Assessing PINK1 and SOD regulation

Human embryonic kidney cells (HEK293T, ATCC^®) and human adenocarcinoma cells (HeLa, ATCC^®) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher) supplemented with 10% fetal bovine serum (Thermo Fisher) at 37°C and 5% CO₂. To assess PINK1 protein levels, HeLa cells were treated with 4 µM MG132 (Sigma-Aldrich) and with 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma-Aldrich) for 24 h and 4 h, respectively, at 37°C before cell lysis. Human neuroblastoma SH-SY5Y cells (IST, Genova, Italy) were cultured in a 1:1 mixture of Ham's F12 and Dulbecco Modified Eagle Medium (Life Technologies) supplemented with 10% fetal bovine serum, in a 5% CO₂ humidified incubator at 37°C. Previously described SOD1 and SOD2 stably overexpressing SH-SY5Y cells were used (17 (link)). When required, SH-SY5Y cells were treated with 10 µM SOD mimetic drug M40403 for 24 h at 37°C.