Figure 1.
BAK assembly into distinct structures correlates with mitochondrial outer membrane permeabilization
(A) Scheme of the assay to monitor Smac release (magenta) from mitochondria to the cytosol due to mEGFP-BAK (green) foci formation.
(B) Representative confocal images of a BAX/BAK DKO HCT116 cell transfected with mEGFP-BAK (green) and Smac-(1-60)-mCherry (magenta) before (0 min) and 16 and 36 min after apoptosis induction with 1-μM STS. Scale bars, 10 μM.
(C) Maximum fluorescent intensity of mEGFP-BAK (green) and mean fluorescent intensity of cytosolic Smac-(1-60)-mCherry (magenta) for individual cells as in (B) (n = 4). Time point 0 min corresponds to the normalized time point of Smac-mCherry release. The release of Smac-mCherry in the cytosol correlates with an increase in the mEGFP-BAK intensity, which is indicative of foci formation. Bold lines represent the mean of individual dataset (light lines).
(D) Percentage of TMRE positive BAX/BAK DKO HCT116 cells transfected with mEGFP-BAK after treatment with 1 μM STS (n = 2). Error bars represent the SD.
(E and F) Representative SMLM images of mEGFP-BAK in BAX/BAK DKO HCT116 cells in healthy (E, mitochondria profile is defined by the dotted lines) and apoptotic conditions (F). Right panels are zoomed regions (indicated by white boxes in the main image). Scale bars: 1 μM in the main images and 500 nm in zoomed regions.