Glass bottoms

U87MG and U251MG cells were seeded in cell culture dishes with glass bottoms (NEST Biotechnology, Jiangsu Province, China), then treated with or without asparaginase (1 IU/mL) for 48 h after complete attachment. Cells treated with autophagy inducer rapamycin (50 mM) for 2 h were served as positive controls. Subsequently, cells were stained with Cyto-ID^® Green dye and Hoechst 33342 for 15 min at 37°C and observed using a confocal microscope (Carl Zeiss LSM710, Carl Zeiss, Germany). All the steps were performed in the dark.

Cells were grown on 35-mm cell culture dishes with glass bottoms (NEST, Wuxi, China). Cells were fixed with 4% paraformaldehyde for 30 min. Fixed cells were washed and permeabilized with 0.1% Triton X-100 for 10 min. Cells were blocked with goat serum for 30 min and incubated with the anti-β-catenin antibody (1:100, 8480S, CST) overnight at 4 °C. After washing with PBS, the cells were subsequently incubated with Alexa Fluor^® 488-conjugated anti-rabbit IgG antibody (1:1000, ab150077, Abcam) for 1 h. The nuclei were stained with DAPI (62248, Thermo Scientific). Images were acquired using a Zeiss LSM710 confocal microscope (Zeiss, Germany) at 400× magnification.

To visualize changes in the transmembrane potential of mitochondria using fluorescence microscopy, cells were seeded into 24-well tissue culture plates with glass bottoms (Wuxi NEST Biotechnology) at an initial density of 10⁵ cells/well in 1 mL of the appropriate medium. Cells were allowed to attach and grow for 48 h prior to treatment. Then, culture media were replaced with the working solutions of tested compounds. At selected time points, tetramethylrhodamine methyl ester (TMRM; Sigma Aldrich, Poznań, Polska), a fluorescent cationic dye accumulating in mitochondria in a potential-dependent manner, was added to culture media (final concentration 100 nM), and plates were placed in the CO₂ incubator for 30 min. Stained cells were observed under the fluorescence microscope and photographed. For cytometric analysis, cells were seeded into tissue culture dishes and cultured for 48 h prior to treatment. After treatment for 3 h and 6 h, cells were incubated with TMRM, detached using trypsin/EDTA (ethylenediamine tetraacetic acid) solution, resuspended in PBS, and analyzed using a flow cytometer (FACS AriaII, Becton Dickinson, San Jose, CA, USA).