Fig. 5

Intracellular NADPH levels determine the oxidative stress resistance via regulating AnCF complex assembly. A Overexpression of gsdA resulted NADPH accumulation in ΔhapC. WT (WT_pyrG), nP.gsdA, ∆hapC, and nP.gsdA/∆hapC were cultivated in NO₃⁻-MM liquid media for 16 h, then the lysates were used for the relative NADPH/NADP⁺ ratio calculation. NADPH/NADP⁺ value of WT was set to 1 (mean ± SD; n = 3, **P < 0.01, one-way ANOVA). B AnCF is involved in impairment of H₂O₂ resistance caused by excess NADPH. Fresh conidia (1 × 10⁸) of four strains were spread on NO₃⁻-MM plates containing the indicated concentrations of H₂O₂. Colonies were counted after a 48-h incubation, and survival rates were expressed as percentages of the CFU for strains incubated without H₂O₂ (mean ± SD; n = 3). C Immunoblot quantification of AnCF complex levels (top) and HapC levels (bottom box) in WT and nP.gsdA during H₂O₂-treatment; H_Flag (WT expressing Flag-tagged HapC) and nP.gsdA/H_Flag (nP.gsdA expressing Flag-tagged HapC). Cell-free lysates (100 µg) from each sample were loaded to native- (top) and SDS-PAGE (bottom box). AnCF and HapC were detected using anti-Flag antibody; actin was used as a control and was detected using an anti-Actin antibody. D Quantitated graph for intracellular AnCF level normalized to actin (mean ± SD; n = 3, *P < 0.05, one-way ANOVA). E Time course analysis of NADPH/NADP⁺ level in H_Flag and nP.gsdA/H_Flag during 1 mM H₂O₂ treatment. Strains were precultivated in NO₃⁻-MM liquid media for 16 h and then exposed to 1 mM H₂O₂ for 30, 60, and 90 min (mean ± SD; n = 3, t-test). F Relative napA mRNA levels in these strains with or without treatment of H₂O₂. Strains were cultivated in NO₃⁻-MM liquid media for 16 h, and then treated by H₂O₂ for 30 min and 60 min (mean ± SD; n = 3, *P < 0.05, **P < 0.01, n.s., not significant, two-way ANOVA)