Ptc 209

Saos-2 cells were cultured overnight to reach 60% confluence and transfected with siRNAs: si-BMI1, si-KLF4, a combination of si-BMI1 and si-KLF4 (si-B+K), or control siRNA (Genepharma, Shanghai, China) at a final concentration of 20 nM in OPTI-MEM for 48 h using a Lipofectamine 3000 kit (Thermo Fisher Scientific) following the instructions. Similarly, the cells were transduced with lentiviral miR-135a (LV-miR-135a; Genechem) or lentiviral control miR-NC in the 5 μg/ml Polybrene for 24 h and treated with 3 μg/ml of puromycin for 3 days. miR-135a expression in generated cell clones was tested for stable expression. Correspondingly, the cells were treated with PTC-209 (an inhibitor of BMI1, purchased from MedChemExpress company, Monmouth Junction, NJ, USA) at a final concentration of 10 μM diluted in DMSO or their DMSO control for 2 days. The cells were grown to 80% confluence after subculture in complete medium for further experiments.

The experiments were conducted on endometrial cancer cell lines HEC-1A (American Type Culture Collection, Manassas, VA, USA) and Ishikawa (European Collection of Aunthenticated Cell Cultures, Wiltshire, UK). Both of the endometrial cell lines were grown in DMEM:F12 media (Biowest, France) containing 10% (HEC-1A) or 5% (Ishikawa) (v/v) FBS in standard condition (37 °C, 5% CO₂).
HEC-1 and Ishikawa cell lines were treated with 0.5, 1, or 5 µM PTC-209 (MedChem Express, Monmouth Junction, NJ, USA) under different conditions. The endometrial cancer cells were treated with PTC-209 in standard culture conditions or low (0.5 mM) or high (30 mM) glucose concentrations. The effect of the treatment was checked after 48 h. Additionally, the cells grown in low or high glucose after 24 h of serum starvation were stimulated with 100 nM insulin (Sigma Aldrich, St. Louis, MO, USA) and were harvested after 2, 4, 6, and 8 h.