Jc 1 dye

Mcf7 cells were plated at the concentration of 20,000 cells/well in 48 wells plate and treated with 5 μmol/L fluoxetine, ebastine, fluspirilene, nefazodone penfluridol, pimozide, spiperone. DMSO 0.05% was used as negative control. After treatment, cells were stained with 10 μg/ml JC-1 dye (Adipogen) in PBS for 30 min in the dark at 37°C. FCCP (Cayman chemicals) was added for 15 min after the staining as positive control. Signals were acquired with a fluorescence microscope (FLoid Cell Imaging Station, Life Technology) and images were analyzed by ImageJ software calculating red/green fluorescence ratio.

To evaluate mitochondrial membrane depolarization, transduced CRC-SC#1 cells were plated at a concentration of 30,000 cells/well in a 48-well plate pre-coated with Geltrex (Thermo Fisher Scientific, Waltham, MA, USA). Cells were stained with 10 μg/ml JC-1 dye (Adipogen, San Diego, CA, USA) in PBS for 30 min in the dark at 37 °C. FCCP (Cayman Chemicals, Ann Arbor, MI, USA) was added for 15 min at the end of the staining as a positive control. Signals were acquired with a fluorescence microscope (FLoid Cell Imaging Station, Life Technology) and images were analyzed by ImageJ software to determine the red/green fluorescence ratio. Mitochondrial morphology was assessed through Mitotracker CMX-Red (Invitrogen, Waltham, MA, USA). CRC-SC#1 cells were seeded at a concentration of 100,000 cells/well in a 24-well plate pre-coated with Geltrex. Subsequently, cells were stained with 10 nmol/L Mitotracker for 30 min at 37 °C. Fluorescence was visualized by DM5500B fluorescence microscope (Leica, Wetzlar, Germany), and analyzed using ImageJ software v 1.52a (Ashland, OR, USA).

HCT116 cells were plated at a concentration of 30,000 cells/well in a 48-well plate and treated with 5 or 10 μmol/L spiperone for the desired time; 0.10% DMSO was used as a negative control. After treatment, cells were stained with 10 μg/mL JC-1 dye (AdipoGen, San Diego, CA, USA) in PBS for 30 min in the dark at 37 °C. FCCP (Cayman chemicals, Ann Arbor, MI, USA) was added for 15 min after the staining as a positive control. Signals were acquired with a fluorescence microscope (FLoid Cell Imaging Station, Life Technology, Carlsbad, CA, USA) and images were analyzed using ImageJ software v 1.52a, calculating the red/green fluorescence ratio. For mitochondrial membrane depolarization rescue experiments, HCT116 cells were pretreated with BAPTA-AM at 10 μmol/L and the MCU inhibitor Ru360 at 10 μmol/L for 30 min, and then treated with spiperone 10 μmol/L for 3 h.