Jc 1 reagent

H and J cybrids were plated in 24-well plates (100,000 cells/well) were incubated for 24 h and treated with 0 or 40 μM of cisplatin for another 48 h. JC-1 reagent (5,5′,6,6′-tetrachloro-1,1′,3,3′- tetraethylbenzimidazolylcarbocyanine iodide) (Biotium, Hayward, CA) was added to cultures for 15 min. Fluorescence was measured using a Gemini XPS Microplate Reader (Molecular Devices) for red (excitation 550 nm and emission 600 nm) and green (excitation 485 nm and emission 545 mm) wavelengths. Intact mitochondria with normal ΔΨm appeared red, while cells with decreased ΔΨm were in a green fluorescent state. Experiments were analyzed in quadruplicate and the entire experiment was repeated three separate times. Cisplatin-treated values were compared to untreated values for statistical significance (P ≤ 0.05, GraphPad Prism Software, Inc.).

¹H- and ¹³C-NMR spectra were recorded on a Bruker Avance 300 FT-NMR spectrometer (Bruker Switzerland AG, Fallanden, Switzerland) operating at 300 MHz (¹H) and 75 MHz (¹³C) using CDCl₃ as the solvent. Specific optical rotations were measured using a Jasco-1020 polarimeter (Jasco Corporation, Tokyo, Japan). Column chromatography and TLC were carried out using Merck silica gel 60 (>230 mesh) and precoated silica gel 60 F₂₅₄ plates, respectively. Spots on the TLC were visualized under UV light (245 or 365 nm) and by spraying with anisaldehyde–H₂SO₄ reagent followed by heating at 80 °C until maximum color formation occurred. Only analytical grade chemicals were used: JC-1 reagent (Biotium, Biotium, Inc., Fremont, CA, USA), Hoechst 33342 (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA), MTT or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich, St. Louis, MO, USA), fetal bovine serum (FBS), and DMEM (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA). DMSO and PI (propidium iodide) reagents were purchased from Merck KGaA, St. Louis, MO, USA.