C59 Cytotoxicity Assay and Wnt Secretion Blockade

SH-SY5Y cells were seeded onto 96-well plates and treated with 1, 10, 100, or 200 μM C59. After 24 h in culture, cytotoxicity was evaluated using an MTT assay. Briefly, the cells were treated with different concentrations of C59 for 24 h. At the end of treatment, the cells were washed with 1× PBS, and 100 μl of fresh 1× PBS was added to each well. Then, 10 μl of thiazolyl blue tetrazolium bromide (MTT, 0.45 mg/ml) was added to each well and incubated for 3 h at 37°C. After the incubation with MTT, DMSO was added as a solubilization solution to dissolve the formazan crystals. The absorbance was measured in an iMark microplate reader (Bio-Rad, Hercules, CA, United States) at 570 nm.

Once we established the non-cytotoxic concentrations of C59, we evaluated the inhibition of Wnt ligand secretion. To do this, we used the trichloroacetic acid (TCA) protein precipitation method to assess Wnt3a levels in the extracellular medium. Briefly, after cells were treated for 24 h with 1 and 10 μM C59, the culture medium was replaced, and 100% TCA solution was added in a 1:4 (TCA:sample) ratio. The samples were incubated at 4°C for 10 min and centrifuged at 14,000 rpm for 5 min. The resulting pellet was washed twice in cold acetone and dried at 95°C for 5 min. Total protein was loaded and resolved by SDS-PAGE using a 10% polyacrylamide gel, and the separated proteins were transferred to a PVDF membrane. The membrane was incubated with a rabbit anti-Wnt3a antibody (overnight, 4°C, 1:1000, ab28472, Abcam, Cambridge, MA, United States) and an HRP-conjugated secondary antibody (1 h, room temperature, 1:5000, cat. no. 31460, Thermo Scientific, Waltham, MA, United States), and Clarity Western ECL Substrate (Bio-Rad) was used for the chemiluminescence reaction. Chemiluminescence was detected using a ChemiDoc-It 515 Imager (UVP, Upland, CA, United States).