Figure 2. SIRT4-eGFP expression leads to an increased autophagic flux upon CCCP-induced mitochondrial uncoupling. (A) HEK293 cell lines stably expressing eGFP, SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(Δ28N)-eGFP were treated with DMSO (control), rapamycin (100 nM), or CCCP (10 µM) for two hours. In addition, during the second hour cells were either untreated (-) or co-treated (+) with BafA1 (Bafilomycin A; 100 nM) that stalls autophagic flux via inhibition of the fusion between lysosomes and autophagosomes. A representative experiment is depicted in which LC3B-I and LC3B-II levels were analyzed by immunoblotting. (B) LC3B-II signals (co-treatment with BafA1) were compared to the protein levels of β-Actin/ACTB as loading control using ImageJ based quantification. Data shown are mean ± s.d. values from four to seven experiments. To evaluate statistical significance (treatment vs. DMSO) two-way ANOVA followed by Tukey’s test was performed (**p<0.01).