Cq1 microscope

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)).

Human paraffin embedded tissue microarray (TMA: #SK2444A and #SKN1001, see Supplementary Table S4 for details) were purchased from US Biomax (Derwood, MD, USA). Antigen retrieval (10 mM sodium citrate buffer, 0.05% Tween20) was performed at 95 °C for 20 min before immunostaining. RHE were prepared for immunofluorescence as previously described [6 (link)]. The following steps are similar for both TMA and RHE sections. After dewaxing and rehydration, tissue sections were permeabilized using PBS 0.1% Triton X-100, 0.1 M glycine during 10 min at room temperature (RT). Samples were then blocked with PBS containing 5% goat serum, 2% BSA and 0.1% Tween20 for at least 1 h at RT. After washing steps, primary antibodies were incubated overnight at 4 °C (Supplementary Table S2). After washings, secondary antibodies were incubated 45 min at RT and nuclear staining was performed using ProLong^TM Glass Antifade Mountant with NucBlue^TM (Thermo Fischer Scientific, #P36981). Negative controls were performed by omitting primary antibodies. Images were visualized using High Content Screening Yokogawa CQ1 microscope (Yokogawa, Tokyo, Japan), digitalized using sCMOS camera (Olympus, Hamburg, Germany) and analysed using ImageJ software (version 2.1.0/1.53c).

HeLa cells expressing doxycycline‐inducible Parkin and stable mCherry‐GFP‐LC3C WT or S93/96A HeLa cells were seeded onto black, clear flat‐bottom 24‐well plates and treated with 1 μg/ml doxycycline and 10 μM CCCP for 16 h. Cells were incubated with Hoechst 33342 (R37605; Thermo Fisher) and washed in PBS before fixation with 4% paraformaldehyde for 15 min at room temperature. Plates were imaged using a Yokogawa CQ1 microscope. After Yokogawa CQ1 plate imaging, the z‐stack images were analyzed using the CellPathfinder software. The cell number was determined by detecting the cell nucleus stained with Hoechst, and mCherry or GFP‐positive puncta were counted by the software.