Figure 8.

Cho1 is required for mitochondrial function. C. neoformans WT H99 and P_CTR4-CHO1 strains were cultured in SC medium alone and in the presence of 1 mm CuSO₄ or 200 μm BCS for 6 h. Cells were then incubated for 10 min in the presence of MitoTracker Red FM at a concentration 0.2 μm and DAPI at a concentration 1 μg before analyzing the mitochondria and mtDNA under the microscope. Scale bar, 10 μm. Among all the cells examined, 99% of them showed identical co-localization. Images with more cells were shown in Fig. S4. A, reduced CHO1 gene expression in the presence of copper prevents MitoTracker Red FM accumulation into the mitochondria. B, mitochondrial DNA stained with DAPI. C, reduced CHO1 gene expression leads to higher levels of intracellular ROS generation. H99 and P_CTR4-CHO1 strains were grown to log phase at 30 °C, loaded with H₂DCFDA, and exposed to BCS and CuSO₄ for 2, 4, and 6 h. Quantification of flow cytometry indicates that there is a significant increase in ROS generation in the P_CTR4-CHO1 strain in the presence of CuSO₄ as compared with BCS or SC medium alone at hour 4 and at hour 6. ROS generation is also significantly higher in P_CTR4-CHO1 as compared with H99 when treated with CuSO₄ at hour 4 and at hour 6. * indicates p < 0.05 and **indicates p < 0.01 based on two-tail t test.