Cell treatments
Induction of steatosis. For the induction of hepatic steatosis, a combination of long-chain FFAs, namely PA and OA was dissolved in methanol (vehicle). Each of the two FFAs was added to the medium, either alone or in combination at different molar ratios (i.e., 1:2 and 2:1, respectively), at 1mM final concentration, which is in the range concentration of FFAs in human plasma (i.e. 0.2-2mM) (Feldstein et al., 2004) for 48 hours (h). PA and OA were chosen as they are the most abundant FFAs in western diets and components of liver triglycerides in both normal subjects and patients with NAFLD (Baylin, Kabagambe, Siles, & Campos, 2002; Gómez-Lechón et al., 2007b). Steatosis was induced by modifying the previously described method (Gori et al., 2014, 2016). Briefly, HepG2 cells were incubated in steatosis medium composed of DMEM (low glucose) supplemented with 2 mM L-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, 1% BSA, 10% charcoal-stripped FBS, and 1 mM FFAs at the above described ratios. Internal controls were represented by steatosis medium without FFAs but with equivalent volume of methanol vehicle. The effects of the FFA treatments in terms of intracellular lipid accumulation, cell viability and oxidative stress were evaluated after 48h in culture via confocal microscopy-based high-content screening (HCS), using fluorescence-based functional assays.
Cell treatments with polyphenols. QUE and HXT were both used at a physiological concentration of 10 μM (Catalán et al., 2015; Hur et al., 2012; Radtke, Linseisen, & Wolfram, 2002; Vidyashankar et al., 2013) for 48h, in the steatosis medium, separately or with the diverse combinations of FFAs.