Prolong gold antifade mountant with dapi nuclear stain

Cells were grown on 4 well glass slides (Millipore) in normal growth medium for 24 h. After fixation the cells were, permeabilized using the fixation/permeabilization kit (BD Biosciences catalogue 554714) and incubated with primary mouse anti‐human LAMP2 (Abcam) or EEA‐1 (BD Biosciences) monoclonal antibodies and an Alexa Fluor 488 conjugated goat anti‐mouse IgG (H + L) antibody. The slides were mounted using ProLong Gold antifade Mountant with DAPI nuclear stain (Thermo Fisher Scientific). The slides were examined using a Leica Sp5 confocal microscope and serial 0.3−0.5 µm Z‐stacks images per field (5−7 cells) were acquired. To analyse acidic lysosomes in situ, the cells were incubated with LysoTracker red DND‐99 (Thermo Fisher Scientific) and examined by confocal imaging. Images were analyzed using ImageJ software.

Total protein lysates from OSCC cells were harvested after 24 h of CXB treatment (0, 25, 50 and 100 µM) using a RIPA lysis buffer (Cell Signaling Technology). Briefly, the samples were examined using anti-PCNA (GeneTex) normalized by β-actin (Millipore) in an immunoblotting analysis. In an immunocytochemistry and immunofluorescence (ICC-IF) assay, OSCC cells were seeded on glass slides coated with poly-L-lysine (Sigma-Aldrich) prior to 24 h treatments (0 and 100 µM). Cells were then fixed with 4% paraformaldehyde (Merk) and blocked with 10% normal goat serum containing 0.3% of Triton X-100, followed hybridization of anti-Ki-67 (Cell Signaling Technology) and a secondary fluorescent antibody conjugated with Alexa Fluor®488 (Thermo Fisher Scientific). The ProLong Gold Antifade Mountant with DAPI nuclear stain (Thermo Fisher Scientific) was applied to the slides prior to visualization under a fluorescence microscope (DM4000 M, Leica Microsystems).

Cells were grown on four-well glass slide (Millipore) in normal growth medium for 24 h. Then the cells were fixed, permeabilized using the fixation/permeabilization kit (BD Biosciences catalogue 554 714) and incubated with primary mouse anti-human LAMP1 (BD Biosciences), LAMP2 (Abcam) or EEA-1 (BD Biosciences) monoclonal antibodies and an Alexa Fluor 488 conjugated goat anti-mouse IgG (H + L) antibody. The slides were mounted using ProLong Gold antifade Mountant with DAPI nuclear stain (ThermoFisher Scientific). The slides were observed on a Leica Sp5 confocal microscope, and ≥10 images each containing ≥3 cells were acquired. Images from three independent experiments were analysed using ImageJ software. The fluorescence intensity per cell was quantified in images of maximum intensity Z-projections. The cellular area, the integrated density and the mean grey values were analysed. Measurements of regions without fluorescence were used for background subtraction. The net average fluorescence intensity per pixel, expressed as corrected total cell fluorescence (CTCF), was calculated for each cell. In addition, the cells were stained with LysoSensor Blue DND-167 1 μM or LysoTracker Red DND-99100 nM (Molecular Probes, ThermoFisher Scientific) for 1 h and observed on a Leica SPE confocal microscope.