Decylubiquinone

P. falciparum parasites were cultured with RPMI 1640 medium (Invitrogen by Thermo Fisher Scientific) supplemented with 5 g/L Albumax I (Invitrogen), 10 mg/L hypoxanthine, 2.1 g/L sodium bicarbonate, HEPES (15 mmol/L), and gentamycin (50 μg/mL). Cultures were maintained in human red blood cells (Type O, Interstate Blood Bank, Tennessee) and kept in a CO₂/O₂ incubator filled with a low oxygen mixture (5% O₂, 5% CO₂, and 90% N₂). Equine cytochrome c, decylubiquinone and atovaquone were obtained from Sigma Aldrich. CCK-8 was obtained from YEASEN. The yeast culture media were: YPD (1% yeast extract, 2% peptone and 3% glucose) and YPGal (1% yeast extract, 2% peptone, 0.1% glucose and 2% galactose). Human hepatocarcinoma cells (HepG2) were cultured in DMEM medium supplemented with 10% fetal bovine serum and penicillin-streptomycin. PEGylated castor oil for animal study was purchased from ACROS (Lot: A0377508).

Mitochondrial complex III activity was assayed as described previously [24] (link). To prepare decylubiquinol, 10 mg decylubiquinone (Sigma, D7911) dissolved in 400 µl of nitrogen-saturated hexane was mixed with 400 µl of 1.15 M sodium dithionite, and vortexed until colorless. The organic phase was collected, and the decylubiquinol was recovered by evaporating the hexane under nitrogen. The decylubiquinol was dissolved in 1 ml ethanol (acidified with 10 mM HCl) and stored in aliquots at −80 °C. Oxidized cytochrome c was from Sigma (C3131). 300 µl reaction mixture (15 µg mitochondrial protein and 60 µM cytochrome c) was added to a cuvette. After the addition of 3 µl decylubiquinol (stock concentration 15 mM, final concentration about 150 µM), reduction of cytochrome c was monitored at 550 nm once every second for 1 min with a SpectraMax M5 Microplate Reader (the chamber temperature was set at 30 °C). The assay was repeated with the addition of 1 µg Antimycin A (Sigma, A8674). Antimycin A-sensitive activity was calculated for the complex III activity. The extinction coefficient of cytochrome c is 21 mM⁻¹ cm⁻¹.