Pcdna3.1 hygro

The cell lines were purchased from ATCC in 2016 and authenticated using STR profiling. All cell lines were routinely cultured in RPMI 1640 supplemented with 10% FBS without antibiotics at 37 °C and 5% CO₂. The plasmids transfected into the BT474 cell line were pcDNA3.1‐P2A‐eGFP containing the sequence of the short Var4 of NR2F1‐AS1 and pcDNA3.1/Hygro(+) containing the sequence of the long Var1 of NR2F1‐AS1; pcDNA3.1‐P2A‐eGFP and pcDNA3.1/Hygro(+) were used as negative controls (GenScript, Piscataway, NJ, USA). Transfection was performed by nucleofection using the same parameters (Nucleofector™ 2b Device, Lonza Bioscience, Basel, Switzerland).

The N-terminal FLAG epitope-tagged human AC isoform 6 (AC6) gene and corresponding site-directed mutants at the ATP and FSK binding interface of AC6 were codon-optimized for expression in mammalian cells and cloned into the KpnI-NotI restriction site of the pcDNA 3.1/Hygro (+) expression vector. The wild-type (WT) and corresponding mutants of AC6 carried by expression vector pcDNA 3.1/Hygro (+) were commercially synthesized by GenScript (Piscataway, NJ, USA). Per the manufacturer’s instructions, the gene construct of AC6 WT and mutants were transiently expressed in HEK293T cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). We generated stable HEK293T cells expressing the WT and mutants in the selection medium containing 200 µg/mL hygromycin as described previously in references [34 (link)]. The stable cell lines used in the study were characterized using Western blot analysis and flow cytometry.