**2.6. Mitochondrial membrane potential (ΔΨm) assay**

hESC-derived neurons cultured on glass coverslips were incubated with 50 nM tetramethylrhodamine ethyl ester (TMRE) (Thermo Fisher Scientific) for 20 min at room temperature. TMRE fluorescent intensity representing ΔΨm was recorded using the confocal microscope (Nikon). Images were obtained from six random fields per coverslip. The data were analyzed using ImageJ software.

#### **2.7. Analysis of release of cytochrome c from mitochondria into cytosol**

NSCs were cultured in neuronal differentiation medium. Ten days later, the differentiated neurons were transduced with the virus CellLight™ mitochondria-green fluorescence protein (GFP) (Thermo Fisher Scientific) for 24 h to label mitochondria. This fluorescent proteinbased reagent contains the leader sequence of E1-α pyruvate dehydrogenase fused to emerald GFP. The transduced neurons expressed GFP within mitochondria. Four days later, the labeled neurons were used for analysis of the effect of ketamine on the cytochrome c translocation. Virus transduction efficiency was calculated as the ratio between GFP-positive cells and total cells. GFP expression in mitochondria was confirmed by the colocalization of GFP and TMRE fluorescence signals within cells. The distribution of cytochrome c in the neurons was analyzed using immunofluorescence staining with antibody against cytochrome c (Abcam).
