Oxygraph 2k instrument

Respiration was evaluated in digitonin-permeabilized cells as previously described^{59 (link)–61 (link)}. For routine respiration, cells were trypsinized, washed with PBS, resuspended in Mir05 medium (0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 110 mM sucrose, 1 g/l essentially fatty acid-free bovine serum albumin, 20 mM Hepes, pH 7.1 at 30 °C) and transferred to the chamber of Oxygraph-2k instrument (Oroboros). The respiration measurements were performed at 37 °C. After closing the chamber, routine respiration on intracellular substrates was recorded. Access of exogenously added substrates was allowed by permeabilizing the cellular plasma membrane with 5 µg digitonin per million cells. Complex II-mediated respiration was assessed by adding 0.5 μM rotenone, 10 mM succinate, 3 mM ADP and 10 µM cytochrome c together with a CII inhibitor (5 mM malonate). Uncoupled state was achieved by CCCP (carbonyl cyanide m-chlorophenyl hydrazone) titration to obtain the maximal respiratory rate. Antimycin A (2.5 μM) was added at the end of each measurement to inhibit ETC and the residual oxygen consumption after antimycin A addition was subtracted from all results to obtain mitochondria-specific rates.

O₂ consumption rate (OCR) was determined by high-resolution respirometry using an Oroboros Oxygraph-2 k instrument (OROBOROS^® INSTRUMENTS GmbH, Innsbruck, Austria). Cells (control or ISL-treated) were seeded at 4 × 10⁶ cells. The cells were centrifuged at 1000 rpm for 4 minutes and resuspended in MIR05 buffer (Oroboros lab). The respiration experiments were conducted at 37°C in MIR05 buffer. A standard protocol using malate (2 mM), glutamate (10 mM), oligomycin (2 μg/ml), FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) (0.45 μM), succinate (10 mM), digitonin (3.68 μM), rotenone (0.5 μM) and antimycin A (2.5 μM) was used for each measurement. Cellular O₂ was calculated from the recorded data as the time derivative of the oxygen content in the chamber; O₂ concentrations were calculated using DatLab software (Oroboros Instruments).

The respiration or oxygen consumption rate of C. albicans cells was measured at 37 °C with an Oxygraph-2k instrument (O2k, OROBOROS Instruments, Innsbruck, Austria). C. albicans cells from overnight culture were inoculated into YPD medium and grown to exponential phase. Cells were harvested and resuspended in 12.5 mM sodium acetate. The basal oxygen consumption level was adjusted to 50 pmol/(s*ml). Cells were then treated with AMPs (P-113: 1 μM, 0.05 μM; P-113Du: 0.1 μM, 0.05 μM; P-113Tri: 0.05 μM, 0.01 μM), and the oxygen consumption rate was recorded until the readings stabilized. After the measurement, cells were collected, spotted (50 μl) onto YPD agar plates, and incubated at 30 °C for 24 h to assess cell viability. The basal oxygen consumption rate of C. albicans was normalized to its survival rate. The respiratory inhibitory activity (RIA) of the AMPs was calculated using the following formula we created: $$\mathrm{Respiratory}\mathrm{inhibitory}\mathrm{activity}(\mathrm{a}.\mathrm{u}.)=1-\frac{Endpoint{O}_{2}consumptionrate}{Basal{O}_{2}consumptionrate\times \left(\frac{\%Survivalrate}{100}\right)}$$
The basal O₂ consumption rate is the routine oxygen consumption rate of cells without any treatment. The endpoint of O₂ consumption rate reflects the stable oxygen consumption rate of cells after AMP treatment. The basal oxygen consumption rate was normalized to the cellular survival rate for each independent run. All experiments were repeated at least three times.