Briefly, the AR42J cells (3×103 cells/well, seeded in a 96-well plate) were pre-incubated with 10, 20, 40 or 80 µM Rg3 at 37°C for 1 h and then exposed to Cn (10−8 M, cat. no. HY-A0190, MedChemExpress) for 24 h. Subsequently, cell viability was determined using a CCK-8 (cat. no. HY-K0301, MedChemExpress), incubating the cells with 10 µl CCK-8 solution at 37°C for 4 h. Cell viability was then determined by measuring the optical density (OD) at 450 nm using a microplate reader (Thermo Fisher Multiskan; Thermo Fisher Scientific, Inc.).
Additionally, to further explore whether Rg3 alleviates AP in Cn-related cell death via ferroptosis, the AR42J cells we pre-incubated with various inhibitors, including a pan-caspase/apoptosis inhibitor (Z-VAD-FMK, 20 µM, MedChemExpress), a ferroptosis inhibitor [ferrostatin-1 (Fer-1), 1 µM, MedChemExpress], a necrosis inhibitor [necrostatin-1 (Nec-1), 20 µM, MedChemExpress] and an autophagy inhibitor [3-methyladenine (3-MA), 20 µM, MedChemExpress], for 2 h at 37°C. Subsequently, the cells were further treated in the presence of 20 µM Rg3 for a further 24 h. Cell viability was determined as described as above.
Additionally, to further explore whether Rg3 alleviates AP in Cn-related cell death via ferroptosis, the AR42J cells we pre-incubated with various inhibitors, including a pan-caspase/apoptosis inhibitor (Z-VAD-FMK, 20 µM, MedChemExpress), a ferroptosis inhibitor [ferrostatin-1 (Fer-1), 1 µM, MedChemExpress], a necrosis inhibitor [necrostatin-1 (Nec-1), 20 µM, MedChemExpress] and an autophagy inhibitor [3-methyladenine (3-MA), 20 µM, MedChemExpress], for 2 h at 37°C. Subsequently, the cells were further treated in the presence of 20 µM Rg3 for a further 24 h. Cell viability was determined as described as above.