Mitophagy Induction in Cell Lines

WT and PINK1 KO HEK293E cells were previously described [13 (link)] and were cultured in Dulbecco’s modified Eagle medium (Thermo Fisher, 11965118) supplemented with 10% fetal bovine serum (Neuromics, FBS001800112). Primary human dermal fibroblasts were collected from a homozygous PINK1^Q456X carrier and a healthy relative to serve as negative control [33 (link)]. All fibroblasts were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% PenStrep (Thermo Fisher, 15140122), and 1% non-essential amino acids (Thermo Fisher, 11140050). WT and pink1 KO MEF cells have been described previously [28 (link)]. MEFs were grown in Dulbecco’s modified Eagle medium supplemented with 20% fetal bovine serum, sodium pyruvate (Fisher Scientific, 11360–070), non-essential amino acids, and PenStrep. All cells were grown at 37°C, 5% CO₂:air in humidified atmosphere. To induce mitophagy, HEK293E cells, MEFs, and human dermal fibroblasts were treated with 20 μM CCCP (Sigma-Aldrich, C2759), 5 μM valinomycin (Cayman Chemicals, 10009152), or 2 μM valinomycin, respectively, for 24 hours. DMSO (Sigma-Aldrich, D4540) was used as vehicle treatment.