Metabolic studies
PMP
Seahorse XFp Cell Mito Stress Tests were performed using Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, Cat. No. 103015-100) according to the manufacturer’s protocol on Seahorse XFe96 Analyzer (for cell lines) and Seahorse XF HS Mini Analyzer (for mouse bone marrow lineage-negative cells and human peripheral blood mononuclear cells) (Agilent Technologies). To perform the metabolic assays on cell lines, Seahorse XFe96/XF Pro Cell Culture Microplates (Agilent Technologies, Cat. No. 103794-100) were coated with Cell-Tak (Corning, Cat. No. 354240) in 0.1 M sodium bicarbonate solution (pH 7.4) for 20 min at room temperature, washed twice with water and stored at 4 °C one day before the assay. Cells were treated with 4OH-TAM for 2 h and washed with XF Mito Stress medium (Agilent XF DMEM Medium (Agilent Technologies, Cat. No. 103335) with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine, pH 7.4). Cells were then maintained in XF Mito Stress medium at 37 ◦C in an incubator for 1 h to pre-equilibrate. For mouse bone marrow lineage-negative cells and human peripheral blood mononuclear cells, cells are seeded in Seahorse XFp PDL Cell Culture Miniplates (Agilent Technologies, Cat. No. 103722-100). In Cell Mito Stress Tests, the concentrations for oligomycin, FCCP and rotenone/antimycin A are 1 μM, 0.5 μM, and 0.5 μM respectively.For the XF Real-Time ATP Rate Assay (Agilent Technologies, Cat. No. 103592-100), cells were washed with the ATP assay medium (Agilent XF DMEM Medium with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine, pH 7.4). In Real-Time ATP Rate Assay, the concentrations for oligomycin and rotenone/antimycin A are 1.5 μM and 0.5 μM respectively. Data analysis was performed using the Seahorse XFe Wave software and statistics were analyzed in Prism 7.0.For permeabilization assay, cells were washed and maintained in pre-warmed mannitol and sucrose buffer (MAS, pH 7.2) (220 mM mannitol, 70 mM sucrose, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, 4 mg/ml BSA), following by adding 1 nM XF Plasma Membrane Permeabilizer (PMP) (Agilent Technologies, Cat. No. 102504-100) before running the assay. After the first injection of tamoxifen and vehicle control, mitochondrial activity was initiated by pyruvate (5 mM), malate (2.5 mM), and ADP (1 mM) for measuring complex I-driven respiration, followed by succinate (10 mM) and ADP (1 mM) for complex II-driven respiration.