Metabolomic Analysis of Huh7 Cells

Cells used for the metabolomic analysis were cultured in a 10 cm dish until Huh7 reached 70% confluence, and the DFO was added to the medium to reach the desired concentration. Three days after DFO addition, adherent cells were detached and collected, washed 3 times with PBS, stored at −80 °C, and sent to Metabolon Inc. for further analysis (n = 3). Metabolomic and statistical analyses were carried out at Metabolon (Morrisville, NC, USA), following previously described methods [17 (link),18 (link)]. In summary, cellular pellets (5 × 10⁵ cells) were subjected to methanol extraction. The resulting extract was then subdivided into portions for analysis using ultrahigh performance liquid chromatography/mass spectrometry (UHPLC/MS,) (Thermo Scientific, Waltham, MA, USA) in positive, negative, or polar ion modes, as well as gas chromatography/mass spectrometry (GC/MS) (Thermo Scientific, Waltham, MA, USA). Identification of metabolites was accomplished by matching ion features against a reference library of chemical standards using an automated process, which was subsequently followed by visual inspection to ensure quality control. For statistical computations and data presentation, any absent values were treated as being below the detection thresholds, and these values were estimated using the lowest compound value available.