Rotenone

We used the CCK-8 assay (Dojindo Laboratories, Tokyo, Japan) to identify the lowest concentration of rotenone that would inhibit cellular survival. Primary neurons were seeded at a concentration of 1 × 10⁶ cells per well in 96-well cell culture plates at 37°C for 7 days. rotenone (Solarbio, Beijing, China) was prepared in 1 mM dimethyl sulfoxide and diluted in the culture medium at concentrations of 0.5, 1, 3, and 5 nM. Cell survival was assessed at 48 hours by adding CCK-8 solution (10 μL per well) directly to the cell suspension and incubating for 2 hours at 37°C. Absorbance values of all wells were measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). The results are expressed as a percentage of values in control cells not exposed to rotenone.

Primary neuronal cells were seeded in 96-well cell culture plates (1 × 10⁶ cells per well) at 37°C for 7 days. Cells were incubated with various concentrations of rotenone (0.5, 1, 3, and 5 nM; Solarbio, Beijing, China) for 2 days, to identify the lowest concentration at which cellular survival was inhibited. CCK-8 solution (10 μL per well; Dojindo Laboratories, Tokyo, Japan) was added directly to the cell suspension and followed by a 2-hour incubation at 37°C. Cell morphology was observed under a light microscope (Olympus, Tokyo, Japan). Optical density was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).