Lc ms uv

Human gastric adenocarcinoma cells CRL-1739 (AGS) were obtained from ATCC (Manassas, VA, USA) and kept in F-12 medium (Gibco, Waltham, MA, USA) enriched with streptomycin (100 μg/mL), penicillin (100 U/mL) (Sigma, St. Louis, MO, USA), and Fetal Bovine Serum (FBS) (10%) (Gibco, Waltham, MA, USA) at 37 °C in a humidified atmosphere of 5% CO₂. In the next step the cells were seeded in 6-well plates (5 × 10⁵ cells/well) and cultivated for 24 h in medium (FBS-free) supplemented with 60 and 120 μM afzelin (≥90% (LC/MS-UV) (Sigma, St. Louis, MO, USA). Stock solution of afzelin was 50 mM (2 mg /50 μL DMSO; Sigma, St. Louis, MO, USA). After washing the wells (Phosphate Buffered Saline, pH 7.4 (PBS)), the cells were lysed 20 min at 4 °C with RIPA buffer (Sigma, St. Louis, MO, USA) containing protease inhibitors (Sigma, St. Louis, MO, USA), diluted in RIPA buffer (1:200). Collected lysates and culture media were centrifuged at 1000× g for 5 min at 4 °C. The obtained supernatants were frozen at −70 °C and then used for Western blot assays. For RT-PCR the monolayers were washed with sterile 10 mM PBS (three times) and sonicated (Sonics Vibra cell; Sonics & Materials, Leicestershire, UK). Aliquots of the homogenate were applied for RNA isolation. Cells without afzelin addition were used as control.