Abt 199

Imatinib, nilotinib and dasatinib were from Selleck Chemicals (Houston, TX, USA). ABT-737, ABT-199, A-1331852 and A-1210477 were kindly provided by AbbVie (North Chicago, IL, USA). Antibodies against BIM, PUMA, BMF, BIK, BAD, BCL-X_L and BCL-w were from Cell Signaling Technology (Danvers, MA, USA), GAPDH and MCL-1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA), NOXA from Calbiochem (Darmstadt, Germany), BCL-2 from Dako (Ely, UK), BAX and BAK from Millipore (Watford, UK), HRK from Aviva Systems Biology (San Diego, CA, USA) and BID and BFL-1 were from Prof J Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands). All other reagents, unless mentioned otherwise, were from Sigma-Aldrich (St Louis, MO, USA).

ABT-199, A-1331852 and A-1210477 were kindly supplied by Abbvie Inc., (North Chicago, IL, USA). Peptides for BIM (MRPEIWIAQELRR IGDEFNA), BAD (LWAAQRYGRELR RMSDEFEGSFKGL), MS-1 (RPEIWMTQGLRRLGDEINAYYAR), HRK (WSSAAQLTAARLKALGDELHQ) and PUMA-2A (EQWAREIGAQARRMAADLNA) were from New England Peptide (Gardner, MA, USA) or GenScript (Piscataway, NJ, USA). Other reagents were from Sigma-Aldrich Co. (St. Louis, MO, USA).

In vivo grade ABBV-075 and ABT-199 for mouse xenograft experiments were kindly provided by AbbVie, Inc. In vitro grade A-1210477 (Catalog No. S7790), ABBV-075 (Catalog No. S8400) and ABT-199 (Catalog No. S8048) were purchased from Selleck Chemicals (Houston, TX) and utilized for in vitro experiments. All compounds were prepared as 10 mM stocks in 100% dimethyl sulfoxide (DMSO) and frozen at −80 °C in 5–10 µL aliquots to allow for single use, thus avoiding multiple freeze-thaw cycles that could result in compound decomposition and loss of activity. Anti-BRD4 (RRID:AB_2620184) antibody was obtained from Bethyl Labs (Montgomery, TX). Anti-c-Myc (RRID: AB_1903938), anti-HEXIM1 (Cat #12604), anti-p21 (RRID: AB_823586), anti-p-Histone H2AX (Ser139) (RRID: AB_2118010), anti-Bcl-xL (RRID: AB_10695729), anti-BAX (RRID:AB_2744530), anti-BAK (RRID:AB_2290287), anti-Cleaved PARP (RRID:AB_331426), anti-BIM (RRID:AB_1030947) and anti-MCL1 (RRID:AB_2281980) antibodies were obtained from Cell Signaling Technologies (Beverly, MA). Anti-CDK6 (RRID: AB_10610066), anti-Bcl2 (RRID: AB_626733), Alexa488-conjugated anti-BAX(6A7) (RRID:AB_626728) and anti-β-Actin (RRID: AB_626630) antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). anti-BAK(NT) (RRID:AB_310159) antibody was obtained from Millipore Sigma (Burlington, MA).

ABT-199 and ABT-737 were provided by AbbVie.

Human leukemic cell lines SU-DHL-6, OCL-LY-19, SU-DHL-16 (DLBCL), and DOHH2 (FL) were obtained from Dr. Eric Hsi and Dr. Neetu Gupta (Cleveland Clinic), Nalm6, Reh (acute lymphoblastic leukemia) were purchased from the American Type Culture Collection (Manassas, VA, USA), Mec-2 from Dr. Y. Saunthararajah (Cleveland Clinic) and MO1040 (both CLL) from Dr. Riccardo Dalla-Favera (Columbia University) and used as before.^{32 (link), 33 (link), 45 (link)} All cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), and antibiotic-antimycotic (Gibco, Life Technologies, Gaithersburg, MD, USA). ABT199-R cells were cultured with 5% FBS. Cell lines were routinely screened for Mycoplasma, variations in growth rates, changes in morphological characteristics, and their response to stress with Annexin V FITC-PI staining; their passage number did not exceed 20. ABT-737 and ABT-199 were obtained from AbbVie (Chicago, IL, USA); NVP-BEZ235, RAD001, and GS-1101 from Selleck Chemicals (Houston, TX, USA); and verapamil and cycloheximide from Sigma-Aldrich (St. Louis, MO, USA).

5-aza (Sigma-Aldrich) was reconstituted in PBS. Cultured cells were treated with 0.5 μM 5-aza. Mice were intraperitoneally injected with 5-aza at 2.5 mg per kilogram body weight per day for 1 week per treatment cycle unless otherwise indicated.
ABT-199 was provided by AbbVie. For in vitro experiments, ABT-199 was dissolved in DMSO and diluted in PBS. Cells were treated with 50 nM ABT-199 unless otherwise indicated. For in vivo experiments, fresh suspensions of ABT-199 in a mix of Phosal 50 PG (Lipoid), polyethylene glycol (PEG) 400 and ethanol (60:30:10) were prepared weekly and stored at 4 °C. ABT-199 was administered by gavage at a dose of 100 mg per kilogram body weight per day.
BMS-345541 was purchased from Selleck Chemicals. For in vitro experiments, BMS-345541 was reconstituted in DMSO and diluted in sterile PBS. Cells were treated with 5 μM BMS-345541. For in vivo experiments, suspensions of BMS-345541 in a mix of DMSO, PEG 300, Tween 80 and water (5:30:5:60) were freshly prepared and stored at RT. BMS-345541 was administered every other day by gavage at 75 mg per kilogram body weight per day for 3 weeks (ten doses total).
Human-recombinant TNF (R&D Systems) was reconstituted in 0.1% FBS/sterile PBS. Cells were treated with 2.5 ng ml⁻¹ TNF.