*5.8. Flow Cytometry Analysis*

PC3 and 22Rv1 cells (1 × 10<sup>5</sup> per line) were treated with 50 μM NCL1 for 48 h, with or without 50 μM CQ, and cell suspensions were then prepared and stained with Guava® ViaCount reagen<sup>t</sup> and propidium iodide according to a Guava® Assay protocol (Guava Technologies, Hayward, CA, USA). CytoSoft Software was used to analyze apoptosis and cell cycle phase distributions on a Guava® PCA Instrument.

### *5.9. Lysosome Localization and Activity Using LysoTracker® and LysoSensor™ Dyes*

22Rv1 cells (3 × 104) were seeded in 8-well chamber slides with RPMI medium and 5% FBS. Cells were incubated for 48 h and treated with 50 μM NCL1 or 50 μM CQ, or a combination of 50 μM NCL1 and 50 μM CQ. Control cells were treated with the same amount of solvent (DMSO and distilled water). We removed the medium from the dish after 48 h of treatment, and add the prewarmed (37 ◦C) probe-containing medium. The cells were then incubated for 60 minutes. Lastly, we replaced the loading solution with fresh medium and observed the cells using an IN Cell Analyzer 6000 (GE Healthcare, Chicago, IL, USA).

### *5.10. Transmission Electron Microscopy (TEM)*

22Rv1 cells were seeded in 6-well plates (1 × 10<sup>5</sup> cells/well) in DMEM containing 10% FBS. After an overnight incubation, cells were treated with or without 50 μM NCL1, and with or without 50 μM CQ for 3 or 72 h. Glutaraldehyde (2.5%) was used to pre-fix cells in 0.1 M phosphate buffer (pH 7.4) at 4 ◦C. Specimens were then post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 45 min. A graded series of ethanol was used to dehydrate specimens, which were subsequently embedded in epoxy resin. An Ultracut-S ultramicrotome (LEICA, Wetzlar, Germany) and a diamond knife were used to cut ultra-thin sections, which were then stained with 2% uranyl acetate in distilled water for 15 min and a lead staining solution for 5 min. A JEM-1011J (JEOL, Tokyo, Japan) electron microscope at 80 KV was used to observe sections.

### *5.11. Ex Vivo Studies Using a Subcutaneous Castration-Resistant PCai1 Model*

Six-week-old male KSN/nu-nu nude mice from Nippon SLC (Hamamatsu, Japan) were maintained as previously described [48,49]. PCai1 cells cultured in T-75 flasks were grown to confluence, trypsinized, and counted. PCai1 cells (1 × 10<sup>6</sup> in 100 μL serum-free DMEM) were subcutaneously injected into the dorsal side of each mouse under isoflurane anesthesia. After 1, 3, and 4 weeks, mice (*n* = 5) were castrated, while other mice (*n* = 5) were left uncastrated as negative controls. Five weeks after implantation, all mice were sacrificed, and the LSD1 expression of PCai1 tumors was analyzed. For the next experiment, all nude mice were castrated, and 1 × 10<sup>6</sup> PCai1 cells resuspended in 100 μL serum-free DMEM were subcutaneously implanted as described above. Ten days later, an intraperitoneal injection of DMSO as a vehicle that was equal in concentration to that used for 1.0 mg/kg NCL1 (*n* = 10), or 1.0 mg/kg (*n* = 10) NCL1 was performed twice per week. Tumor size (determined by caliper measurement) and body weight were measured twice per week. Mice were sacrificed 5 weeks after the implantation of cells.

All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Nagoya City University Graduate School of Medical Sciences; the approval number was H24M-58.
