Extracellular flux (Seahorse) assay

Control and NCoR1 KD DC line (30,000 cells/well) and BMDCs (0.1 × 10⁶ cells/well) were seeded in culture miniplates for the assay. Mito-stress test was performed using serial injections of Oligomycin (2μM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (CCCP-2μM) and Rotenone+Antimycin A (0.5μM each). The WAVE software was used to calculate respiratory characteristics such as baseline oxygen consumption rate (OCR), Coupled ATP, maximal respiratory capacity, and spare respiratory capacity. Serial doses of Glucose (10mM), Oligomycin (2μM), and 2-DG (50mM) were used in the Glyco-stress test. A pre-injection ECAR experiment was performed to determine the dependency of stimulated control and NCoR1 KD cells on mTOR and HIF-1α using rapamycin (20nM) and KC7F2 (10μM) respectively. ECAR values were determined similar to a mito-stress test using the WAVE software report generator. To establish pyruvate and glutamine dependency, the Mito fuel flex test was used. During pyruvate oxidation measurement, the first injection consisted of UK-5099 (2μM) and the second a combination of etomoxir (4μM) + BPTES (3μM). In a similar way, glutamine oxidation was measured with sequential injections of BPTES (3μM) followed by UK-5099 (2μM) + etomoxir (4μM) together. All the chemicals were prepared according to the manufacturer’s protocol (Seahorse bioscience, Agilent technologies). Fatty acid oxidation index was calculated from control and NCoR1 KD CpG samples by sequential injections of etomoxir (50μM) and Oligomycin (2μM).