Rhodamine 123

WY14643, GW501516, rosiglitazone, MTT, H₂O₂, cytotoxicity detection kit PLUS (LDH), Hoechst 33342, and 2,7-dichlorofluorescein diacetate (DCFDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rhodamine 123 was purchased from Enzo Life Sciences, Inc. (Burlington, ON, Canada). The SOD and CAT assay kits were purchased from DoGenBio (Guro-gu, Seoul, Korea). Monoclonal rabbit antibodies for cleaved-caspase 3, 7, and 9; cleaved-PARP; caspase 3 and 7; and PARP, as well as monoclonal mouse antibodies for caspase 9 were purchased from Cell Signaling Technology (Beverly, MA, USA) and the dilution was 1:1000. High performance liquid chromatography (HPLC) was performed using a Gilson 307 pump, Shodex RI-71 detector, and ODS column (YMC-Triart C18, 250 × 10.0 mm, i.d. 5 μm). ¹³C NMR spectra were obtained using a Varian UNITY 400 spectrometer and ¹H NMR spectra were recorded using a Varian INOVA 500 spectrometer. Optical rotation was detected using a Jasco P-1020 polarimeter.

∆ψm level was measured with rhodamine 123 and JC-1 dyes (Enzo Life Sciences, Inc., Farmingdale, NY, USA). Briefly, 1 × 10⁵ cells in 12-well culture plates (SPL) were incubated with Rk1. After 24 h, the supernatant was removed, and cells were stained with 0.1 μg/mL rhodamine 123 or 5 μg/mL of JC-1 for 30 min at 37 °C. The cell pellet was suspended in PBS and imaged. The intensity of rhodamine 123 staining was determined by Accuri C6 flow cytometry. The lack of rhodamine 123 staining showed loss of mitochondrial membrane potential (∆ψm).