Fig. 1. Isolation of GFP-BAK containing SMALPs.

A Loss of TMRE fluorescence due to mitochondrial depolarization is used as a proxy for MOMP progression in the cell population. U2OS BAK KO cells stably transfected with mEGFP-BAK were incubated with 100 nM of TMRE prior to apoptosis induction with ABT-737 (1 µM) and S63845 (1 µM) and measured every 5 min by confocal microscopy. B Redistribution of mEGFP-BAK into foci over time is also used as a proxy for MOMP progression upon apoptosis induction as in (A). C Representative images of healthy (control) and apoptotic cells expressing mEGFP-BAK (green) from 4 independent experiments. Scale bar, 10 µM. D Strategy to isolate SMALPs enriched in mEGFP-BAK from apoptotic cells. U2OS BAK KO cells stably transfected with mEGFP-BAK were incubated with ABT-737 (1 µM) and S63845 (1 µM) for 50 min. Isolated mitochondria from these cells were solubilized with 0.5% SMA co-polymer, and the resulting mEGFP-BAK-containing SMALPs were enriched by affinity purification using GFP-Trap©. “This method adapted from article was published in Molecular Cell, Volume 82, Cosentino et al., The interplay between BAX and BAK tunes apoptotic pore growth to control mitochondrial-DNA-mediated inflammation, Pages 933-949. CC BY license http://creativecommons.org/licenses/by/4.0/ (2022).” E Analysis of SMALP size and homogeneity using negative-stain TEM (left representative images from 2 independent experiments) and dynamic light scattering (DLS) (right). The diameter of the nanoparticles varies between 10 and 12 nm in EM images. Scale bar, 50 nm. A diameter of 12 ± 2 nm was calculated from DLS data. F Western blot (WB) analysis of fractions immunoprecipitated from purified mEGFP-BAK-SMALPs from healthy and apoptotic cells. Immunoblots show total input (T), flow through (FT), wash (W) and elution (E) fractions probed with antibodies against GFP. Representative of three independent experiments. Source data are provided as a Source Data file.