Polarstar galaxy

Microbead coating, binding measurements, and the calculation of the amount of lipid coating the microbeads were done as described previously (51 (link)). Binding experiments at different salt concentrations (50, 300, and 500 mM KCl) were conducted using MatP-Alexa 488 (125 nM Alexa 488, 250 nM MatP) and 20 g/liter beads (35 μM accessible lipids). Experiments in the presence of matS were performed by adding, prior to or after incubation with the attached lipids, unlabeled matS (0.1 or 1 μM) to the samples containing MatP-Alexa 488 (0.250 μM). Additionally, matS-Fl (0.1 μM) was added to samples containing unlabeled MatP (0.250 μM) and lipids. After 20 min of incubation of the protein or the nucleoprotein complex with the coated beads, samples were centrifuged, and free protein/nucleoprotein complex remaining in the supernatant was quantified using a fluorescence plate reader (Varioskan Flash, Thermo Scientific, or POLARstar Galaxy, BMG Labtech) as described previously (51 (link)). Assays were performed in triplicate, and the binding isotherm was constructed by plotting the concentration of bound MatP as a function of the concentration of free MatP. The linearity of the signal of the labeled protein with its concentration was verified.

The production of ROS was measured with DCFH-DA (Molecular Probes, USA) as previously described [20 (link)]. Briefly, exponentially growing C. albicans cells were collected by centrifugation, washed twice with PBS and adjusted to 1 × 10⁷ cells/ml. Then, the cells were treated with 20 μg/ml of DCFH-DA and incubated with shaking at 30°C for 30 min. After washing the cells with PBS for three times, 8 μg/ml MG, 0.125 μg/ml CAS alone or in combination were added. To test the impact of polyamines on ROS production, putrescine, spermidine, and spermine were all added at the concentration of 2.5 mM. The cell suspensions were cultured with shaking at 30°C. At each designated time points, portions of the cell samples were harvested and 100 μl of the supernatant was transferred to the wells of a flat-bottomed 96-well microplate to detect fluorescence intensity using a fluorescence spectrometer (POLARstarGalaxy; BMG Labtech, Offenburg, Germany) with an excitation wavelength at 488 nm and an emission wavelength at 525 nm.

Cell viability was determined by measuring the adenosine triphosphate (ATP) concentration in viable cells according to manufacturers’ recommendations (PerkinElmer, Waltham, MA, USA) using a luminescence reader (LumiSTAR Galaxy; BMG Labtech, Ortenberg, Germany). ATP concentrations were calculated using an eight-point calibration curve. Cytotoxicity effects were determined by measuring the release of LDH according to manufacturers’ recommendations (Promega, Gutenbergring, Walldrof, Germany) using a microplate reader at 490 nm (POLARstar Galaxy; BMG Labtech, Allmendgrün, Ortenberg, Germany). LDH release was calculated as fold change using the untreated control as a reference set to 1.

Fluorescence of carboxyfluorescein was determined by using a microplate reader (polarstar galaxy, BMG Labtech, Offenburg, Germany) at an excitation wave length of 485 nm and an emission filter at 520 nm. Triplicates per sample were measured and blank medium values were substracted. Fluorescence of total APTS-dextran was measured as described above, separation and fluorescence measurement of single APTS-dextran fractions by capillary electrophoresis were carried out as previously published [22] (link).