Fig. 3. Fate of BID following BSB stimulation.

a Colocalization of pDsRed2-BID with the specific mitochondrial (MitoTracker Deep Red FM) and lysosomal (LysoTracker green DND-26) dyes was analyzed by time-lapse imaging confocal microscopy. pDsRed2-BID-transfected HeLa cells were stained with the probes, stimulated with 80 μM BSB and monitored for 3 h. One hour after addition of BSB, the uniformly distributed red fluorescence in the cytosol, spatially distinct from the blue granular mitochondrial and the green granular lysosomal ones, appeared translocated and colocalized with mitochondria (purple) and lysosomes (yellow), as clearly appreciable at the level of the single optical slices. Three hours after addition of BSB, the apoptotic features were clear: the uniform red pattern was completely replaced by red clusters; blue and green fluorescences (and respective colocalizations) decreased as a consequence of dye leakage. Scale bar: 17 μm. b Key frames selected from Supplementary Movie 1. 80’ stimulation with BSB led to the overlap between green-stained mitochondria and red fluorescent BID, generating yellow signals (white arrows). Scale bar: 17 μm. c Kinetic western blot demonstrating the appearance of tBID and, to a lesser extent, full-length BID in the mitochondrial fraction. The positive control (C+) of BID cleavage is etoposide. The shown experiment is representative of three.