Figure 5.

Pharmacological manipulation of the Ras signaling pathway of FCZ-resistant C. albicans NBC099. (A) NBC099 cells grown on glass cover slides and treated with H₂O of 0.2% DMSO (as the vehicle control), dibutyryl cAMP (db.cAMP; in water), caffeine (in water), forskolin (in DMSO), or lovastatin (in DMSO). After incubation for 30 min at 37 °C, cells were further treated with 10 µg/mL of rsAg@NCs for 24 h. Immunofluorescence detection of apoptosis was observed by using TUNEL, AO, and DAPI staining assays. For the TUNEL assay, cells were washed and permeabilized. DNA ends of cells were labelled by using the APO BrdU TUNEL Assay Kit, and cells were observed under a fluorescence microscope. Cells were stained with 5 µg/mL and 1 µg/mL of AO and DAPI, respectively. Subsequently, cells were analyzed by fluorescence microscopy. (B) To quantify apoptotic cell death, treated or untreated cells were also analyzed by flow cytometry, using the TUNEL assay kit. Water or DMSO (final concentration of <0.2%) was used as a vehicle control for drug treatments. Each reported value represents the mean ± SE from three independent experiments (* p < 0.001, compared with the untreated control).