Cell viability assay

Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. C6 (2×10⁴ cells/well) and U251 (1×10⁴ cells/well) cells were seeded into a 96-well plate respectively overnight in 5% CO₂ at 37℃. And the cells were pretreated with CQ (15 μM) for 1 h before being exposed to MOX. Next, Cells were incubated with 5 mg/mL MTT reagent for another 4 h at 37°C. After the medium was carefully removed, 150 μL of DMSO was added and agitated to dissolve the formazan crystals. The absorbance was recorded at 490 nm on an enzyme-linked immunosorbent assay reader (HUADONG-Y, China).

The long-term effects of MOX on tumor cell proliferation were analyzed with a colony formation assay. U251 and C6 cells were seeded into a 6-well plate. Next the cells were treated with CQ (15 μM) for 1 h before being exposed to MOX and cultured in medium for 13 days. The medium was refreshed every three days. Thereafter, the cells were washed with PBS three times, fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Sigma, USA). Quantification of colony formation was also performed using ImageJ software.