CCCP induces Bif-1 and LC3 translocation to fragmented mitochondria.myc-Parkin/Bif-1^−/− mouse embryonic fibroblasts were transduced with lentiviruses encoding Bif-1–AcGFP and Mito-mCherry. (A-B) The cells were treated with 30μM CCCP for 4 hours (A) or 20 hours (B) and then analyzed by time-lapse fluorescent microscopy at 5-minute (A) or 1-minute (B) intervals. The time stamps delineate the incubation time periods in hours (°) and minutes (′) after the initiation of time-lapse imaging. (C) Cells treated with 30μM CCCP for 20 hours, immunostained for LC3, and analyzed by fluorescence deconvolution microscopy. Magnified images are shown in the insets. Arrows indicate colocalization of Bif-1–AcGFP, Mito-mCherry, and LC3. (D) The fluorescence intensities along the dotted line in panel D were quantified using SlideBook Version 5.0 software. The values of the vertical axis represent fluorescence intensity units (ADU). The horizontal axis represents distance (S indicates start point; and E, end point). The scale bars represent 10 μm in the left panels in panels A and B and 5 μm in the right panels of time-lapse images in panels A through C.