3 hydroxybutyric acid 3 oba

Human uveal melanoma cells (92.1) were purchased from ATCC Company (Milan, Italy). Briefly, cells were cultured in RPMI1640 medium with 10% fetal bovine serum (FBS) (cat. no. 10082147), 100 U/mL penicillin, and 100 U/mL streptomycin (cat. no. 15070063; all from Gibco, Waltham, MA, USA) and expanded once they reached 80% confluency using trypsin-EDTA solution (0.05% trypsin and 0.02% EDTA). Lactate (Sigma-Aldrich, Milan, Italy), AZD3965, 3,5-dihydroxybenzoic acid (3-5-DHBA) (Sigma-Aldrich, Milan, Italy), and 3-hydroxybutyric acid (3-OBA) (Sigma-Aldrich, Milan, Italy) were added to cell culture at final concentrations of 20 mM, 10 μM, 150 μM, and 3 mM, respectively, when needed.

Human breast cancer cell line (MDA-MB-231) was purchased from ATCC Company (Milan, Italy). Cells were suspended in DMEM-F12 (Gibco, cat. no. 11965092) culture medium containing 10% fetal bovine serum (FBS, Gibco, cat. no. 10082147), 100 U/mL penicillin and 100 U/mL streptomycin (Gibco, cat. no. 15070063). At 80% confluency, cells were passaged using trypsin-EDTA solution (0.05% trypsin and 0.02% EDTA, Gibco, cat. no. 25300054). Lactate, 3,5-dihydroxybenzoic acid (3-5-DHBA), 3-hydroxybutyric acid (3-OBA) and IGFBP6 (Sigma–Aldrich, Milan, Italy) were added to cell culture at final concentrations of 20 mM, 150 μM, 3 mM and 400–800 ng/mL, respectively, dissolved in cell culture media for different times (4 to 48 h). Same amount of cell culture media was used for control cultures.