2.3.1. Long-Chain Fatty acid Oxidation Stress Test

Huh7 and AML12 cells were seeded in a 6-well plate (350,000 cells per well) and transfected as described in Section 2.2.2. At 24 h after transfection, cells were replated in a 96-well Seahorse Agilent plate at 40,000 cells per well. At 24 h after the replating, Seahorse XF Long Chain Fatty Acid Oxidation Stress Test (103672-100 kit, Agilent, Santa Clara, CA, USA) was performed precisely as described by the Seahorse Agilent user guide (n = 3, in triplicate). The concentrations of the drugs used were as follows: etomoxir—4 μM, oligomycin—1.5 μM, FCCP—2 μM, and rotenone/antimycin A—0.5 μM. The assay was performed using the Seahorse XFe96 Analyzer with the standard template for the protocol for Seahorse XF Substrate Oxidation Stress Test. After running the assay, Hoechst 33,342 (1 µg/mL final concentration) was added to the medium and the cell number in each well was determined using a Cytation 5 cell imaging multimode reader (BioTek, software Gen5™, Agilent, Santa Clara, CA, USA). Data from the Seahorse analyzer were then normalized to cell number and represented as described in the Seahorse Agilent user guide.