Jc 1 staining

Mitochondrial membrane potential was measured using JC-1 staining (Cayman). Cells were pre-infected with indicated adenovirus before receiving 20 Gy of X-ray irradiation. 24 h after irradiation, the cells were incubated for 30 min in the dark with JC-1 dissolved in serum-free medium at 37°C. Nuclei were counterstained with DAPI.

Mitochondrial stability was assessed using a mitochondrial membrane potential (ΔΨ_m) assay kit with JC-1 staining (Cayman Chemical, Ann Arbor, MI). HCC70 cells (1 × 10⁴) were stimulated with IL-26 (30 ng/ml) in the presence or absence of Gefitinib (40 μM), signal inhibitors, and neutralizing antibodies on flat-bottom plates 96-well (Corning) for 24 h. After stimulation, cells were stained for JC-1 staining Solution. The ΔΨ_m was assessed using a fluorescence microscope (Nikon, Tokyo, Japan) at wavelengths of 530 and 590 nm, respectively. The ΔΨ_m depolarization displayed green fluorescence. Stained cells were quantitated using Image-J software (NIH). Images were captured using objectives of ×40.

Mitochondrial function and integrity of ARPE-19 cells were evaluated using ATP Detection Assay Kit-Luminescence (Cayman) and JC-1 staining (Cayman), respectively. In the ATP detection, the luciferin in assay reagent was converted to oxyluciferin with light emission that was quantitatively proportional to the amount of intracellular ATP (n = 6 per treatment condition). JC-1 staining (1 μM 30 min, n = 6 per treatment condition) was performed as per the manufacturer's protocol. Both luminescence and fluorescence were detected using the Hidex Sense Microplate Reader (Hidex).