13 hode

The medium used to prompt the adipogenic differentiation of C‐MSC consists of IMDM supplemented with 10% FBS (Euroclone, Milan, Italy), 0.5 mmol/l 3‐isobutyl‐1‐methylxanthine (Sigma‐Aldrich, St. Louis, Missouri, USA), 1 µmol/l hydrocortisone (Sigma‐Aldrich, St. Louis, Missouri, USA), 0.1 mmol/l indomethacin (Sigma‐Aldrich, St. Louis, Missouri, USA), 10,000 U/ml penicillin (Invitrogen, Carlsbad, California, USA), 10,000 µg/ml streptomycin (Invitrogen, Carlsbad, California, USA), and 20 mmol/l L‐Glutamine (Sigma‐Aldrich, St. Louis, Missouri, USA). For the preparation of AM, refer to (Pilato et al, 2018 ).
C‐MSC were plated in AM at a concentration of 20,000 cells/cm² and treated with 150 μg/ml oxLDL (see the paragraph “oxLDL preparation”) (Asmis & Begley, 2003 ; Hamel et al, 2008 (link); Lee et al, 2010 (link)), 20 μg/ml 13HODE (Cayman Chemicals, Ann Arbor, Michigan, USA) (Nagy et al, 1998 (link); Kim et al, 2013 (link)), or 5 mmol/l NAC (Sigma‐Aldrich, St. Louis, Missouri, USA) (Zolkipli et al, 2011 (link); Wang et al, 2015 (link); Mao et al, 2016 (link); Pieralisi et al, 2016 (link)), a known thiolic antioxidant (Sunitha et al, 2013 (link)). After 72 h, treatment effects were evaluated by ORO (Sigma‐Aldrich, St. Louis, Missouri, USA) staining and Western blotting analysis.

Cells were seeded at 3 × 10³ for U251-MG and U87-MG and at 5 × 10³ for A172 in 96-well plates. After 24 h, cells were treated with 0.1 µM, 0.5 µM, and 1 µM of the following (S)-form oxylipins: 13-HODE, 9-HODE, 15-HETE, 5-HETE, 12-HETE, 8-HETE, 9-HETE, 20-HETE (all Cayman Chemical, USA) and 100% ethanol (control). At 24, 48, and 72 h of treatment, each well was incubated for 4 h with 0.25 mg/mL of tetrazolium at 37 °C in a humidified atmosphere with 5% CO₂. At the end of incubation, cells were washed with warm PBS and lysed with 100 µL of 0.04-M HCl in isopropanol to solubilize the formazan. The absorbance was read at 590 nm in an Epoch microplate reader (BIOTEK, Weinusky, VT, USA).