*4.3. Cell Treatments*

For CPZ treatment, a stock solution of 30 mM CPZ (C9012-25G, Sigma-Aldrich, St. Louis, MO, USA) was prepared freshly. For this, CPZ powder was dissolved in 50% ethanol/medium and shaken at 225 rpm at 60 ◦C for 15–20 min until its complete dissolution. Working solutions were prepared by diluting the stock in the specific medium for each cell type in a series of sequential solutions to reduce the ethanol concentration [72,73]. After 24–48 h of plating (30−40% cellular confluence), cellular toxicity was induced by the addition of CPZ in growing concentrations (50–1000 μM; see corresponding figure legends), for 24, 48, or 72 h. In order to prove that results are only attributable to CPZ not ethanol, the vehicle effect was also tested for each sequential solution in the cell models. As it can be observed in the graphs (Figure A3), we demonstrated that even the highest concentration of ethanol used to dissolve CPZ did not negatively affect, in a statistically significant way, cell viability of HOG and SH-SY5Y cells after 24 and 48 h of treatment.

To investigate the effect of the antipsychotic drug CLO (C6305-100G, Sigma-Aldrich, St. Louis, MO, USA) in the Apo D expression, cells were treated with different concentrations (0.1–5 nM; see corresponding figure legends) for 24 h, before fresh addition of CPZ. For the exogenous addition of Apo D, hApo D purified from BCF or hrApo D derived from human cells (P05090, Novoprotein, Summit, NJ, USA) were added (0.05–1000 nM; see corresponding figure legends) to cell cultures 24 h prior to CPZ.

For inhibition of endocytic mechanisms, SH-SY5Y cells were treated with different chemical inhibitors, cytochalasin D (8 μg/mL; C2618, Sigma-Aldrich, St. Louis, MO, USA), chlorpromazine hydrochloride (5 μg/mL; C8138, Sigma-Aldrich, St. Louis, MO, USA) and dynasore (80 μM; 324410, Sigma-Aldrich, St. Louis, MO, USA). Stock solutions were prepared in dimethyl sulfoxide (DMSO) and diluted in serum-free medium supplemented with 30 mM HEPES on the day of the experiment. The final DMSO concentration added was kept <0.1%. Cells were washed in serum-free medium and treated with the respective inhibitors (see corresponding figure legends) for 30 min before Apo D addition. H2O2 was used as positive control.

Drug concentrations and times of treatments were based on the bibliography and on our previous experience [75,76].

#### *4.4. MTT Assay*

Cell viability was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, a method based on the activity of mitochondrial NAD dependent oxidoreductases as indicator of the functional state of mitochondria. For this, 3000–5000 cells/well were seeded in 96-well plates and grown in 100μL/well of complete medium. Once treatments were completed, 10 μL of MTT (5mg/mL in phosphate buffered saline (PBS; 10010-023, Gibco, Invitrogen, Paisley, Scotland, UK)) (M5655; Sigma-Aldrich, St. Louis, MO, USA) were added to each well. Four hours later, 100 μL of lysis solution (20% sodium dodecyl sulfate (SDS); 50% dimethylformamide; pH 4) were added to the culture and incubated overnight at 37 ◦C. Absorbance at 570 nm was measured using a Multiskan EX Microplate Reader (ThermoFisher Scientific, Waltham, MA, USA). Values from blank wells, containing only medium, were subtracted from the values of the samples. Cell viability was expressed as the percentage of the controls.
