FIGURE 8: The lithium-mimetic drug ebselen inhibits capsule formation, and its activity is potentiated by combination with lithium or bortezomib. (A) DIC microscopy images of the C. neoformans H99S wild type and the pkr1D mutant grown in CIM (control) or CIM supplemented with the indicated concentrations of LiCl and/or ebselen for 48 h and stained with India ink to visualize capsule. EBS, ebselen. Scale bar, 5 µm. (B) Quantification of cell diameter and capsule thickness for cells from panel A. The experiment was performed twice, and at least 64 cells were analyzed per strain and condition. Grey squares and blue circles indicate individual data points from both experiments, and the black bar indicates the median ± interquartile range. The dotted black lines indicate the median capsule sizes of the Wt or pkr1D mutant grown under control conditions. ***P < 0.001, and ****P < 0.0001 by t-test. (C) Relative capsule thickness data from panel B. Results were plotted relative to the untreated controls. Note that capsule formation by the pkr1D mutant is less affected by EBS but more strongly reduced by LiCl compared to the Wt. (D) DIC microscopy images of the C. neoformans H99S wild type and the pkr1D mutant grown in CIM (control) or CIM supplemented with the indicated concentrations of bortezomib and/or ebselen for 48 h and stained with India ink to visualize capsule. BTZ, bortezomib; EBS, ebselen. Scale bar, 5 µm. (E) Quantification of cell diameter and capsule thickness for cells from panel D. The experiment was performed twice, and at least 100 cells were analyzed per strain and condition. Small grey squares and blue circles indicate individual data points from both experiments, and the black bar indicates the median ± interquartile range. The dotted black lines indicate the median capsule sizes of the Wt or pkr1D mutant grown under control conditions. ns, not significant. ****P < 0.0001 by t-test. (F) Relative capsule thickness data from panel E. Results were plotted relative to the untreated controls. Note that capsule formation by the pkr1D mutant is less affected by BTZ and EBS compared to the Wt.