Description(s):

 

The GAL4-UAS system is a powerful technique for studying gene expression and function. It has also been adapted to study nuclear receptor chemical-binding functions. Our GR LBD-driven GAL4 reporter HEK 293 stable cell line is a reliable, rapid, and sensitive method for analyzing the intracellular status of NRs upon exposure to drug candidates or other stimuli. These functional assays allow for measurement of receptor activation or inhibition by compounds and can be used to determine compound potency and selectivity.

Principle

Nuclear hormone receptors (NHRs) are a group of ligand-binding transcription factors (TFs).  More than 350 NHRs are available in the PDB. Like other TFs, they are able to regulate gene expression by binding to specific DNA regulatory elements, but their activities are modulated by the corresponding ligands. They play many important physiological functions such as embryonic development, organ physiology, cell differentiation and homeostasis. They are also associated with human diseases including cancer, obesity/diabetes, arthritis and asthma. Members of this family are often chosen to be drug targets.

Conventionally, the response element-luciferase reporter assays are used to study endogenous or exogenous receptors, and analyze the receptor signaling pathway in a native biological context.  Due to their low sensitivity of these assays and the similar response element shared by multiple NHRs, their applications are limited.  Signosis has developed LDB-driven GAL4 reporter HEK 293 stable cell lines by delivering two vectors, one expressing 8 copies of GAL4 upstream activator sequences (UAS) in front of the luciferase reporter gene, another expressing Gal4 DNA Binding Domain (DBD) and GR ligand binding domain(LBD). Upon addition of a corresponding ligand, the DBD-LBD fusion protein is activated, and binds to GAL promoter binding site, leading to the induction of luciferase.  The stable clones were selected with hygromycin and G418, The functional assay was subsequently conducted by DEX treatment, and the clone with the highest induction fold (50) has been chosen for this product. The advantages of these stable cell lines are low cross-reactivity with other nuclear receptors and less toxicity of the chimeric receptors to the cells when overexpressed.  These cell lines can be used to screen drug compounds for agonist or antagonist hit identification.

 

 

Data

Analysis of GR Luciferase Reporter Cell Line in response to Dex treatment.   
The cells were seeded on  a 96-well plate for overnight with DMEM including 10% FBS. The cell then were treated with 10 nM Dex in DMEM + 0.1% FBS for 16 hours.

Literature

View user manual

Citations

 

NRF2/ARE Luciferase Reporter MCF7 Stable Cell Line  SL-0010

Withaferin A induces Nrf2-dependent protection against liver injury: role of Keap1-independent mechanisms. DL Palliyaguru, DV Chartoumpekis, N Wakabayashi. Free Radical Biology and Medicine 2016. http://dx.doi.org/10.1016/j.freeradbiomed.2016.10.003

Luciferase Stable Expressing Hela Cell Line SL-0102

Redox-responsive, reversibly-crosslinked thiolated cationic helical polypeptides for efficient siRNA encapsulation and delivery.N Zheng, Z Song, Y Liu, R Zhang, R Zhang, C Yao. Journal of Controlled Release.Volume 205, 10 May 2015, Pages 231–239

Targeted delivery of cisplatin to tumor xenografts via the nanoparticle component of nano-diamino-tetrac. Thangirala Sudha, Dhruba J Bharali, Murat Yalcin, Noureldien HE Darwish, Melis Debreli Coskun, Kelly A Keating, Hung-Yun Lin, Paul J Davis, Shaker A Mousa. February 2017 ,Vol. 12, No. 3, Pages 195-205 , DOI 10.2217/nnm-2016-0315.

NFkB Luciferase Reporter Stable Cell Lines  SL-0001

Thymoquinone Modulates Blood Coagulation in Vitro via Its Effects on Inflammatory and Coagulation Pathways. V Muralidharan-Chari, J Kim, A Abuawad, M Naeem.  Int. J. Mol. Sci. 2016, 17(4), 474; doi:10.3390/ijms17040474

Drug repurposing screen identifies lestaurtinib amplifies the ability of the poly (ADP-ribose) polymerase 1 inhibitor AG14361 to kill breast cancer associated gene-1 …G Vazquez-Ortiz, C Chisholm, X Xu, TJ Lahusen, C Li… - Breast Cancer Research  2014,  Jun 24;16(3):R67. doi: 10.1186/bcr3682.

NFkB Luciferase Reporter A549 Stable Cell Lines  SL-0014

Alcohol promotes migration and invasion of triple-negative breast cancer cells through activation of p38 MAPK and JNK†M Zhao, EW Howard, AB Parris, Z Guo, Q Zhao, X Yang.  Molecular Carcinogenesis, 2016, DOI: 10.1002/mc.22538

HEK293 TCF/LEF Luciferase Reporter Stable Cell Line SL-0015

Active β-catenin is regulated by the PTEN/PI3 kinase pathway: a role for protein phosphatase PP2A. Amit Persad,1,* Geetha Venkateswaran,1,* Li Hao,1 Maria E. Garcia,1 Jenny Yoon,1 Jaskiran Sidhu,1 and Sujata Persad1. Genes Cancer. 2016 Nov; 7(11-12): 368–382., doi:  10.18632/genesandcancer.128

Diallyl trisulfide inhibits proliferation, invasion and angiogenesis of glioma cells by inactivating Wnt/β-catenin signaling. Qingxia TaoCuiying WuRuxiang XuLijun NiuJiazhen QinNing LiuPeng ZhangEmail authorChong Wang. Cell and Tissue Research, 2017, doi:10.1007/s00441-017-2678-9

BCN057 induces intestinal stem cell repair and mitigates radiation-induced intestinal injury. Payel Bhanja, Andrew Norris, Pooja Gupta-Saraf, Andrew Hoover and Subhrajit Saha. Stem Cell Research & Therapy, vol. 9, no. 1, Feb. 2018, doi:10.1186/s13287-017-0763-3.

(Nrf2/ARE) luciferase reporter cell line HEK293

Direct Antioxidant Properties of Methotrexate: Inhibition of Malondialdehyde-Acetaldehyde-Protein Adduct Formation and Superoxide Scavenging. Matthew C. Zimmermana, Dahn L. Clemensb, c, Michael J. Duryeeb, Cleofes Sarmientoa, Andrew Chioub, Carlos D. Hunterb, Jun Tiana, Lynell W. Klassenb, c, James R. O’Dellb, c, Geoffrey M. Thieleb, c, Redox Biology,2017.07.018, doi: 10.1016/j.redox.2017.07.018

Active β-catenin is regulated by the PTEN/PI3 kinase pathway: a role for protein phosphatase PP2A. Amit Persad1,*, Geetha Venkateswaran1,*, Li Hao1, Maria E. Garcia1, Jenny Yoon1, Jaskiran Sidhu1 and Sujata Persad1.Genes & Cancer. January 30, 2017 . https://doi.org/10.18632/genesandcancer.128