Fig. 6: MacroD1-mediated regulation of Ndufb9 MARylation and its influence on mitochondrial complex I activity in response to LPS challenge. | Nature Communications

Fig. 6: MacroD1-mediated regulation of Ndufb9 MARylation and its influence on mitochondrial complex I activity in response to LPS challenge.

From: Cardiomyocyte mitochondrial mono-ADP-ribosylation dictates cardiac tolerance to sepsis by configuring bioenergetic reserve in male mice

Fig. 6

A Co-IP assay for Ndufb9 MARylation in the hearts of mice injected intraperitoneally with and without LPS (10 mg/kg) for 18 h. B Co-IP assay for Ndufb9 MARylation in AC16 cells transfected with MacroD1 siRNA (Si-M) or scrambled control (NC) for 36 h and then treated or untreated with LPS (1 μg/ml) for 18 h. C Co-IP assay for Ndufb9 MARylation in AC16 cells transfected with Si-M, His-MacroD1, or His-MacroD1 mutant plasmids for 48 h. D Mass spectrum identification of the MARylation site of Ndufb9 based on mitochondrial lysates isolated from mice hearts with anti-MARylation antibody. E Sequence conservation display on the MARylation site of Ndufb9 from Homo sapiens, Mus musculus, and Rattus. The red dotted box indicates the MARylation site of Ndufb9. F MARylation site mutation display of the Ndufb9 sequence. The red dotted box indicates the MARylation site mutation (Ndufb9R173A). G Co-IP assay for Ndufb9 MARylation in AC16 cells transfected with Flag-Ndufb9 or Flag-Ndufb9R173A plasmid for 36 h and then treated with/without LPS (1 μg/ml) for 18 h. H Co-IP assay for Ndufb9 MARylation in AC16 cells transfected with the indicated siRNA or plasmids for 48 h. I–L Mitochondrial complex I activity (I; n = 5 cell samples from independent experiments), ATP levels (J; n = 5 cell samples from independent experiments), and MitoSOX fluorescent staining analysis (K, L; n = 5 cell samples from independent experiments) in AC16 cells transfected with indicated siRNA or plasmids for 36 hours and treated with/without LPS (1 μg/ml) for 18 h. The targeted sequence of the Ndufb9 plasmid corresponding to Si-Ndufb9 was synonymously mutated to avoid the interference of Si-Ndufb9 (Flag-SM-Ndufb9). Flox: MacroD1flox/flox controls; cKO: MacroD1flox/flox/αMHCMerCreMer mice. Data are shown as the mean ± SD. Data analysis was conducted utilizing one-way ANOVA with Tukey’s multiple comparison test. Source data are provided as a Source Data file.

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