20–50 μg of the protein extracts were separated in 10% Tris–glycine SDS-PAGE (Invitrogen). For LC3B, 4–20% Tris–glycine SDS-gel was used. The following primary antibodies were used: PCNA (sc-56, Santa Cruz), lamin (sc-6215, Santa Cruz), COX IV (AMB-7487, BioSite), VDAC-1 (sc-58649, Santa Cruz), Ku86 (sc-1485 Santa Cruz), DNA ligase III (611877, BD Transduction Laboratories), LC3B (NB600-1384, Novus Biologicals), cleaved PARP-1 antibody (1074-1, Epitomics), Polγ (ab128899, Abcam), TFAM (B01P, Abnova). TOPOIIα antibody was a home-made rabbit polyclonal antibody. The secondary antibodies, polyclonal rabbit-mouse IgG/HRP or peroxidase-labeled polyclonal swine-rabbit IgG were from Dako Cytomation.
For autophagy, the experiments were carried out as described previously [27 (link)]. In brief, 1 × 106 cells were seeded on 100 mm dishes. 24 h later, cells were treated with 5 μM rotenone. Cells were incubated for 24 h, harvested by trypsination and lysed in 0.15 ml lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM DTT and protease inhibitor cocktail, Roche) for 30 min on ice. The lysate was briefly sonicated three times at 5 W for 5 s on ice. The extracts were centrifuged at 16,000 × g for 10 min and the supernatant collected.
For autophagy, the experiments were carried out as described previously [27 (link)]. In brief, 1 × 106 cells were seeded on 100 mm dishes. 24 h later, cells were treated with 5 μM rotenone. Cells were incubated for 24 h, harvested by trypsination and lysed in 0.15 ml lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM DTT and protease inhibitor cocktail, Roche) for 30 min on ice. The lysate was briefly sonicated three times at 5 W for 5 s on ice. The extracts were centrifuged at 16,000 × g for 10 min and the supernatant collected.