Fig. 5: MOCCI reduces CIV activity, membrane potential, and ROS production. | Nature Communications

Fig. 5: MOCCI reduces CIV activity, membrane potential, and ROS production.

From: Coding and non-coding roles of MOCCI (C15ORF48) coordinate to regulate host inflammation and immunity

Fig. 5

a (Top) Schematic uncoupled electron flow assay on Agilent’s Seahorse platform to measure CIV activity in isolated mitochondria using TMPD/ascorbate as electron donor (see Methods). (Bottom) Seahorse uncoupled electron flow analysis of AAV-MOCCI and AAV-GFP isolated mouse heart mitochondria. Oxygen consumption rate (OCR) was normalized with citric synthase (CS) activity. Dotted line shows the compounds injected at each time point. CIV activity = maximal activity after TMPD addition − minimal activity after azide injection. AA antimycin A, TMPD N,N,N′,N′-Tetramethyl-p-phenylenediamine dihydrochloride. Data were presented as mean and SEM of 3 biological replicates. b CIV activity of AAV-MOCCI and AAV-GFP isolated mouse heart mitochondria as measured by uncoupled electron flow assay on Seahorse shown in (a). Each column represents one mouse, and each dot represents one technical replicate. Data were presented as mean ±  SEM; P = two-tailed unpaired Student’s t test; n = 3 biological replicates with 6 technical replicates each. c CIV activity of digitonin (0.0025%) permeabilized MOCCI and control cells as measured by Seahorse. Data were presented as mean ±  SEM; P = two-tailed unpaired Student’s t test; n = 6 technical replicates each. d CIV activity of MOCCI and control A549 cells as measured by respiratory chain enzyme assay (RCA). Data were presented as mean ±  SEM; P = two-tailed unpaired Student’s t test; n = 8 biological replicates each. e Membrane potential of MOCCI and control cells as measured by flow cytometry of TMRE or JC-1 dye incorporation. Data are presented as mean ±  SEM; P = one-way ANOVA; n = 4 biological replicates per condition from two repeats. f Total cellular ROS levels of MOCCI and control cells as measured by flow cytometry of DCF dye reaction. Data were presented as mean ±  SEM; P = one-way ANOVA; n = 3 biological replicates per condition. g Total cellular ROS levels of MOCCI and control cells as measured by Amplex Red assay. Data were presented as mean ±  SEM; P = one-way ANOVA; n = 4 biological replicates per condition.

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