 96 well plates (Greiner) and grown to confluence. 10.000 primary cancer cells or 2.000 knockdown PC053M cells were seeded per well on top of the GGMs in serum free medium (DMEM, Lonza) with or without the addition of 100 nM KAAD-cyclopamine (Merck Millipore). After 3 days of coculture, images were taken on a Zeiss fluorescence microscope and percentage of GFP positive cells was determined by flow cytometry on a FACSCanto II.
Damhofer H., Ebbing E.A., Steins A., Welling L., Tol J.A., Krishnadath K.K., van Leusden T., van de Vijver M.J., Besselink M.G., Busch O.R., Henegouwen M.I., van Delden O., Meijer S.L., Dijk F., Medema J.P., van Laarhoven H.W, & Bijlsma M.F. (2015). Establishment of patient-derived xenograft models and cell lines for malignancies of the upper gastrointestinal tract. Journal of Translational Medicine, 13, 115.
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7

Shh Pathway Regulation by Autophagy

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Recombinant Shh was synthesized, purified, and tested as previously described. KAAD-cyclopamine was purchased from EMD Millipore (Billerica, MA). Bafilomycin A1, chloroquine, doxycycline, and anti-β-actin antibody (AC-74) were from Sigma-Aldrich (St. Louis, MO). Antibodies targeting HA(C29F4), myc (9B111), ATG101, p62, LC3B, FIP200, ULK1, phospho-ULK1(Ser757), ATG13, and GLI1 (L42B10) were from Cell Signaling Technology (Danvers, MA). Secondary anti-rabbit-HRP and anti-mouse-HRP antibodies were purchased from BioRad (Hercules, CA). Anti-GFP, secondary anti-rabbit-HRP and anti-mouse-HRP Alexa 488 and Alexa 568 antibodies were from Invitrogen (Waltham, MA).
Chen L.X., Morales-Alcala C.C, & Riobo-Del Galdo N.A. (2018). Autophagic Flux is Regulated by Interaction Between the C-terminal Domain of Patched1 and ATG101. Molecular cancer research : MCR, 16(5), 909-919.
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