Lysotracker deep red dye

The Lysotracker Deep Red dye (Molecular Probes, L12492) and the fluorescent DQ-Red-BSA^{36 (link)} dye (Molecular Probes, D12051) were used to quantify lysosome morphology and proteolysis, following the manufacturer’s instructions.

To investigate lysosome morphology, we utilized the Lysotracker Deep Red dye (Molecular Probes, L12492) following the manufacturer’s instructions, as in ref. ^{38 (link)}. To study the proteolytic activity of lysosomes, the fluorescent DQ-Red-BSA dye (Molecular Probes, D12051) was used following manufacturer’s instructions, as in ref. ^{38 (link)}. To investigate autophagic vesicles we used the CYTO-ID® Autophagy Detection Kit in live cells, which uses a novel dye that selectively labels accumulated autophagic vacuoles. The assay was carried out following manufacturer’s instructions. Briefly, 2 μl of CYTO-ID® Green Detection Reagent were diluted in the assay buffer and then incubated for 30 min in the incubator. Imaging was carried out in complete media. In Lysotracker and Cyto-ID experiments, the number of vesicles per cell was obtained by using Cell Profiler software to assess the number of fluorescent puncta and divide it by the number of nuclei (representing the number of cells) in the same image field.

PC3, DU145 and LNCaP cells were seeded in 12-well plates at a concentration of 1.0 × 10⁵ cells/mL and allowed to grow for 42 h before treatment. Depending on the solvent used to dilute drugs, DMSO or water was added as vehicle control. After 4 h treatment the media was removed from cells and Lysotracker Deep Red dye (50 nM, Invitrogen, Carlsbad, CA, USA) was diluted in fresh media and added to cells for 15 min at 37 °C in the dark. Cells were collected, together with the media, and resuspended in PBS and analyzed using the Novocyte flow cytometer (Acea Biosciences). An amount of 20,000 events were collected from each sample and gated using the PER-CP channel. Data was analyzed using CellQuest software.