T175 cell culture flasks

Human HCT-116 WT (CRC cells) and human MRC-5 (fibroblasts) were from the European Collection of Cell Cultures (Salisbury, UK). Human p53-deficient HCT-116 p53^-/- (CRC cells) were a generous gift from Prof. Dr. Ajay Goel (CA, USA). Furthermore, human Jurkat (T-lymphocytes) were bought at the Leibniz Institute (Braunschweig, Germany). HCT-116 WT, HCT-116 p53^-/-, and MRC-5 were grown adherently, while Jurkat cells were grown non-adherently in T175 cell culture flasks from Thermo Fisher Scientific (Schwerte, Germany). All cells were cultivated at 5% CO2, at 37°C and in cell culture medium from Sigma-Aldrich (Taufkirchen, Germany): DMEM F-12 (1:1), supplemented with 10% fetal bovine serum (FBS), 1,2% penicillin/streptomycin, 1% L-glutamine, 1% ascorbic acid, 1% essential amino acids and 0,5% amphotericin B as used before (19 (link)).

Mitochondrial pellets were isolated from T₁₇₅ cell culture flasks or 15-cm Petri dishes (ThermoFisher, Madrid, Spain). Pre-cast NativePAGE^TM 3–12% Bis-Tris gels (ThermoFisher, UK) were loaded with 60–80 μg of mitochondrial protein and processed for blue native electrophoresis (BN-PAGE) and in-gel activity (IGA) assays as described before [48 (link)]. After electrophoresis, proteins were transferred to nitrocellulose membranes at 40 V overnight and probed with antibodies.