Acridine orange

To detect the autophagy and apoptosis cells, H441, H1975, and CL152 cells were plated in 6-cm dishes at a density of 300,000 cells per dish; tested drugs were added the day after seeding. Acridine orange staining was used to detect the autophagic cells, and lung cancer cells were stained with 1 μg/mL Acridine orange (Sigma) for 15 min, trypsinized, and resuspended in cold PBS before detection. A FITC Annexin V apoptosis detection kit (BD Pharmingen, San Jose, CA, USA) was used to detect the apoptotic cells, and the experimental steps were followed cautiously, according to the protocol described. Unstained cells and cells stained with Annexin V or PI were used to gate the population of early and late apoptotic cells, respectively.

The following methods were used to assess drug-induced autophagic cell death: (1) Western blot analysis of microtubule-associated protein 1 light chain 3 (LC3 II) as described previously [2 (link), 44 (link)]; and (2) electron microscopy. Samples were fixed with 2.5% glutaraldehyde solution buffer in PBS at 4°C for 1 h, postfixed in 1% osmium tetroxide solution at 4°C for 3 h, dehydrated in graded concentrations of ethanol and embedded in LR white resin (Sigma-Aldrich, 62661). Ultrathin sections (70 nm) were examined with a JEOL JEM-1400EX electron microscope at 120 Kv. Representative areas were recorded at 20,000, 40,000 magnification; (3) Immunofluorescence of acridine orange (Sigma-Aldrich, A6014). HCC cells were grown on coverslips. After being washed with PBS, cells were treated with 2 μM SC-2001 or SC-2001+3-MA for 48 h, fixed with ice-cold 4% paraformaldehyde (Sigma-Aldrich, P6148) for 30 minutes at room temperature then stained with acridine orange (5 μg/ml) for 5 minutes at room temperature. Cells were examined under a Leica DM2500 fluorescence microscope.

Caco-2 monolayers were gently rinsed with ice-cold PBS and incubated with 0.2% (v/v) trypan blue solution (Sigma) for 15 minutes at 4°C, or with 10 μg/ml acridine orange (Sigma) for 15 minutes at 37°C. After rinsing with ice-cold PBS, monolayers were detached from the fibrillar collagen pre-coated inserts. For acridine orange staining, monolayers were mounted with Fluoromount (Sigma) and red fluorescence was imaged under a fluorescence microscope with an excitation wavelength of 550 nm. For Trypan blue stainings, the monolayers were mounted with PBS on slides for optical microscope imaging. Trypan blue positive cells in a monolayer were quantified by summarizing the blue area of four fixed independent sections imaged under 100x magnification. The results of three monolayers or more were averaged in a single experiment for each condition. Caspase-dependent DNA fragmentation of Caco-2 monolayers was measured using the Cell death detection ELISA (Roche) based on nucleosome immunoreactivity according to the manufacturer’s instructions.

Acridine Orange (AO): Acridine Orange (Sigma Aldrich), a weak base that accumulates in acidic organelles, was used to label the lysosomes. After treatments, cells were incubated with 5 µM AO for 10 min at 37˚C and washed twice with PBS. Red fluorescence (corresponding to acidic vesicle staining) was measured using a Varioskan Flash Multimode Microplate Reader with excitation/emission at 480/630 nm. Representative pictures were taken with an epifluorescence microscope (DM600 microscope, Leica).
Lysosensor: Lysosomal acidification was measured in cells loaded with 2µM LysoSensor yellow/blue DND-160 (Invitrogen) for 15 min at 37˚C. The LysoSensor dye is a ratiometric probe that produces yellow fluorescence in acidic environments but changes to blue fluorescence in more neutral environments. pH calibration was performed following the protocol previously established by Diwu et al 45 (link). Briefly, cells were incubated in MES buffer (5 mM NaCl, 115 mM KCl, 1.3 mM MgSO₄, 25 mM MES) supplemented with 10 µM nigericin and 10 µM monensin, for 10 min, with pH adjusted within 3 to 7. The samples were read in a Varioskan Flash Multimode Microplate Reader with excitation at 360 nm. The emission 440/540 nm ratio was then calculated for each sample.

For the rhodamine-phalloidin staining, embryos fixed in 4% PFA/PBS were permeated through incubation with PBTX 2% O/N at 4°C. Blocking was done with 1% DMSO, 1% BSA in PBS for 30 minutes at room temperature. Embryos were stained through incubation with phalloidin-TRITC (P1951, Sigma-Aldrich) 1:1000 in PBTX 1%. For acridine orange staining, alive embryos were incubated with 15 μg/ml acridine orange (A6014, Sigma-Aldrich) for 30 minutes at 28,5°C, washed twice in fish water and examined immediately after.

Cells grown on cover slips were treated as described. 24 h following initial treatment, cells were incubated with medium containing 2 μg/mL acridine orange (Sigma-Aldrich) for 20 min. Subsequently, medium containing acridine orange was removed and cells were washed twice with PBS and fixed using 4 % PFA (Merck) for 15 min at room temperature. Pictures were obtained using a Zeiss LSM 780 confocal laser scanning microscope with ZEN microscopy software at 63x magnification.