Figure 4. Oxygen consumption is reduced in HEK293 cells expressing SIRT4-eGFP. Respirometric measurements of oxygen consumption were performed in HEK293 cell lines stably expressing SIRT4-eGFP (A), SIRT4(H161Y)-eGFP (B), or SIRT4(Δ28N)-eGFP (C) as previously described [52,64]. Oxygen consumption was measured under basal and stressed conditions (leak: treatment with oligomycin A, 2 µg/ml; ETS: mitochondrial uncoupling of the electron transport system using CCCP, 400-500 nM; residual oxygen consumption, ROX: treatment with 500 nM rotenone and 2.5 µM antimycin A) as compared to control cells expressing eGFP. To evaluate statistical significance two-way ANOVA followed by Tukey’s test was performed [**p<0.01, ***p<0.001; SIRT4-eGFP: n=5; SIRT4(H161Y)-eGFP, n=4; SIRT4(Δ28N)-eGFP: n=4].