Lysotrackertm red dnd 99

PC3 cells (3 × 10⁵) were seeded per 60 mm plate on glass coverslips and treated for 4 h with DMSO as a negative control, 0.5 μM lapatinib, 10 μM siramesine or a combination of siramesine and lapatinib in 2 mL DMEM F12 (5% FBS, 1% penicillin-streptomycin) media. Cells were stained for 30 min at 37 °C in the dark with 100 nM LysoTracker^TM Red DND-99 (Invitrogen) in 2 mL media, washed with PBS, and fixed for 20 min with 1 mL paraformaldehyde at room temperature. Cells were mounted with 10 μL mounting media containing DAPI. Images were obtained by confocal microscopy.

The intracellular localization and co-localization of the formulations with lysosomes were evaluated by confocal microscopy. Cells were seeded at 5 × 10⁵ cells per ml onto Poly-D-Lysine coated coverslips in a 24-well plate and treated with FAM-labelled siRNA contatining formulations. The incubation periods varied from 6, 12 to 24 h. LysoTracker^TM Red DND-99 (Invitrogen, Invitrogen, Waltham, MA, USA), a red-fluorescent dye for labelling and tracking acidic organelles, was prepared at 100 nM in serum-free media. The Syto^TM Deep Red Nucleic Acid Stain (Invitrogen, Invitrogen, Waltham, MA, USA), which stains the nuclei of cells, was added to the Lysotracker working solution to give a final nucleic acid stain of 1 µM. After incubation, the media was removed carefully, and the dyes were added to each well (500 µL dye solution/well); after 150 min the dye solution was withdrawn. The cells were fixed with 4% paraformaldehyde for 10 min at room temperature followed by wash with PBS. The coverslips were removed and mounted with Fluorescent Mounting Media (Dako, Agilent, Santa Clara, CA, USA); the cells were imaged using an OLYMPUS FV1000 Confocal Laser Scanning Biological Microscope.

The cells were seeded and were treated with erastin or other agents with or without 30 nM Wortmannin (W1628; Sigma-Aldrich). All cells were stained with LysotrackerTM Red DND-99 (L7528; Thermo Fisher Scientific) to assess the later process of autophagy. Co-localization of LC3-GFP-puncta and lysosome was confirmed using the ZEISS LSM 880 confocal microscope. Also, autophagy-related molecules were confirmed by immunoblotting.

HCT116 and RPE1 cells and their aneuploid derivatives were seeded to a 96-well plate (HCT116: 10⁴ cells per well, RPE1: 10³ cells per well) and incubated at 37 °C and 5% CO₂ on the day before experiments. For cell fixation, a 3% formaldehyde solution was used for 15 min at room temperature. Next, cells were permeabilized with 0.1% Triton for 20 min at room temperature. Then, cells were preblocked with 3% bovine serum for 30 min at room temperature. Primary antibodies were diluted in blocking solution (Supplementary table 2) and incubated overnight at 4 °C. The next day, cells were washed and secondary antibodies were added in a concentration of 1 mg per ml followed by incubation for 1 h in the dark at room temperature. SYTOX® green (Invitrogen) or DAPI (Invitrogen) were used to localize the nucleus. For cytoplasmic staining, the HCS Cell Mask (H327 12 Component, 701618, Invitrogen) was applied. The cells were covered with SlowFade Gold Antifade (Invitrogen). To localize mitochondria, MitoTracker^TM Red CMXRos (M7512, Invitrogen) was used (100 nM for 45-h incubation at 37 °C). For lysosome localization, we also used LysoTracker^TM Red DND-99 (L7528, Thermofisher Scientific) according to the manufacturer’s protocol. Information regarding the used antibodies can be found in Supplementary table 2.

Cells were cultured in 12-well plates and treated with the corresponding reagents for the designed times, respectively. Thereafter, cells were stained with LysoTracker^TM Red DND-99 (L7528; Thermo Fisher Scientific), and FA-induced changes of autolysosomes activity were detected by flow cytometry [29 (link)]. In addition, hepatocytes of yellow catfish were co-stained with Hoechst (blue) and Lyso Tracker (red). The intensity of fluorescence was visualized by a laser-scanning confocal microscope (Leica Microsystems, Wetzlar, Germany), and quantified on a CytoFLEX Flow Cytometer (Beckman Coulter, WA, USA) and data analysis was performed using FlowJo VX software (Beckman Coulter, WA, USA).