Umi 77

Pools of BIMs2A or empty vector (EV) cells were made by routine cloning into an all-in-one tetracycline inducible vector (SH570MK as detailed in Additional file 1) and selected using Puromycin. BIMs2A expression was induced with 2 μg/ml DOX or vehicle control daily in the media and cells were harvested at the time points indicated in the figures. Annexin V PI staining was performed using the Annexin V-FITC Apoptosis Kit (Biovision, CA, USA) as per the manufacturer’s instructions. ABT-263 (5 μg/ml) was added to the media at the indicated times. For siRNA experiments, 5 nM siRNAs targeting MCL-1 (DHA-L-004384-00-0005) or non-targeting control siRNA (D-001206-14-05) was premixed with RNAiMAX (ThermoFisher) and cells were transfected the day after plating at a density of 1 × 10⁵ cells per well with ON-TARGETplus SmartPools of MCL-1 or nontargeting controls siRNAs as per the manufacturer’s instructions. A1210477 and UMI-77 (Selleckchem, MA, USA) was added to the media at 5 or 10 μM respectively as indicated in the figures. Dasatinib (Bristol-Myers Squibb, Princeton, NJ, USA) was added to the media in 2D and 3D at a concentration of 1 μM and 200 nM respectively. 3D collagen I/fibroblast models were performed as described previously [20 ].

Doxorubicin was obtained from the Pharmacy at the Leiden University Academic Hospital, ABT-737, UMI-77 and HA14-1 were from SelleckChem (Huissen, Netherlands). WEHI-539 was from ApexBio (Texas, U.S.A.). The pan-caspase inhibitor z-VAD-fmk was obtained from Bachem (Weil am Rhein, Germany). The Bcl-xL antibody (clone 54H6) used for immunohistochemistry, the Bcl-xL antibody (2762s) used for Western blot and Bak antibody were from Cell Signaling (Bioké, Leiden, The Netherlands). LC3 antibody was from Novus Biologics (Cambridge, England) and Ki67 was from Abcam (Cambridge, England). Chloroquine was bought from Sigma Aldrich (Zwijndrecht, The Netherlands). Hoechst 33342 was purchased from Fischer Scientific (Bleiswijk, The Netherlands).

S55746 was obtained from Servier; A1210477 from Active Biochem; and ABT-199, UMI-77, and Z-VAD-fmk from Selleck Chemicals.

Stock solutions (10 mM) of trametinib, ABT-199, ABT-263, AT-101, sabutoclax, and UMI-77 (Selleckchem, Houston, TX, USA) were prepared in DMSO and stored at −80 °C. Appropriate concentrations of each drug were prepared in DMSO prior to use. Cell lines were transfected with siRNAs to BCL-2 (ON-TARGETplus Human BCL2 SMARTpool; Dharmacon, Lafayette, CO, USA), BCL-XL (Hs_BCL2L1_2, QIAGEN, Hilden, Germany), MCL-1 (Hs_MCL1_6, QIAGEN), BIM (Hs_BCL2L11_5, QIAGEN), and BAD (Hs_BAD_3, QIAGEN) using 4D-Nucleofector system (Lonza, Basel, Switzerland). AllStars Negative Control siRNA (QIAGEN) was the negative control.
The BIM-GFP expression plasmid was constructed by cloning the human BIM_EL cDNA (BCL2L11-001, ENST00000393256) into a pMCEF vector and moving the BamHI and AgeI restriction fragment into the pEGFP-N1 vector. The plasmid was transfected into SD1 cells using 4D-Nucleofector system (Lonza) and GFP⁺ cells were sorted by flow cytometry after 48 h.

Cell lines were plated at 2000 cells per well in 384 well plates for 24 h at 37 °C. Detailed information on the cell lines and their growth conditions is provided in Additional file 2: Sheet 1. All cell lines were obtained from American Type Culture Collection (ATCC). Drugs were diluted to six doses in media containing 5% FBS (Gibco/Life technologies) and 1% anti–anti (Gibco/Life technologies). Erlotinib, trametinib, UMI-77, obatoclax, doxorubicin, and neratinib were purchased from Selleckchem, and bafilomycin and AKT1/2 inhibitor were from Sigma-Aldrich. Drugs were dissolved in 100% DMSO and stored at −80 °C. Detailed information on drug doses is provided in Additional file 2: Sheet 2. Cell viability and growth was measured using CellTiter-Glo (Promega) 72 h post-treatment. All treatment doses were performed in four replicates. The Drug Discovery Core Facility, a part of the Health Sciences Cores at the University of Utah, performed the dose response assay. EC50s (concentration of each drug that provides half of the maximum response) were determined and converted to drug sensitivity values defined as the negative log of the EC50s (−logEC50) (Additional file 2: Sheet 3). EC50 values were calculated from dose response data by plotting in GraphPad Prism 4 and using the equation Y = 1/(1 + 10ˆ((logEC50 − X) × HillSlope)) with a variable slope (Y_min = 0 and Y_max = 1).

Doxorubicin (S1208), docetaxel (S1148), navitoclax (S1001), A-1155463 (S7800), venetoclax (S8048) and UMI-77(S7531) were purchased from Selleckchem. Cyclophosphamide monohydrate was purchased from Sigma-Aldrich (C0768). RTqPCR probes are listed in the key resources table with further detail in Table S3. Antibodies are listed in the key resources table with the dilutions used detailed in Table S4.