Fig. 1.

Reduced persulfidation in the hearts of CSE and 3MST knock out mice affects NDUFB7 persulfidation and NADH/NAD⁺ ratio. (A-B) Volcano plots showing persulfidated cysteines in proteins, in hearts from wild type (WT) compared to (A) CSE or (B) 3MST knock out (KO) mice and respective GO enrichment analysis of the modified proteins. n = 4/group. Unpaired Student's t-test. Red/Green: persulfidated cysteines in the respective proteins with a p < 0.1 enriched in the WT samples, blue: persulfidated proteins with a p < 0.1 enriched in the CSE (A) or the 3MST (B) samples. (C) Persulfidated NDUFB7 S-SH detected with a dimedone switch method and plotted as the relative fluorescence intensity of Daz2:Cy5/NBF in whole hearts from mice as in panels A and B. n = 6/group. one-way ANOVA, Tukey's multiple comparisons test. (D) NADH/NAD⁺ ratio in whole heart homogenates from mice as in panels A and B. n = 4/group. Kruskal-Wallis, Dunn's multiple comparisons test. (E) Representative Western blot analysis for HA tagged NDUFB7, GAPDH and persulfidated NDUFB7 S-SH detected with a dimedone switch method and plotted as the relative fluorescence intensity of Daz2:Cy5/NBF in HEK293 cells transfected with wild type (WT) NDUFB7, or NDUFB7 mutated at Cys80 to alanine (C80A), Cys90 to alanine (C90A) or double mutated and co-transfected with pcDNA or a CSE and a 3MST expressing plasmid. Dithiothreitol (DTT) was used as a negative control for the removal of endogenous persulfidation. n = 6/group. two-way ANOVA, Tukey's multiple comparisons test. (F) Representative Western blot analysis for CSE, 3MST, HA tagged NDUFB7, Vinculin and persulfidated NDUFB7 S-SH detected as the relative fluorescence intensity of Daz2:Cy5/NBF in murine HL-1 cardiomyocytes infected with lentiviruses expressing a wild type (WT) NDUFB7, or NDUFB7 mutated at Cys80 to alanine (C80A), Cys90 to alanine (C90A) or double mutated. DTT was used as a negative control. n = 6/group. one-way ANOVA, Tukey's multiple comparisons test.