Jc 1 staining

Cellular ATP generation was measured to reflect mitochondrial function. Firstly, A549 cells were washed three times with cold PBS at room temperature. Subsequently, a luciferase-based ATP assay kit (CellTiter-Glo® Luminescent Cell Viability Assay; cat. no. G7570; Promega Corporation, Madison, WI, USA) was used to analyze ATP content, according to the manufacturer's protocols (10 (link)). ATP production was measured using a microplate reader at a wavelength of 570 nm (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA) (26 (link)). To observe the mitochondrial potential, JC-1 staining (cat no. M34152; Thermo Fisher Scientific Inc.) was used. Briefly, 10 mg/ml JC-1 was added to the medium for 10 min at 37°C in the dark, in order to label mitochondria. Images were captured under an Olympus IX81 microscope (Olympus Corporation). Normal mitochondrial potential was indicated by red fluorescence, whereas damaged mitochondrial potential was indicated by green fluorescence (27 (link)).

The mitochondrial membrane potential was measured by JC-1 staining (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Briefly, 1×10⁵ cells attached to HAS-G were washed with ice-cold PBS twice and incubated with the JC-1 working solution at 37°C in the dark for 30 min. Subsequently, the supernatant was removed, the cells were washed twice with PBS and imaged using a fluorescence microscope (Olympus Corporation; magnification, ×100).

Changes in ΔΨ m were measured using JC‐1 staining (Thermo Fisher Scientific). H9c2 cells were seeded in 6‐well plates, and after the cells reached about 80% confluence, they were treated with 100 µg/mL PNS or 50 µg/mL PNS for 24 hours, followed by the addition of 300 μmol/L PA. After 24 hours, the medium was discarded, and 2 μg/mL JC‐1 was added and incubated at 37 °C for 20 minutes, and the cells were washed with PBS and photographed using a fluorescence microscope (Olympus).