Dynamic BH3 profiling

DBP experiments were performed as previously described^22,23. In brief, 3 × 10⁴ cells/well in a 96-well plate were used for cell lines. 25 μL of BIM BH3 peptide (final concentration of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM), 25 μL of BAD BH3 peptide (final concentration of 10 μM), 25 μL of HRK BH3 peptide (final concentration of 100 μM), and 25 μL of MS1 BH3 peptide²⁴ (final concentration of 10 μM) in MEB (150 mM mannitol, 10 mM hepes–KOH pH 7.5, 150 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1% BSA, 5 mM succinate) with 0.002% digitonin were deposited per well in a 96-well plate (3795, Corning, Madrid, Spain). Single cell suspensions were stained with the viability marker Zombie Violet (423113, BioLegend, Koblenz, Germany) and then washed with PBS and resuspended in MEB in a final volume of 25 μL. Cell suspensions were incubated with the peptides for 1 h following fixation with formaldehyde and staining with cytochrome c antibody (Alexa Fluor^® 647 anti-Cytochrome c—6H2.B4, 612310, BioLegend). Individual DBP analyses were performed using triplicates for DMSO, alamethecin (BML-A150-0005, Enzo Life Sciences, Lorrach, Germany), the different BIM BH3 concentrations used, BAD, HRK, and MS1 BH3 peptides. The expressed values stand for the average of three different readings performed with a high-throughput flow cytometry SONY instrument (SONY SA3800, Surrey, UK). % priming stands for the % of cytochrome c release obtained from different BH3 peptides, and Δ% priming represents the difference between treated cells minus non-treated cells for a given peptide.