4.3. BH3 Profiling—Plate-Based Assay

Cells were incubated at 0.5 × 10⁶ cells/mL for 18 h with AZD1775 (WEE1 inhibitor, Selleckchem No.S1525, Houston, TX, USA) (Figure S8), palbociclib (CDK4/6 inhibitor, Selleckchem No.S1116), RO3306 (CDK1 inhibitor, Selleckchem No.S7747), roscovitine (CDK2 inhibitor, Selleckchem No. S1153), nocodazole (M1404, Sigma Aldrich, St. Louis, MO, USA), or ultraviolet (UV) radiation (IBL 637, CisBioInternational, Gif-sur-Yvette, France). After incubation, cells were washed with mannitol experimental buffer (MEB) (150 mM D-mannitol (M9647, Sigma Aldrich,), 10 mM HEPES (H3375, Sigma Aldrich), 50 mM KCl (1.04936, Merck, Darmstadt, Germany), 20 nM EGTA (E4378, Sigma Aldrich), 20 nM EDTA (11280, Serva Electrophoresis, Heidelberg, Germany), 0.1% BSA (11930, Serva Electrophoresis), 5 mM succinate (1.00682, Merck) in dH2O, pH 7.5) and resuspended at 3.2 × 10⁶ cells/mL in MEB. A cell suspension was mixed 1:1 with 4 µM JC-1 permeabilization/staining solution ((ENZ-52304, Enzo Life Sciences, Farmingdale, NY, USA), 0.004% digitonin (1500 643, Boehringer Mannheim, Mannheim, Germany), 20 mM β-mercaptoethanol (8.05740, Merck) and 40 µg/mL oligomycin (O4876, Sigma Aldrich) prepared in MEB) and incubated at room temperature in the dark for 10 min. BIM, PUMA, BAD, NOXA, MS1, HRK, BMF, and PUMA2A (JPT Peptide Technologies, Berlin, Germany) were prepared in MEB in a black flat-bottom non-treated polystyrene 96-well plate (3915 Costar, Corning Incorporated, Kennebunk, ME, USA). Peptide sequences used for the assay were identical to those described in Ryan and Letai [18]. Plates were either used directly or sealed (Silverseal sealer ref 676090, Greiner-Bio-One, Frickenhausen, Germany), frozen at −80 °C and thawed for 1 h at room temperature before use. Cells in permeabilization/staining solution were added to the plate 1:1 at a final volume of 100 uL and shaken for 15 s, followed by measurement of fluorescence (excitation 545 nm, emission 590 nm) every 5 min for 2 h at 30 °C (Varioskan). All experiments were performed in triplicates or quadruplicate. The area under the curve (AUC) was calculated as a percentage of mitochondrial outer membrane permeabilization (MOMP) and normalized to PUMA2A (negative control) and FCCP (positive control) with the formula: 1 − ((AUC sample − AUC FCCP) ÷ (AUC PUMA2A − AUC FCCP)) × 100%. The dynamic BH3 profile (ΔMOMP) was calculated by subtracting the percentage treated MOMP from percentage untreated MOMP. Based on the specific interacting partners of the BH3 peptides [18], the cell specific anti-apoptotic dependency of cells can be established. BH3 profiling with a mean ΔMOMP ≥ 20% were classified as biologically relevant, even if they were not always statically significant. ΔMOMP changes for the BIM peptide indicates cells have become more primed for apoptosis or closer to their apoptotic threshold. Similarly, ΔMOMP changes for NOXA, HRK, or BAD indicate cells have become more dependent on MCL-1, BCL-XL, or BCL-2/XL/W, respectively. Together, these data can predict on which anti-apoptotic protein cells are dependent, and how this changes upon inhibitor treatment.