EC Cell Line Transfection Studies

Downregulation of FXYD5/Dys expression in HGE was achieved by transient transfection of a FXYD5/Dys small interfering RNA (siRNA) (#45745, three target-specific 19–25-nt siRNAs designed to knock down gene expression; SCBT) with Lipofectamine® 2000 (Thermo-Fisher Scientific) following the protocol suggested by the manufacturer. Briefly, 5 × 10⁵ cells were seeded in each well of a sterile six-well plate and incubated overnight. Then, cells were transfected with 100 pmol/ml of FXYD5/Dys or control scramble siRNA (#37007, SCBT). In addition, Hec1a cells were transfected with pcDNA3-FXYD5/Dys or pcDNA3 (control) plasmids. Cells numbering 5 × 10⁵ were transfected with 1 μg of plasmid DNA with Lipofectamine® 2000. After a 5-h incubation period, the transfection mixture was replaced by a complete culture medium. Cells were subjected to transcript and protein expression analysis as well as to functional tests 24–72 h post-transfection, as specifically indicated.