Mccoy5a

Human colonic epithelial cells (NCM460-NTR1) and colonic cancer HCT-116 cells were maintained in M3D medium (Incell, San Antonio, TX) and McCoy5a (ATCC, Manassas, VA) respectively supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 1% L-glutamine, and 10 units/ml penicillin, and 100 µg/ml streptomycin at 37°C in air supplemented with 5% CO2. For HIF-1α silencing, NCM460-NTR1 cells were seeded (6-well plates, 3 × 10⁵ cells per well) and 24 h later, were transfected with si-RNA against HIF-1α or siRNA-A control (Santa Cruz, Santa Cruz, CA) using LipofectamineTM RNAiMAX (Thermo Fisher Scientific, Waltham, MA); 48 h post-transfection, were treated as indicated with NT. For miR-210 silencing, HCT-116 or NCM460-NTR1 cells were transfected with antisense miR-210 (as-miR-210), using Lipofectamine RNAiMAX. Cells transfected with antisense control miRNA (as-miR-control) served as controls. Where indicated, NCM460-NTR1 cells were treated with NT (10⁻⁷ M, 6 h) +/− 18 h of pretreatment with PX-478 (40 × 10⁻⁶ M) or their vehicles (1% BSA in PBS and 0.9% NaCl in water respectively).