Unlabeled inositol

The inositol uptake assay was performed following a previously published method (31 (link)). In brief, the Cryptococcus strains were grown in YPD liquid cultures overnight at 30°C. Cells were diluted in YPD to an OD₆₀₀ of 1.0, grown at 30°C, and collected at an OD₆₀₀ of 5.0 by centrifugation at 2,600 × g for 5 min. Cells were then washed twice with PBS at 4°C and resuspended in 2% glucose to reach a final concentration of 2 × 10⁸ cells/ml as determined by the use of a hemacytometer. For the uptake assay, the reaction mixture (200 μl) contained 2% glucose, 40 mM citric acid-KH₂PO₄ (pH 5.5), and 0.15 μM myo-[2-³H]-inositol (MP Biomedicals) (1 μCi/μl). An additional 200 μM concentration of unlabeled inositol (Sigma-Aldrich) was added to the reaction mixtures for competition assays. Equal volumes of the reaction and cell mixtures (60 μl each) were warmed to 30°C and mixed for the uptake assay, which was performed for 10 min at 30°C. As negative controls, mixtures were kept at 0°C (on ice) during the 10-min incubation. Aliquots of 100 μl were removed and transferred onto prewetted Metricel filters (1.2-μm pore size) on a vacuum manifold. The filters were washed four times each with 2 ml of ice-cold water. The washed filters were removed and added to liquid scintillation vials for measurements on a PerkinElmer TRI-CARB 2900TR scintillation counter.