Figure 3. SIRT4-eGFP expression results in overall decreased mitochondrial recruitment of mCherry-Parkin upon CCCP-induced mitochondrial uncoupling. (A) HEK293 cell lines stably expressing eGFP, SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(Δ28N)-eGFP (GFP signal in white) were transfected with an expression construct for mCherry-Parkin (red). After one day, cells were treated with CCCP (10 µM) for two hours followed by co-treatment with BafA1 (100 nM) during the second hour. Cells were fixed and subjected to confocal microscopic analysis. Mitochondria were detected using the marker MTC02 (green). (B) Quantification of the mitochondrial content via MTC02 staining analysis using ImageJ software (Material & Methods and suppl. Material & Methods). (C) Quantification of mCherry-Parkin/MTC02 double-positive dots was performed based on the mitochondrial content as determined in (B) using ImageJ software (Material & Methods and suppl. Material & Methods). Numbers of cells analyzed in (B) and (C) from four experiments: eGFP, 57; SIRT4-eGFP, 55; SIRT4(H161Y)-eGFP, 43; SIRT4(Δ28N)-eGFP, 48. To evaluate statistical significance two-way ANOVA followed by Tukey’s test was performed (*p<0.05).