Genotoxicity Evaluation of Test Compounds

To assess if the test compounds induce some level of genotoxicity, cells were seeded as previously described and treated with 10 μM test compounds. Since γ-H2AX signal can decrease over time due to DNA repair, this assay captured SCQ-induced DNA damage after a short treatment period of 4 h. Celastrol was used as a positive control [87 (link)]. After fixation, permeabilization and blocking, cells were exposed to mouse monoclonal anti-phospho-Histone H₂AX antibody (1:1000 in blocking buffer, 15 µL, overnight). After exposure to goat anti-mouse Alexa Fluor 488 secondary antibody (1:10,000, 15 µL, 1 h), cells were stained using DAPI and stored in PBS (Figure S2), images were aquired using an INCell 2200 analyzer (10× magnification) and analyzed using IN Carta image analysis software as described above (GE Healthcare, Rydalmere, NSW, Australia). The number of γ-H₂AX-positive cells was automatically quantified for all acquired images. Percentage γ-H₂AX-positive cells was expressed as mean ± SD of at least quadruplicates from one assay. At least 500 cells were analyzed for each treatment.