In vitro activity: WEHI-539 hydrochloride is a selective inhibitor of Bcl-XL, it augments carboplatin induced caspase 3/7 activity, PARP cleavage and annexin V labelling. As a single agent, WEHI-539 causes noticeable PARP cleavage in Ovcar-4 (5 μM in Ovcar-4.) and Ovsaho (1 μM in Ovsaho) cells.

Kinase Assay: The assay buffer (stock 50 mM HEPES and 100 mM NaCl, pH 7.5) is prepared fresh daily and adjusted to 5 mM DTT, casein (0.1 mg/mL sodium salt; aliquots stored at -20 °C) and Tween 20.

Cell Assay: WEHI-539 is dissolved in DMSO (20 mM) and stored, and then diluted with appropriate media before use. Ovcar-8, Ovcar-3, Ovcar-4 and Ovsaho cells are grown in the RPMI, Igrov-1, Cov-362 and Cov-318 cells are grown in DMEM and Fuov-1 cells are grown in DMEM/F-12 nutrient mixture. ABT-737, ABT-199 and WEHI-539, are prepared as a 20 mM solution in DMSO. For cell growth assays, cells are plated in 96 wells plate (5,000 cells/well for all cell lines except Ovcar-8 which is plated at a density of 2,500 cells/well). The next day, cells are treated with drugs. After 72 h the culture medium is removed and the cells are fixed with 100 μL of cold 10 % Trichloroacetic acid (TCA), incubated on ice for 30 min and stained with 0.4 % sulforhodamine B (SRB). The data are analysed by using Graphpad Prism 4 software. Non-linear regression is used to fit a four parameters Hill equation. For drug combinations studies the cells are exposed simultaneously to a range of concentrations of carboplatin combined with fixed concentration of BH3 mimetics that is expected from the single agent studies to cause 5 % growth inhibition: ABT-737, 1 μM in Ovcar-8, Ovcar-3 and Igrov-1, 2 μM in Ovcar-4 and Ovsaho and 6 μM in Cov-362; ABT-199, 1 μM in Ovcar-4, 2 μM in Ovcar-3, Igrov-1, Cov-362 and Ovsaho and 3 μM in Ovcar-8; WEHI-539, 0.2 μM in Igrov-1, 0.3 μM in Ovcar-8, 1 μM in Ovcar-3 and Ovsaho, 3.1 μM in Cov-362 and 5 μM in Ovcar-4. Surviving cell number is assessed by SRB staining. A combination index (CI) is calculated.