Fig 1.
(A) Chemical structure of DD. (B) The purity of DD as determined by Agilent 1260 system equipped with a ZORBAX SB-C18 column in 76% MeOH-H2O solvent with a flow of 1.8 mL/min. (C) Time-killing kinetics of DD and AMB at the concentrations of 8 mg/L against C. albicans SC5314. Bars indicate standard deviations.
Table 1.
Gene-specific primers used for relative quantification of genes expressions by RT-PCR.
Table 2.
The IC50 values of DD against human cell lines.
Table 3.
The MIC80 values of DD against Candida species.
Fig 2.
Effect of DD on the cell membrane integrity.
(A) showed the effect of DD on the cell membrane dynamic. SC5314 cells were treated with various doses of DD or 8 mg/L of AMB (positive control) for 3 h following with staining of DPH for spectrofluorophotometer detection. (B) showed the change of membrane permeabilization after cells were treated with DD or 8 mg/L of AMB (positive control). Treated cells were stained with PI and analyzed by flow cytometry. The cells with more than 101 fluorescence intensity were regarded as stained cells. Bars indicate standard deviations.
Fig 3.
Effect of DD on the cell morphology and intracellular glycerol.
(A) The alterations of cell morphology were analyzed by flow cytometry. FSC (y-axis) is an indicator of size, SSC (x-axis) is an indicator of granularity and the z-axis represents the cellular population intensity. The values of x-axis and y-axis are linear and provide relative values for comparison among different treatments. (B) The contents of intracellular glycerol were measured using Glycerol Assay Kits after treated with various doses of DD for 3 h. (C) The total RNAs were extracted by the hot phenol method and the expressions of HOG1 and RHR2 were measured using qPCR. Bars indicate standard deviations.
Fig 4.
The ultrastructure of DD-treated C. albicans cells and the expressions of genes associated with cell membrane synthesis and cell wall assembly.
(A) and (B) showed the transmission electron micrographs of C. albicans with 12 mg/L of DD-treatment or not. The destruction and fragmentation of plasma membrane were observed in DD-treated cells indicated by arrow in B. (b) is the magnification of (a). (C) showed the effect of DD on the expression of cell membrane related genes. After treatment with 8 mg/L of DD for 3 h, SC5314 cells were harvested for the total RNAs extraction and then the expressions of indicated genes were measured using qPCR. Bars in (C) indicate standard deviations.
Fig 5.
Effect of DD on intracellular ROS production.
SC5314, preincubated with or without Tu or NaN3, was exposed to the increasing concentrations of DD or 8 mg/L of AMB (positive control) for 3 h. After staining with 40 mg/L of DCFH-DA, the samples were detected by flow cytometry and visualized by CLSM with 488 nm of excitation and 525 nm of emission. (A) showed the flow cytometry results and (B) showed the CLSM observation. The bars in (A) indicate standard deviations and in (B) indicate 5 μm.
Fig 6.
Effect of DD on the mitochondria function.
SC5314, preincubated with or without Tu or NaN3, was treated with various concentrations of DD and 8 mg/L of AMB (positive control) for 3 h before staining with 5 μM of Rh 123 to indicate the alteration of mitochondrial membrane potential (mtΔψ). The fluorescence intensity was analyzed by flow cytomerty and shown at (A). C. albicans CAI4-TOM70-GFP, preincubated with or without Tu or NaN3, was treated with various concentrations of DD. The cells were visualized by CLSM and shown at (B). The bars in (A) indicate standard deviations and in (B) indicate 5 μm.
Fig 7.
Effect of Tu and NaN3 on the fungicidal activity of DD.
SC5314, pretreated with or without Tu or NaN3, was exposed to 8 mg/L of DD for 3 h at 30 °C. The number of viable cells was then determined by colony counting method. The bars indicate standard deviations. Statistical significances were determined by Student’s t-test. *P < 0.05. **P < 0.01.