Fig. 2: Azole resistant mechanisms of the C. glabrata ipi1R70H mutant.
a Sterol analysis. C. glabrata wild-type (WT) and ipi1R70H strains were grown in synthetic defined (SD) broth or SD broth with 8 μg/mL fluconazole ( + FLC). Sterol contents were analyzed by reverse-phase HPLC as described in the methods. The means and standard errors for three independent experiments are shown (ns, not significant; ordinary one-way ANOVA with Sidak’s multiple comparison test). b qRT-PCR. C. glabrata wild-type (WT) and the ipi1R70H mutant were grown as described in (a) and total RNA was extracted. mRNA abundance of ERG11 was measured by qRT-PCR and normalized using ACT1 as an internal control. Data are expressed as the means ± standard deviations (ns, not significant; ordinary one-way ANOVA with Sidak’s multiple comparison test). qRT-PCR was repeated on three independent occasions with similar results. c qRT-PCR. Total RNA was extracted from C. glabrata wild-type, Cg50, and two different clones of ipi1R70H mutant strains. mRNA abundance was measured by qRT-PCR and normalized using ACT1 as an internal control. Expression of wild-type strain was defined as 1 in each assay, and relative mRNA abundances in other strains were calculated. Data are expressed as the means ± standard deviations (ordinary one-way ANOVA with Dunnett’s multiple comparison test). qRT-PCR was repeated on three independent occasions with similar results. Source data for panels (a–c) are provided as a Source Data file. d Rhodamine 6 G (R6G) accumulation assay. Intracellular concentration of the fluorescent dye R6G, a substrate of azole efflux pumps, was measured by flow cytometry in three different wild-type strains, two different clones of the ipi1R70H mutant, and a mutant lacking Cdr1 efflux pump (cdr1Δ). Fluorescence intensity of R6G is shown on the X-axis. Left panel: Cells were incubated with R6G under normal growth conditions to examine the activity of R6G efflux. Right panel: Cells were exposed to R6G under the de-energized condition to examine the levels of R6G infused passively into the cells. These experiments were repeated twice with similar results.
