PC-3 and HEK-293T cells were transfected by using the TransIT-2020 (Mirus Bio LLC) and TurboFect reagents (Thermo Scientific), respectively, per instruction of the manufacturer. DU145, C4-2, and LNCaP cells were transfected by using the Lipofectamine 2000 and Plus reagent (Invitrogen) as described (27 (link)). Reporter gene assay was performed as previously described (28 (link)) with either an androgen-responsive element-luciferase plasmid (ARE-luc) containing three ARE regions ligated in tandem to the luciferase reporter or a luciferase construct driven by three repeats of an AR-V-specific promoter element of the ubiquitin-conjugating enzyme E2C (UBE2C) gene (UBE2C-luc). To ensure an even transfection efficiency, we conducted the transfection in bulk, and then split the transfected cells for luciferase assay.
Protocol detail
Transfection and Reporter Assay for Androgen Receptor Activity
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293t cells
Androgen
Ar element
Assay
Bulk
Gene
Lipofectamine 2000
Luciferase
Plasmid
Reporter gene
Transfection
Ube2c
Ubiquitin conjugating enzyme
Corresponding Organization : Jilin University
Other organizations : University of Wisconsin–Madison, Stanford University, New York University, Purdue University West Lafayette
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Variable analysis
independent variables
- Transfection method used for each cell line (PC-3 and HEK-293T cells were transfected using different reagents)
- Reporter gene activity (measured using luciferase assay)
- Transfection efficiency (ensured by conducting the transfection in bulk and then splitting the transfected cells for luciferase assay)
- Positive control: Androgen-responsive element-luciferase plasmid (ARE-luc) containing three ARE regions ligated in tandem to the luciferase reporter
- Positive control: Luciferase construct driven by three repeats of an AR-V-specific promoter element of the ubiquitin-conjugating enzyme E2C (UBE2C) gene (UBE2C-luc)
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