Figure 1. Catalytically active SIRT4-eGFP increases mitochondrial ROS production after CCCP-induced mitochondrial uncoupling. HEK293 cell lines stably expressing eGFP, SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(Δ28N)-eGFP were treated with CCCP (10 nM) for two hours either with or without pretreatment with MitoQ (100 nM; 16 hours). Thereafter cells were stained with the cell-permeant dyes MitoTracker® Deep Red and MitoSOX™ Red followed by flow cytometric analysis. Shown are mean ± s.d. values from nine experiments. To evaluate statistical significance (comparison of eGFP vs. wild-type or mutant SIRT4 or comparisons indicated by brackets) two-way ANOVA followed-up by Tukey’s test were performed (**p<0.01; ***p<0.001). The effect of MitoQ treatment was evaluated using a paired t-test (#p<0.05).