Evaluating Metabolic Function of 2D HconF Cells

For the bio-cellular function of the 2D HconF cells, the oxygen consumption rate (OCR) and the extracellular acidification (ECAR) of 2D HconF cells were evaluated by a Seahorse XFe96 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), as described in our previous studies [14 (link),15 (link)]. Briefly, 20 × 10³ 2D HconF cells were placed in wells of a 96-well assay plate as follows: (1) non-treated control (NT), (2) treated with TGF-β2, (3) treated with ATRA, and (4) treated with TGF-β2 and ATRA. After replacing the culture medium with Seahorse XF DMEM assay medium (pH 7.4, Agilent Technologies, #103575-100) supplemented with 5.5 mM glucose, 2.0 mM glutamine, and 1.0 mM sodium pyruvate, the basal OCR and ECAR values were determined using a Seahorse XFe96 Bioanalyzer (San Francisco, CA, USA), and thereafter, the samples were further analyzed after supplementation with 2.0 μM oligomycin, 5.0 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 1.0 μM rotenone and antimycin A, and 10 mM 2-deoxyglucose (2-DG). The OCR and ECAR values were normalized to the amount of protein per well.