Pg5luc

HEK293 cells grown to 70% confluence were transfected with the expression plasmid pBIND-ERα (50 ng/μL; Promega, Madison, WI, USA) and reporter plasmid pG5-Luc (50 ng/μL; Promega) using PEI MAX (50 µg/mL; Polysciences, Warrington, PA, USA) and Opti-MEM (Thermo Fisher Scientific). After incubation of the plasmids with the transfection reagent for 20 min at room temperature, HEK293 cells were transfected with the plasmid-reagent solution for 6 h at 37 °C. Transfected cells were seeded in a 96-well plate at 2 × 10⁴ cells/well and treated with screening sample (10 µg/mL) or various concentrations of SRE or 1 nM 17β-estradiol (positive control; Sigma-Aldrich, St. Louis, MO, USA) in DMSO at 37 °C for 24 h. After incubation, the medium was then removed, and luminescence was measured using a luciferase assay system (Promega) with a FLUOstar Omega microplate reader (BMG LABTECH, Ortenberg, Germany) according to the manufacturer’s instructions.

HEK293FT cells were seeded in 48-well plates at 6.0 × 10⁴ cells/well using steroid-free medium and cultured overnight. For determination of CRE- or androren response element (ARE)-mediated transcriptional activity, cells were transfected with β3-AdR expression vector (pcDNA3.2-β3-AdR-V5 (74 (link))), AR expression vector (pcDNA3.1-AR (68 (link)), pcDNA3.1-AR (K630A/K632A/K633A), pcDNA3.1-AR (C619Y) (32 (link), 75 (link)), pcDNA3.1-AR (L26A/F27A) (76 (link)), pcDNA3.1-AR (E897Q) (76 (link)), pcDNA3.1-AR (ΔpolyQ+Q6+Q5) (35 (link)), pcDNA3.1-AR (C806A)), luciferase reporter vector (p4xCRE-TATA-Luc2P (74 (link)), pGL4-ARE2-TATA-Luc (77 (link)), or pUCP1-pro-Luc2P), and Renilla luciferase reporter vector (pGL4.74[hRluc/TK] (Promega)) with PEI MAX (Polysciences Inc) and Opti-MEM (Thermo Fisher Scientific) for 24 h. For the mammalian two-hybrid assay, cells were transfected with pGAL-CREB, pcDNA3.1-AR or pACT-AR (NTD) (76 (link)), pG5luc (Promega), and pGL4.74[hRluc/TK] with PEI MAX and Opti-MEM for 24 h. Cells were incubated in the presence of 10 nM DHT for 16 h and then stimulated with 10 μM CL316243 or 10 μM forskolin for an additional 4 h. Cells were treated with the AR antagonist bicalutamide (10 μM) 30 min prior to DHT treatment. Luciferase reporter activity was determined as described previously (68 (link)). Data are expressed as relative light units.

For ERE-Luc assays, HeLa cells were transfected with pERE-E1b-Luc [39 (link)] and ERα expression vector using Lipofectamine LTX (Invitrogen), and lysed in Glo Lysis Buffer (Promega). For GAL4-Luc assays, cells were transfected with pBIND-YAP1 and pG5luc (Promega). Luc activity was measured on a Berthold luminometer as described [64 (link)]. Cells were co-transfected with pERE-E1b-luc, ERα expression plasmids, and siRNAs using Trans-IT-TKO (Mirus Corp.) or RNAimax (Invitrogen). siRNAs are listed in Supplementary Table 3.

Interaction of proteins was determined using a modification of the Checkmate mammalian two-hybrid system (Promega). Two vectors were used, pAct and pBind3-D. pAct (Promega) contains the herpes simplex virus VP16 activation domain followed by a multiple cloning site. pBind3-D [64 (link)] is a modification of pBind (Promega) in which the DNA-binding domain of the yeast GAL4 gene followed by an altered multiple cloning site and the vector lacks the Renilla luciferase module.
N2a cells were seeded on 96-well plates and transfected in parallel with pAct, pBind3-D, peGFP (Clontech), and a pG5luc (Promega) expressing firefly luciferase under control of GAL4 [64 (link)]. The next day fluorescence generated by eGFP was determined for normalization and light emission generated by luciferase activity was detected after adding Bright-Glo (Luciferase Assay System, Promega) with a Multilabel Counter (PerkinElmer). Luciferase activity was normalized to eGFP-fluorescence. All transfections and analysis were performed in septuplicate and experiments repeated three times. Average relative luciferase light units and S.D. were determined using the Prism software.

293 T cells were transfected with luciferase reporter plasmid pG5-luc (Promega Corp., Madison, WI, USA), test plasmids, negative control plasmids and positive control plasmids respectively. Forty-eight hours post transfection, cells were harvested and luciferase activities were assayed by using the luciferase reporter assay system (Promega Corp., Madison, WI, USA) according to the manufacturer’s recommendations.

Plasmids carrying either a native 3ABCD ORF (p3ABCD) or a 3ABCD ORF containing mutated 3C^pro with Cys142Ser and Cys163Gly (pmu3ABCD) were generated as previously described [45 (link)] with slight modification. The primer sequences for PCR cloning are presented in Supplementary Materials Table S1. Plasmids pBIND-VP16 and pG5luc were purchased from Promega, Madison, WI, USA [46 (link)]. To produce pBIND_FMDV 3C^pro plasmids for use in the protease assay, the 3ABCD and mu3ABCD DNA fragments in p3ABCD and pmu3ABCD were subcloned into pBIND-VP16 plasmid (Promega, Madison, WI, USA) between a GAL4-binding domain and a VP16 activation domain at the BamH I and Mlu I sites, resulting in pBV_3ABCD and pBV_mu3ABCD, respectively. The pG5luc containing the GAL4-binding site upstream of the firefly luciferase gene was used as a reporter system while pBV_3ABCD and pBV_mu3ABCD also carried the Renilla luciferase gene, which served as an internal luciferase control.