BH3 profiling and chemosensitivity methods

BH3 profiling was conducted by flow cytometry according to published protocols (^{Fraser et al, 2018}). Briefly, cells in culture were trypsinized and added to wells of prepared 96 well plates containing the indicated peptide conditions and .001% digitonin in mannitol experimental buffer (MEB; 10 mM HEPES (pH 7.5), 150 mM mannitol, 50 mM KCl, 0.02 mM EGTA, 0.02 mM EDTA, 0.1% BSA, and 5 mM succinate). Peptide treatments were carried out for 60 minutes at 28 degrees C, then cells were fixed for 10 minutes in 2% PFA. Fixation was quenched with N2 buffer (1.7 M tris base and 1.25 M glycine (pH 9.1)), then cells were stained overnight with DAPI and an Alexa Fluor 647-conjugated anti-cytochrome c antibody (Biolegend, clone 6H2.B4). Stained cells were analyzed using an Attune NxT flow cytometer, with gates drawn based on cytochrome c staining in the negative and positive control treatments (PUMA2A and DFNA5 peptide). The percentage of cytochrome c negative cells was reported for each peptide treatment condition.

For chemosensitivity assays, cells were plated at 10⁴ cells per well in 100 μl culture medium on 96-well flat-bottom plates (Denville). They were treated with the following drugs at specified concentrations: etoposide 10 μM, staurosporine 0.1 μM, and doxorubicin 1 μM. After 24 hours in standard tissue culture conditions, cells were stained with Alexa Fluor 488-conjugated Annexin V in 10× Annexin binding buffer [0.1 M Hepes (pH 7.4), 1.4 M NaCl, and 25 mM CaCl₂ solution]. Alexa Fluor 488-conjugated Annexin V was added to the solution at a 1:500 dilution. The staining solution was added to the cells at a 1:10 dilution, and the cells were allowed to stain for 20 minutes on ice in the dark. Annexin V positivity was measured by Attune flow cytometer equipped with an autosampler (Thermo Fisher Scientific). Biological replicates indicate assay performed on different flasks of MEF cell culture grown under the same condition and passage number. Statistical analysis was performed using a two-way ANOVA with Holm-Sidak’s correction for multiple hypothesis since the data were normally distributed (^{Fraser et al, 2022; Singh et al, 2023}). All statistical analyses were performed in Prism 9 GraphPad software.