MTT assay

To study the effect of GSK-J4 on neuroprotection, MTT assays were performed to assess cell viability as previously described⁴⁹. In brief, SH-SY5Y cells were cultured in the 96-well plates at a density of 1 × 10⁵ cells per well at 37 °C. Two days later, cells were placed in a fresh medium with different treatments. After a particular treatment, cell viability was assessed by the MTT assay (11465007001 ROCHE, Cell Proliferation Kit I (MTT)). First, the supernatant in each culture well was removed and the wells were washed with PBS twice. 20 μl MTT solution (5 mg/ml in PBS) was added to each well (containing 100 μl culture medium). After incubating for 4 h, the medium was removed carefully. Then, 150 μl DMSO was added to each well and shaken for 10 min to allow the crystals to be fully melted. Finally, the absorbance intensity was measured at 490 nm with a microplate reader (Bio-RAD 680, USA) and together with a reference wavelength of 620 nm. Control wells (cells with the same concentration of drug in medium) were set at the same time. The cell viability (%) was expressed as a percentage relative to the control group.