Fig. 4

IBC improved the susceptibility of C. neoformans to cell stressors and fluconazole and decreased virulence factor production. (A) Growth phenotypes of C. neoformans were assessed in the presence of various cell stressors and different concentrations of IBC. Cells were normalized by OD₆₀₀ and subsequently diluted. 5 µL aliquots of serial dilutions were subsequently spotted on YPD agar medium supplemented with calcofluor white (1.5 mg/mL), caffeine (0.5 mg/mL), and NaCl (0.5 M), 0.03% SDS and 0.2% Congo red in the presence of various concentrations of IBC at 30 °C, respectively. For temperature stress, the YPD plates were incubated at 37 °C in the presence of various concentrations of IBC. Experiment was repeated three times to ensure reproducibility and representative images are shown. (B) Fluconazole susceptibility was assessed for the IBC-treated C. neoformans cells. Cells were normalized by OD₆₀₀ and were subsequently grown on YPD medium agar plates in the presence of fluconazole and different concentrations of IBC. Cells were incubated at 30 °C and plates were imaged at the indicated times. (C, E) Representative images of various concentrations of IBC-treated C. neoformans grown in Dulbecco’s Modified Eagle’s Medium (DMEM) for 48 h at 37 °C. Capsules were visualized by India ink staining and examined under a microscope, and the capsule diameter was evaluated. (D, F) C. neoformans cells were grown on L-DOPA agar plates for 72 h at 30 °C with or without the addition of IBC. Reduced amounts of melanin formation in the colonies were observed in a concentration-dependent manner