Determination of mitochondrial function

The skin of mice in P1-3 was cut with sterile scissors and placed in a medium composed of DMEM, FBS, and penicillin-streptomycin. After removing the excess tissue, the fibroblasts were cultured in the medium. Two strains of WT and KI cells were kept at 37°C in a 5% CO₂ incubator.

Mitochondrial function was assessed using ROS ({"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}}M36008, Invitrogen) and mitochondrial membrane potential (ab112134, Abcam) assays, according to the manufacturer’s instructions and the reported guidelines [50]. Cells were stained with 100 nM of MitoTracker ({"type":"entrez-nucleotide","attrs":{"text":"M22425","term_id":"197105","term_text":"M22425"}}M22425, Invitrogen) and live cells were imaged using an LSM980 laser scanning confocal microscope.