*2.6. Oxygraph*

Mitochondria from 40 flies were harvested and resuspended in 100 μL MiRO5 buffer (mmol/L, 0.5 EGTA, 3 MgCl2, 60 K-lactobionate, 20 taurine, 10 KH2PO4, 20 HEPES, 110 sucrose and 1 g/<sup>L</sup> BSA essentially fatty acid-free adjusted to pH 7.1). The assay was performed using the OROBOROS® Oxygraph-2k (O2k, Oroboros Instruments, Innsbruck, Austria) similar to previously published methods [21]. The oxygen electrodes were calibrated with air-saturated respiration medium (MiRO5) at 25 ◦C as per manufacturer instructions. SUIT protocol was used to test the activities of Complex I (malate and pyruvate) and Complex II (rotenone and succinate). Following substrates and inhibitors were added sequentially: malate (2 mM) and pyruvate (5 mM), succinate (10 mM), rotenone (0.5 μM), malonic acid (5 mM), and antimycin A (2.5 μM). ADP (1–5 mM) was added at distinct steps after the addition of Complex I and II substrates. FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) titrations

(0.05 μM steps) were carefully performed to obtain maximum electron transport capacity. Cytochrome c (10 μM) test was performed in each experiment to make sure that the mitochondrial membrane integrity was not compromised. The rate of oxygen consumption (oxygen flux) as a function of time was normalized to the total protein concentration for each experiment. Background calibration and air calibration were performed as suggested by the manufacturer prior to the experiments. Data were analyzed by DatLab software (v5.0, Oroboros Instruments, Innsbruck, Austria). n = 5 independent experiments from 40 flies each. The protein content was used to normalize the amount of mitochondria from the same extracts.
