Oligomycin

All cell lines were cultured in DMEM (Gibco) containing 10% FBS, 4.5 g l⁻¹ glucose, 1 mM l-glutamine, 0.11 g l⁻¹ pyruvate and maintained at 37 °C in 5% CO₂ atmosphere. For mitochondria depletion, cells were treated with 1 μM antimycin A (Sigma A8674) and 1 μM oligomycin (Sigma O4876) every 12 h for the indicated time periods as previously described [18] (link). We recommend that a titration of these drugs is undertaken in different cell systems in order to determine the most effective concentration to cause mitochondrial damage and depletion in any given system.
In order to activate autophagy, cells were treated with 100 nM rapamycin (LCL laboratories R-5000) or 150 μM of deferoxamine mesylate (DFO) for 2 h prior treatment with oligomycin and antimycin A. For the inhibition of the autophagic flux cells were treated with 10 μM hydroxychloroquine (Sigma) or 100 nM bafilomycin A1 (Sigma) together with antimycin A and oligomycin.

Bone marrow-derived macrophages (BMDM) were cultured according to described protocols 32 . Briefly, mice were euthanatized and cells from femurs were collected. 4 x 10⁵ cells were plated per sterile plastic petri dish in 10 mL of DMEM/F-12 (ThermoFisher Scientific, #11320033) supplemented with 10% fetal bovine serum (Sigma-Aldrich, #F2442), 1% penicillin/streptomycin (Sigma-Aldrich, #P4333), and 100 ng/mL of recombinant M-CSF (R&D Systems, #416-ML). Cells were incubated at 37 ºC and 5% CO₂ and the medium was changed at days 3, 5 and 7. At day 7, cells were exposed to conditioning conditions: (i) 100 ng/mL of LPS (Sigma-Aldrich, #L2880) + 20 ng/mL of rINFγ (R&D Systems, #485-MI), (ii) 20 ng/mL of rIL-4 (eBioscience), (iii) 20 ng/mL of rIL-13, (iv) 7.9 µg/mL of Oligomycin (Sigma-Aldrich, #75351) + 2.1 µg/mL of Antimycin (Sigma-Aldrich, #A8674), (v) 20 ng/mL of rIL-4 + 7.9 µg/mL of Oligomycin + 2.1 µg/mL of Antimycin, and (vi) 20 ng/mL of rIL-13 + 7.9 µg/mL of Oligomycin + 2.1 µg/mL of Antimycin. Concentrations were based on previous publications 33 (link). After 24 h, supernatants (conditioned mediums) were collected and stored at -20 ºC until use.
Motor neuron-like NSC-34 cells were cultured in DMEM (ThermoFisher Scientific, #10566016) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Sigma-Aldrich, #P-0781) in a 37 ºC and 5% CO₂ incubator.