Tangeretin

Human hepatoma HepG2 cells were purchased from the Korean Cell Line Bank (KCLB, Korea) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and streptomycin (Gibco, USA) in a humidified atmosphere under 5% CO₂ at 37°C. HepG2 cells were seeded in 96-well plates. After reaching confluence, the cells were serum-starved overnight and exposed to 0.4 mM PA (Sigma-Aldrich, USA), in the presence or absence of 5 μM simvastatin (Sigma-Aldrich), 2 mM metformin (Sigma-Aldrich), 50 μM of hesperidin (LKT Laboratories, USA), narirutin (Sigma-Aldrich), nobiletin (Sigma-Aldrich), sinensetin (Sigma-Aldrich), or tangeretin (Sigma-Aldrich) for 24 h. The cells treated with simvastatin or metformin were used as the positive control.

NaSH was purchased from Sigma and prepared freshly for each experiment. Briefly, a starting solution of NaHS powder dissolved in distilled water was prepared and then diluted 5000 times and incubated with DNTB [1 mM 5,5′-dithio-bis(2-nitrobenzoic acid)] (Sigma-Aldrich) for 1 to 2 min, yielding a colored product. H₂S concentration was determined by reading the absorbance of this product with a spectrophotometer set at 412 nm using the formula $[{\mathrm{H}}_2\mathrm{S}]= \text{cuvette volume}*\text{dilution}*{\text{OD}}_{412\text{nm}}$ Working solutions were prepared in Ringer buffer [140 mM NaCl, 2 mM CaCl₂, 1 mM MgSO₄, 1.5 mM K₂HPO₄, and 10 mM glucose (pH 7.4)]. Cells were washed twice with phosphate-buffered saline (PBS) to remove all FBS to avoid quenching, incubated with different concentrations of NaHS for 30 min at room temperature, and washed twice before confocal analyses. In most experiments, the final concentration was 100 or 500 μM for untransfected or AQP8-overexpressing cells, respectively.
Extracellular catalase (CAT, 5000 U/ml in Ringer buffer), DPI (10 μM in DMEM for 3 hours), DTT (5 mM in Ringer buffer), H₂O₂ (50 μM in Ringer buffer), MESNA (10 mM in Ringer buffer), tangeretin (200 μM in complete medium), and CHX (0.5 mM, see below) were all purchased from Sigma-Aldrich. Heat shock was performed as described (21 (link)).