Cytotoxicity assay

The cytotoxicity of harmaline and harmine was measured by CCK-8 assay. In brief, the cells were seeded on 96-well plates at 1 × 10⁵ cells/mL and cultured in 100 μL of culture medium at incubator for 24 h. The medium in each well was then replaced with 100 μL of medium containing harmaline or harmine at the following concentrations: 0.5, 1, 2.5, 5, 25, and 100 μM. After 12 h incubation, 10 μL of CCK-8 dye was added to each well, and cells were incubated for another 2 h. Cell viability (%) was calculated using the following Equation (1):

OD_sample refers to the absorbance of a well with treated cells and CCK-8. OD_blank refers to the absorbance of a well with medium and CCK-8 but without cells. OD_control refers to the absorbance of a well with untreated cells and CCK-8. The absorbance at 450 nm was measured by a microplate reader (Biotek Instrument; Gene Co., Ltd., VT, USA), and the results are presented as mean ± SD from triplicate wells.