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. 2022 Mar 3;82(5):933–949.e9. doi: 10.1016/j.molcel.2022.01.008

Figure 4.

Figure 4

BAX and BAK foci assembly proceeds via a different mechanism of oligomerization

(A) Representative confocal images of a BAX/BAK DKO U2OS cell transfected with mEGFP-BAX (green), Tomm20-BFP (blue) and Smac-1-60-mCherry (magenta) and treated with ABT-737, S63845 and qVD-Oph for apoptosis induction. Time points are after Smac-1-60-mCherry release from mitochondria. Scale bars, 10 μm. Inset: apoptotic foci of mEGFP-BAX growing over time on mitochondria. Scale bars, 3 μm.

(B) Representative photon-counting confocal images of BAX/BAK DKO U2OS cells transfected with mEGFP-BAK, mEGFP-BAX, or mEGFP-BAX(S184V) and treated for apoptosis induction. Scale bars, 10 μm.

(C) Average mEGFP expression level per mitochondrial area in individual cells (represented by individual dots in the plot) transfected with mEGFP-BAK, mEGFP-BAX, or mEGFP-BAX(S184V) detected by single-cell fluorescent intensity analysis of mEGFP signal. The error bars correspond to the SD from the different experiments.

(D–F) Representative distribution of foci intensity, in stoichiometric units, at different time points in individual apoptotic cells overexpressing mEGFP-BAX (D), mEGFP-BAK (E), or mEGFP-BAX(S184V) (F).

(G) Average foci intensity obtained from the fitting of foci distributions as in (D–F) at each time point for individual apoptotic cells expressing mEGFP-BAX (yellow, n = 9), mEGFP-BAK (blue, n = 9) or mEGFP-BAX(S184V) (purple, n = 4). Lines in the graph correspond to the average values from all measured cells and colored areas correspond to data variability from single cells (mean ± SD).

(H and I) Number of foci with molecularity higher than 200 (H) or 400 (I) normalized to the total number of foci in the cell at different time points after Smac release. Average total n° foci over analyzed cells is ranging from 67 (at 5 min) to 517 (60 min) for mEGFP-BAX (n = 9 cells) and from 250 (at 5 min) to 481 (60 min) for mEGFP-BAK (n = 9 cells). The error bars correspond to the SD from the different experiments.

(J) Number of foci per mitochondrial area over time for apoptotic cells as in (G). See also Figures S5 and S6.