Geneticin

The pHAGE TRE dCas9-VP64 lentiviral expression vector (Addgene) was electroporated into 4523T cells using NEPA21 Electroporator as described previously [23 (link)]. Briefly, 1.2 × 10⁶ of 4523T cells were resuspended in 96 μL Opti-MEM medium (Thermo Fisher Scientific, Waltham, MA, USA) and mixed with 10 μg of pHAGE TRE dCas9-VP64 in 4 μL Opti-MEM to make a total volume of 100 μL. The cells were then electroporated with optimized conditions at 275 V and a pulse width of 1.5 ms of poring pulse. The transfected cells were selected with geneticin (Sigma-Aldrich, Burlington, MA, USA) 48 h (h) post electroporation at a concentration of 0.45 µg/mL. geneticin selected cells were then single cell sorted using a fluorescent activated cell sorter (FACS), the FACSAria U3 (BD Bioscience, Wokingham, Berkshire, UK). The positive single cell clones were identified via PCR using forward primer 5′-CCTGAATGCAGTGGTAGGCA-3′ and reverse primer 5′-CATTCGTTTCCGGCCGTTTT-3′ from the dCas9-VP64 sequence. For all further studies, clone 4 of 4523T-dCas9-VP64 (C4) cells was used.

[³H]-estradiol 17 β-D-glucuronide (E₂17βG) (specific activity 37.2 Ci/mmol) and [³H]-cholecystokinin-8 (CCK-8) (specific activity 98.4 Ci/mmol) were purchased from Perkin Elmer Life Science (Waltham, MA, USA). Nicardipine was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) Unlabeled CCK-8, E₂17βG, dimethyl sulfoxide (DMSO), Dulbecco’s Modified Eagle Medium (DMEM), Hank’s Balanced Salt Solution (HBSS), antibiotic antimycotic solution, trypsin–EDTA solution, Triton X-100, and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Poly-L-lysine was purchased from Trevigen Inc. (Gaithersburg, MD, USA). Geneticin^® and HEPES were purchased from BD Biosciences (Bedford, MA, USA). Bio-Safe II liquid scintillation mixture was purchased from Research Products International (Mt. Prospect, IL, USA).

Human breast cancer cells (MCF‐7 and MDA‐MB‐231) undergoing the logarithmic growth phase were seeded onto a 6‐well plate. When the cells reached 80% confluence, they were transfected with the pGL4.51 plasmid (Promega Corporation, USA). Twenty‐four hours after transfection, a single‐cell suspension containing 400 μg/ml of G418 (Geneticin) (BD Bioscience Clontech, San Diego, CA, USA) was prepared and seeded into a new 6‐well plate for extended culture. The media (G418) was refreshed every 2–3 days until monoclonal cells were detected. We selected monoclonal cells with good tolerance from the cell culture plates and continued to grow these cells in 24‐well plates in G418 medium. When the degree of fusion was >90%, the cells were transferred into 96‐well plates for culture and were then assayed for luciferase activity. Cell lines with the highest clonal luminescence value were inoculated on a 96‐well plate, and different concentration gradients were generated. After 24 h, we added thee luciferase substrate (Promega Corporation, USA) and measured bioluminescence intensity with a small animal live imaging system (Merck KGaA, Darmstadt, Germany).