Map lc3β g 9

To prepare whole cell extracts from exponentially growing cultures, cells were lysed in RIPA lysis buffer [50 mM Tris, pH 8.0; 150 mM NaCl, 5 mM EDTA (pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (Pierce)]. Lysates were cleared by centrifugation at 12,000 rpm for 15 min and the supernatant was used for Western Blotting. Antibodies for immunoblotting were: TFAM [D5C8] (Cell Signaling), Drp1 [6Z-82] (SCBT), phospho-Drp1-Ser616 [D9A1] (Cell Signaling), cyclin B1 [GNS1] (SCBT), cyclin A [BF683] (SCBT), Ran [610340] (BD Biosciences), MAP LC3β [G-9] (SCBT), Mfn1 [D-10] (SCBT), GAPDH [GA1R] (Invitrogen), Opa1 [612606] (BD Biosciences), Nrf1 [147.1] (SCBT) and Nrf2 [A-10] (SCBT). Image quantification was performed in ImageJ (version 1.53a; freely available at http://imageJ.nih.gov/ij)^{90 (link)}. Full-size membranes of Western blots from which panels in Figs. 2–5 were prepared are shown in Supplemental Fig. S5–S8.

Cells cultured on glass coverslips (Corning) were washed with PBS and fixed using 4% PFA and permeabilized using 0.25% Triton X-100. For imaging after sorting, cells were suspended in PBS at concentrations of ~ 5 × 10⁶ cells/ml and fixed to coverslips by centrifugation. After blocking in 5% BSA, cells were incubated with primary antibodies at 4 °C, washed with PBS, and incubated with highly cross-adsorbed, AlexaFluor-conjugated secondary antibodies at room temperature. Coverslips were mounted to slides using Vectashield hard-set mounting medium with DAPI (Vector Laboratories). Images were acquired using a PerkinElmer UltraVIEW ERS spinning disc confocal imager mounted on a Zeiss AxioVert 200 inverted microscope equipped with a 63×/1.4 Oil DIC Plan-Apochromat 0.19/0.17. Primary antibodies used for fluorescence microscopy were: TFAM (Cell Signaling), cyclin B1 [GNS1] (SCBT), 8-oxo-dG [15A3] (SCBT), COXIV (Cell Signaling), and MAP LC3β [G-9] (SCBT).