α cytochrome c

Mitochondrial membrane potentials of dAdo- or dGuo-exposed (160 μM; 24 h) U937-derived macrophages were analyzed by using the Orange Mitochondrial Membrane Potential Assay Kit (Abcam) according to the manufacturer’s instructions. Mitochondria of live (non-apoptotic) cells accumulate the MitoOrange Dye in this approach while the fluorescence intensity decreases following the collapse of the mitochondrial membrane potential in apoptotic cells. Stained cells were examined via flow cytometry and the FlowJo software (BD Life Sciences) according to standard laboratory protocols. For the detection of cytochrome c in deoxyribonucleoside-exposed cells, cell lysates were generated as described above and fractionated using the Cell Fractionation Kit (Abcam). Mitochondrial and cytosolic fractions were subjected to immunoblotting as described before by using the following rabbit primary antibodies: α-Cytochrome c (α-Cytochrome c, 11940, Cell Signaling) and α-GAPDH (α-GAPDH, ab181602, Abcam; loading control). Immuno-reactive signals were revealed with a secondary antibody conjugated to horseradish peroxidase as indicated before.

Antibodies used in this work include: α-QIL1 (Sigma SAB1102836), α-MINOS1 (Aviva, San Diego, USA, ARP44801-P050), α-CHCHD3 (Aviva ARP57040-P050), α-CHCHD6 (Proteintech, Chicago, USA, 20639-1-AP), α-IMMT (Abcam, Cambridge, UK, ab110329), α-TIMM23 (BD-Biosciences, Franklin Lakes, USA, 611222), α-SAMM50 (Proteintech 20824-1-AP), α-APOOL (Aviva OAAF03292), α-PCNA (Santa Cruz, Dallas, USA, sc-56), α-HSP90 (Epitomics, Burlingame, USA, 3363-1), α-TOMM70A (Epitomics T1677),α-Flag (Sigma SLB6631), α-HA (Covance, Princeton, USA, D13CF00834), α-ATP5A (Abcam ab14748), α-DLD (Santa Cruz sc-271569), α-TMEM11 (Proteintech 16564-1-AP), α-Cytochrome C (Cell Signaling, Danvers, USA, 4272S), α-TOMM20 (Santa Cruz sc-11415), Phalloidin (Invitrogen A22287), and α-APOO (Novus Bio, Littleton, USA, NBP1-28870).