Phytosphingosine

For analysis of sensitivity to myriocin and aureobasidin A, overnight yeast cultures in YPD were diluted to an OD₆₀₀ of 0.5 in YPD and further incubated at 30°C for 4–6 h. Sensitivity of the yeast cultures to myriocin (Sigma-Aldrich, St. Louis, MO) was assayed by spotting 5-μl samples of 10-fold serial dilution of these yeast cultures on YPD plates containing 0.5 μg/ml myriocin. Growth was assessed after 48 h of incubation at 30°C. To determine sensitivity of the yeast cultures to aureobasidin A, 0.5 ml of these cultures was used to inoculate an YPD agar plate on which Whatman paper disks were placed containing 5 μl of 1 mg/ml aureobasidin A (Takara Bio, Shiga, Japan) in water. After 3 d of incubation at 30°C the size of the halo was measured. To assess sensitivity of yeast cultures to phytosphingosine (Sigma-Aldrich), cells were grown to stationary phase (>5 d) in YPD and subsequently diluted to an OD₆₀₀ of 0.05 in YPD containing 5 or 15 μM phytosphingosine and 0.0015% Nonidet P-40. The OD₆₀₀ of the different cultures was measured at regular time points. For PI staining, yeast cells growing exponentially in YPD medium were treated for 2 h with 0.5 μg/ml myriocin. Next cells were collected, washed in phosphate-buffered saline, and stained with PI using the Annexin-V-Fluos Staining Kit (Roche Diagnostics, Vilvoorde, Belgium) according to the provided protocol.