Tbh70x

The MTT assay to evaluate CuET toxicity under oxidative conditions in cancer cells was modified from previous protocols^{63 (link)}. Briefly, cells were plated on 96-well tissue culture plates (Fisher, FB012931) at 5000 cell per well and grown at 5% CO₂ at 37 °C. The next day, oxidative stress was achieved by treating the cells with growth medium containing 0, 25, or 50 μM TBH70X (Sigma, 458139). After 6 h, the TBH70X-containing medium was removed and replaced with medium containing 0, 0.1 or 0.25 μM CuET. The DMSO control contained 100 μL culture medium with 0.05 μL DMSO (Fisher, BP231100). After 18 h of CuET treatment, 20 μL of 5 mg/mL MTT (Sigma, M2128) was added to each well and the cells were further incubated for 3.5 h in 37 °C incubator. To dissolve the formazan crystals, the culture medium was carefully removed and 150 μL of MTT solvent (4 mM HCl, 0.1% NP-40 in isopropanol) was added to each well with 15 min shaking at room temperature. The absorbance was measured at 590 nm with a reference wavelength of 620 nm with a SAFIRE II plate reader (Tecan, Männedorf, Switzerland). Each experimental group contained four biological replicates with the error bar indicating standard deviation. The cell survival rate was calculated by the following equation: cell survival rate (%) = (absorbance of experimental group/absorbance of control group) × 100%.

FCCP, oligomycin, rotenone, antimycin A, TBH70X, tert-Butyl hydroperoxide solution (Luperox), taxol, and Poly-l-Lysine (mol wt 70,000–150,000, 0.01%) were obtained from Sigma. Hoechst 33342, Mitotracker DeepRed, MitoSOX, and SYTOX Green were from ThermoFisher Scientific. TMRE was from Abcam. All compounds were stored at −20 °C except taxol and Hoechst (4 °C). Dyes were stored protected from light. FCCP, rotenone, antimycin A, Hoechst, MitoTracker DeepRed, and SYTOX were stored at −20 °C, TMRE was pre-diluted at 10 μM in media (10×) and aliquoted for single use. MitoSOX was likewise aliquoted for single use³⁰ .