Oligomycin

Real-time oxygen consumption and extracellular acidification rates were measured by using an XF96 oximeter (Seahorse Biosciences, North Billerica, MA, USA). SGBS cells were seeded onto 96-well XF96 cell culture microplates. Cells were kept in growth medium at the longest for 24 hours and then the formerly-described differentiation process started. After recording the baseline oxygen consumption, cells received a single bolus of dibutyril-cAMP at 500 µM concentration to induce adrenergic stimulation. Then, stimulated oxygen consumption was measured in every 30 minutes. The final reading took place at 6 h post-treatment. Differentiated adipocytes were treated with 2 mM ß-guanidinopropionic acid (Sigma) to block the creatine-driven substrate cycle^{21 (link)}. In addition, proton leak respiration was measured by oligomycin (Enzo, USA) treatment at 2 µM concentration, which blocks the ATP synthase. For baseline correction, cells received a single bolus of Antimycin A (Sigma) treatment at 10 µM concentration. After the measurements, oxygen consumption rate was normalized to protein content^{27 (link),28 (link),56 (link)}.

Cell imaging was performed in 384-well plates. For living cells, 30 min prior imaging, 25 μl of culture medium were removed and replaced by 25 μl of 2× mix of dyes, to a final concentration of 200 ng/ml of Hoechst H33342 (nuclear staining; Life Technologies), 10 nM of TMRM (mitochondrial membrane potential; Life Technologies), and/or 1/100 Annexin-V-Alexa Fluor 647 (early apoptosis; Life Technologies). If chemical inhibitors of the ETC were used in the experiments, they were added to hMDMs at the indicated times points at the following concentrations: 5 μM oligomycin (Enzo), 100 μM DCCD (Sigma), 10 μM FCCP (Tocris), and 50 μM BTB06584 (Sigma). Once the assay was performed, cells were fixed with 4% PFA, permeabilized with 0.1% Triton-X100, blocked with 1% BSA, and stained with primary mouse antibodies against Xpress tag (1:100, Invitrogen) and secondary anti-mouse Alexa Fluor 488 antibodies (1:1000, Invitrogen). Image acquisitions of multiple fields (9–25) per well were performed on an automated confocal microscope (Opera Phenix, PerkinElmer) using ×40 or ×60 objective, excitation lasers at 405, 488, 561, and 640 nm, and emission filters at 450, 540, 600, and 690 nm, respectively.

Hela cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal bovine serum (Sigma-Aldrich). Cells were incubated in a humidified atmosphere at 5% CO₂ and 37 °C. HeLa cells were transfected using Fugene (Promega) or PEI MAX 40000 (24765, PolySciences, Inc., Warrington) according to the manufacturer’s recommendations. HeLa cells were treated with 0 to 50 µM carbonyl cyanide m-chlorophenyl hydrazine (CCCP – Sigma-Aldrich), 10 µM oligomycin (Enzo Lifesciences), 1 µM antimycin (Enzo Lifesciences), 100 nM bafilomycin (Sigma-Aldrich) or 1 mM 1,2-dimethyl-3-hydroxy-4(1 H)-pyridon (deferiprone - DFP for the indicated time (Sigma-Aldrich).