Rhodamine 123 rh123

Perfluorooctane sulphonate (PFOS; potassium salt, purity ≥ 98%, Sigma-Aldrich, Shanghai, China) was dissolved in dimethyl sulphoxide (DMSO; Sigma-Aldrich, Shanghai, China) and stored at 20°C. N-acetyl-cysteine (NAC), 3-methyladenine (3-MA), and chloroquine (CQ) were purchased from Sigma-Aldrich (St. Louis, MO, USA; A7250-10G, M9281-100MG, and C6628-25G, respectively). Methyl thiazolyl tetrazolium (MTT) was purchased from Amresco (Solon, OH, USA, 0793-5g). A ROS assay kit was purchased from Beyotime Biotech (Nanjing, China, S0033). An AV-FITC/PI kit was purchased from KeyGen Biotech (Nanjing, China, #KGA108). Monodansyl cadaverine (MDC) and rhodamine 123 (Rh123) were purchased from Beyotime Biotech (P6659-20 μg and C2007, respectively). A BCA kit was purchased from Beyotime (Shanghai, China, P0012S). Anti-p62 was purchased from Proteintech (Shanghai, China, 55274-1-AP). Anti-LC3 was purchased from Sigma-Aldrich (Shanghai, China, L8918-200 μL). Anti-Bcl-2 and anti-Bax were purchased from CST (Shanghai, China, #3498 and #5023, respectively). Anticleaved-caspase-3 and β-actin were purchased from Santa Cruz, Paso Robles (CA, USA). Horseradish peroxidase- (HRP-) conjugated secondary antibodies and the SuperSignal West Pico Kit were purchased from Thermo (Waltham, MA, USA).

Mitochondrial membrane potential (MMP) was measured by Rhodamine 123 (Rh123) staining (Beyotime, Nantong, China). Rh123 can selectively enter the mitochondria at full membrane potential and remain in the mitochondria [13 (link),14 (link)]. After indicated treatment, cells were incubated with 5 µg/mL Rh123 at 37 °C for 30 min in the dark. The samples were immediately analyzed at excitation wavelength of 507 nm and emission wavelength of 529 nm using a fluorescence microscope.