Hypoxia Modulates Fatty Acid Effects

Cells (approximately 1 × 10⁶ cells per sample) were seeded and after a 24-h pre-incubation period (allowing cells to attach), the culture medium was replaced with a serum-free medium containing 2% BSA with or without FA(s) (SA, a combination of SA and OA, or OA alone) at required concentrations. Cells were placed inside a standard incubation cabinet (20% oxygen level, external normoxia) or inside chambers of a specific incubation cabinet [31 (link)] in which oxygen levels were 4% (moderate hypoxia) or 1% (strong hypoxia). After relevant time of incubation, cells were harvested and the western blot analysis was performed as described previously [4 (link)]. All primary antibodies were used in a 1:1000 dilution. The chemiluminescent signal was detected using a Carestream Gel Logic 4000 PRO Imaging System equipped with Carestream Molecular Imaging Software (Carestream Health, New Haven, CT, USA), which was used for image acquisition. Image Master^TM 2D Platinum 6.0 software (GE Healthcare, Uppsala, Sweden) was used to obtain data for densitometric analysis.