Fig. 1: Development and phenotypic characterization of the C. glabrata ipi1R70H mutant.
a A schema representing sequential exposure of C. glabrata wild-type (CBS138) cells to increasing concentrations of micafungin. C. glabrata cells were spread on YPD plates containing micafungin at the indicated concentrations, incubated at 30 °C for 2 days, and subjected to antifungal susceptibility tests. b, c Spot dilution assay. C. glabrata strain Cg50 was obtained from a plate containing micafungin at the concentration of 0.03 µg/mL as described in (a). Strain Cg51 was obtained after the repeated subculture of Cg50 in YPD broth without micafungin for 20 days. An ipi1R70H mutant was constructed as described in the methods. Serial 10-fold dilutions of C. glabrata cells were spotted onto an SC plate containing an antifungal agent at the indicated concentrations. Plates were incubated at 30 °C or the indicated temperatures for 2 days. All susceptibility tests were performed on at least three separate occasions. d Doxycycline-mediated transcriptional repression of IPI1 resulted in a growth defect in C. glabrata. Logarithmic-phase cells of the C. glabrata wild-type (ACG22) and tet-IPI1 strain, in which the native IPI1 promoter was replaced with the tetracycline regulatable promoter, were adjusted to ~ 1 × 105 cells/mL in SD broth and incubated at 30 °C for 24 h with or without 20 µg/mL of the tetracycline analog doxycycline (Dox). Data are expressed as mean ± standard deviation for biological triplicates (****P < 0.0001; ns, not significant; one-way ANOVA with Dunnett’s multiple comparison test). The experiment was repeated twice with similar results. Source data are provided as a Source Data file. e Northern blotting. Logarithmic-phase C. glabrata cells grown at 30 °C were subsequently incubated at 42 °C, a non-permissive temperature for the ipi1R70H mutant. Total RNA was extracted from each strain at the indicated time points. Top, northern hybridization was performed using an ITS2 probe. Bottom, total RNA was analyzed using 1.0% agarose gel with ethidium bromide, and 25S and 18S rRNA are indicated. The experiment was repeated twice with similar results. Source data including uncropped and unprocessed scans with molecular weight markers are provided as a Source Data file.
