Bc1440

Approximately 1 mL of mitochondrial extract was introduced to a population of 5 × 10⁶ cells. The sample underwent ice-cold homogenization using either a homogenizer or mortar. Subsequently, the mixture was centrifuged at 4 °C and 600× g for 10 min.
Mitochondrial complex I (NADH-Q reductase) (BC0510, Solarbio, Beijing, China), II (FADH2-Q reductase) (BC3230, Solarbio, Beijing, China), III (cytochrome reductase) (BC3240, Solarbio, Beijing, China), IV (cytochrome oxidase) (BC0940, Solarbio, Beijing, China), V (adenosine triphosphate (ATP) synthase) (BC1440, Solarbio, Beijing, China) activities, and ATP content (BC0300, Solarbio, Beijing, China), were measured using their respective analytical kits according to the manufacturer’s protocols [58 (link),59 (link),60 (link)].

Mitochondria were isolated from SH‐SY5Y cells using a cell mitochondria isolation kit (SM0020, Solarbio). The cells were resuspended in precooled lysis buffer. The cell suspension was transferred to a small‐volume glass homogenizer and ground 30 times within an ice bath. The cell suspension was homogenized and centrifuged at 1000 × g for 10 min at 4°C. The resultant supernatant was then centrifuged at 12,000 × g for 10 min at 4°C. The obtained mitochondrial precipitates were resuspended in storage buffer and used immediately. Protein concentrations were determined by BCA assay (Thermo Fisher). Mitochondrial respiration complex activity was measured using the complex I–V enzyme activity microplate assay kit (BC0510, BC3230, BC3240, BC0940, and BC1440; Solarbio). The enzyme activity assays were conducted according to the manufacturer's instructions. Briefly, mitochondrial homogenates were added to the respective reaction buffer. The reaction mixture was transferred to a pre‐warmed quartz cuvette and immediately placed into a spectrophotometer. Mitochondrial complex activity was expressed as nmol/min/mg protein.

A mitochondrial respiratory chain complex I activity assay kit (BC0515, Solarbio, Beijing, China) was used to measure mitochondrial OXPHOS complex I enzyme activity. Complex II, III, IV and V activities were measured by assay kits (BC3230, BC3240, BC940, BC1440, Solarbio, Beijing, China), respectively. Absorbance values were determined using a SpectraMax iD5 multifunctional microplate reader (Molecular Devices, USA). All experiments were repeated three times.