Anti mir 199a 5p

The anti-miR-199a-5p was purchased from Ambion. The delivery of anti-miR-199a-5p in PA1 cells was performed using oligofectamine (Thermo Fisher Scientific). Briefly, PA1 cells at 70% confluency in 96-well or 6-well plates were transfected with anti-miR-199a-5p (2.5 nM) in Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific). The following day, the cells were treated with ST09 for 24 h. Post incubation, MTT assay was performed to evaluate the cell viability in the 96-well plate and cells in the 6-well plate collected for protein.

The human esophageal squamous cancer cell lines TE7 and TE10 are kind gifts from Dr. Nishihira (Tohoku University, Sendai Japan). They were derived from esophageal squamous cell carcinomas from separate patients and are characterized in the Cell Resource Center for Biomedical Research at Tohoku University. These cell lines were cultured in RPMI media (Mediatech Inc, Herndon, VA) supplemented with 10% heat-inactivated FBS. hESO is an esophageal epithelial cell line derived from esophageal specimens harvested at the time of donor lung procurement. These cells were cultured in BEBM media (Lonza Corp, Walkersville, MD) supplemented with 20% heat-inactivated FBS and the BEGM bullet kit. All cells were maintained in a 37°C incubator with 5% CO2 humidified air. Pre-miR-199a-5p, anti-miR-199a-5p, and scrambled control-miRNA were purchased from Ambion (Austin, TX).