Mitochondrial Protein Import Efficiency

HeLa cells were transiently transfected with the MTS-EGFP plasmid (gift from David Chan, Addgene # 23214) for 24 h (Chen et al., 2003 (link)). After 4 h of transfection, medium was exchanged and cells were treated with 10 μM CCCP (Sigma-Aldrich, C2759) or DMSO overnight. Cells were stained with 200 nM Mitotracker Red FM (Thermo Fisher Scientific, M22425) for 30 min prior to imaging, washed once with 1x PBS and incubated in medium during live imaging on a Yokogawa CQ-1 microscope. Images were acquired at 488 nm excitation and 525/50 nm emission for GFP, and 561 nm excitation and 685/40 nm emission for Mitotracker Red FM. One-hundred cells per biological replicate were manually categorized in one of four categories to assess the efficiency of mitochondrial protein import of MTS-EGFP, using ImageJ 1.53c (Schneider et al., 2012 (link)).