Fig. 3. ATP synthesis in C. albicans lacking the δ subunit.
a Intracellular ATP concentrations in WT, atp16Δ/Δ and atp16Δ/ATP16 cultured in YNB + 2% glucose medium for 12 h were measured by using targeted metabolomics analysis. b The intracellular ATP content in WT, atp16Δ/Δ and atp16Δ/ATP16 (2 × 106 CFU) cultured in YPD medium for 12 h was measured by using luciferase/luciferin analysis. c, d Oxygen consumption rate (OCR) in WT, atp16Δ/Δ and atp16Δ/ATP16 in response to dicyclohexylcarbodiimide (DCCD) (100 μM), Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (2 μM) and rotenone (Rot)/antimycin A (AA) (0.5 μM) were measured by a Seahorse XFe96 analyser (c) and bioenergetic parameters (basal respiration and OXPHOS ATP synthesis) were calculated (d). e ROS production in WT, atp16Δ/Δ and atp16Δ/ATP16 were measured by a fluorometric assay. The ratios of the fluorescence intensities of the ROS-sensitive dyes were normalized to those in WT. f Mitochondrial membrane potential (ΔΨm) in WT, atp16Δ/Δ and atp16Δ/ATP16 were measured by a fluorometric assay and the red/green mean fluorescent intensity ratio was calculated. g The expression levels of proteins involved in OXPHOS were assessed by proteomics analysis. The subunits of complex I, complex II, complex III, complex IV, F1Fo-ATP synthase and others in atp16Δ/Δ were markedly downregulated compared with those in WT, fold change >1.5 (p < 0.05). The three columns for each strain represent three experiments were performed with 3 biological replicates. h F1Fo-ATPase activity in WT, atp16Δ/Δ and atp16Δ/ATP16, were measured by an EnzChek Phosphate Assay Kit. i Glycolytic rate in WT, atp16Δ/Δ and atp16Δ/ATP16. j, k, Extracellular acidification rate (ECAR) in WT, atp16Δ/Δ and atp16Δ/ATP16 in response to the glucose (10 mM), Rot/AA (0.5 μM) and 2-DG (50 mM) were measured by Seahorse XFe96 analyser (j) and bioenergetic parameters (glycolysis) were calculated (k). l Intracellular ATP content in WT, atp16Δ/Δ and atp16Δ/ATP16 (2 × 105 CFU) in the absence (−) or presence (+) of the glycolysis inhibitor 2-DG (50 mM) after cultured in YNB + 0.2% glucose medium at 30 °C for 12 h. m Glycolytic metabolites concentrations in WT, atp16Δ/Δ and atp16Δ/ATP16 were measured by targeted metabolomics analysis. n, o, p The activities of the rate-limiting enzymes of glycolysis, Hk2 (n), Pfk1 (o), and Pyk1 (p), in WT, atp16Δ/Δ and atp16Δ/ATP16. q, r G6PD activity (q) and NADPH level (r) of the PPP pathway in WT, atp16Δ/Δ and atp16Δ/ATP16. In a, m six independent experiments were shown. In b–f, h–l, and n–r three independent experiments are shown. In a–f and h–r data were expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant; by two-tailed unpaired Student’s t-test (a, b, d–i, k, m–r), or two-way ANOVA (l).