Abstract
The measurement of the transcriptional activity of the HH signaling pathway is widely used as an indication of pathway activation. Luciferase reporter assays are powerful tools to measure the specific ability of a transcription factor to bind to its consensus sequence and to activate transcription of target genes. Here, we describe a protocol to measure the transcriptional activity of the HH pathway in normal and cancer cells. This technique allows studying the activity of GLI transcription factors and their modulation by drugs and/or other factors.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Ingham PW, McMahon AP (2001) Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15:3059–3087
Rohatgi R, Scott MP (2007) Patching the gaps in Hedgehog signalling. Nat Cell Biol 9:1005–1009
Jiang J, Hui CC (2008) Hedgehog signaling in development and cancer. Dev Cell 15:801–812
Kinzler KW, Vogelstein B (1990) The GLI gene encodes a nuclear protein which binds specific sequences in the human genome. Mol Cell Biol 10:634–642
Hahn H, Wicking C, Zaphiropoulous PG, Gailani MR, Shanley S, Chidambaram A et al (1996) Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 85:841–851
Xie J, Murone M, Luoh SM, Ryan A, Gu Q, Zhang C et al (1998) Activating Smoothened mutations in sporadic basal-cell carcinoma. Nature 391:90–92
Stecca B, Ruiz I, Altaba A (2010) Context-dependent regulation of the GLI code in cancer by HEDGEHOG and non-HEDGEHOG signals. J Mol Cell Biol 2:84–95
Wood KV, de Wet JR, Dewji N, DeLuca M (1984) Synthesis of active firefly luciferase by in vitro translation of RNA obtained from adult lanterns. Biochem Biophys Res Comm 124:592–596
de Wet JR, Wood KV, Helinski DR, DeLuca M (1985) Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc Natl Acad Sci U S A 82:7870–7873
Matthews JC, Hori K, Cormier MJ (1977) Purification and properties of Renilla reniformis luciferase. Biochemistry 16:85–91
Sasaki H, Hui C, Nakafuku M, Kondoh H (1997) A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro. Development 124:1313–1322
Chen JK, Taipale J, Young KE, Maiti T, Beachy PA (2002) Small molecule modulation of Smoothened activity. Proc Natl Acad Sci U S A 99:14071–14076
Taipale J, Chen JK, Cooper MK, Wang B, Mann RK, Milenkovic L et al (2000) Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406:1005–1009
Pandolfi S, Montagnani V, Penachioni JY, Vinci MC, Olivito B, Borgognoni L, Stecca B (2013) WIP1 phosphatase modulates the Hedgehog signaling by enhancing GLI1 function. Oncogene 32:4737–4747
Acknowledgments
This work was supported by grants from AIRC (Associazione Italiana per la Ricerca sul Cancro, projects IG-9566 and IG-14184) and Regional Health Research Program 2009. S.P. was supported by an AIRC fellowship.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Pandolfi, S., Stecca, B. (2015). Luciferase Reporter Assays to Study Transcriptional Activity of Hedgehog Signaling in Normal and Cancer Cells. In: Riobo, N. (eds) Hedgehog Signaling Protocols. Methods in Molecular Biology, vol 1322. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2772-2_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2772-2_7
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2771-5
Online ISBN: 978-1-4939-2772-2
eBook Packages: Springer Protocols