Fig. 3.

Reduced NDUFB7 persulfidation during aging impairs NADH/NAD⁺ ratio. (A) Representative immunoblotting analysis for CSE and 3MST and relative densitometric analysis normalized to the DNA content in primary murine isolated cardiomyocytes from 3 or 18 month old wild type mice. n = 4/group, Unpaired Student's t-test. (B) Representative confocal images and quantification showing the results of the dimedone switch method for the detection of persulfidation plotted as the intensity of the Daz2:Cy5 persulfidation signal (blue to red, upper panel) and DAPI (grey), NBF–Cl (red) and a-actinin (blue) in the lower panel in samples as in A. n = 4–6/group, Unpaired Student's t-test. (C) Representative immunobloting and respective quantification for NDUFB7 and VINCULIN for samples as in panel A. n = 4–5/group. (D-E) Persulfidated NDUFB7 S-SH detected with a dimedone switch method and plotted as the relative fluorescence intensity of Daz2:Cy5/NBF (D) and NADH/NAD ratio (E) in samples as in panel A, treated for 10 min with solvent (Sol.) or 100 μmol/L NaHS. n = 6/group. two-way ANOVA, Bonferroni's posthoc analysis.