To analyze stress sensitivity, 5 μl of a yeast cell suspension (2 × 107 cells/ml) was added to 195 μl medium in a 96-well plate (tissue culture test plate; TPP Techno Plastic Products AG) containing liquid YPD or YPD and different concentrations of fluconazole (10, 25, 35, 50, 75, and 100 μg/ml) and tunicamycin (0.5, 1, 1.5, 2, and 3 μg/ml). For the ER stress analysis, yeasts were incubated in YPD and tunicamycin (1.5 μg/ml). The growth was monitored by measuring the absorbance at 600 nm every 30 min for 150 cycles at 37°C, using a Tecan reader (Infinite M200 PRO plate reader; Tecan Group GmbH) with orbital shaking. All experiments were done in independent biological triplicates on different days and are shown as the mean with standard deviation (SD) for each time point.
The MIC50 was determined in a 96-well plate (tissue culture test plate; TPP Techno Plastic Products AG) containing minimum medium (1% yeast nitrogen base, 1% amino acids, 0.5% ammonium sulfate, and 2% glucose) with increasing concentrations of fluconazole (FL) (4, 8, 16, 32, 64, 128, and 256 μg/ml), voriconazole (VC) (0.5, 1, 2, 4, 8, and 16 μg/ml), and clotrimazole (CL) (1, 2, 4, 8, and 16 μg/ml) at 37°C.
The MIC50 was determined in a 96-well plate (tissue culture test plate; TPP Techno Plastic Products AG) containing minimum medium (1% yeast nitrogen base, 1% amino acids, 0.5% ammonium sulfate, and 2% glucose) with increasing concentrations of fluconazole (FL) (4, 8, 16, 32, 64, 128, and 256 μg/ml), voriconazole (VC) (0.5, 1, 2, 4, 8, and 16 μg/ml), and clotrimazole (CL) (1, 2, 4, 8, and 16 μg/ml) at 37°C.