Venetoclax abt 199

Antibodies used were to ACTIN (AC-15, Sigma #A1978), MCL-1 (Rockland #600-401-394S), BAX (N-20 Santa Cruz Biotechnology #sc-493), BAK1 (aa23-38, #B5897 Sigma), BCL-2 (#610539, BD Biosciences), Cytochrome c (#556433, BD Biosciences), VDAC2 (M.T. Ryan, Monash University), Cleaved CASP3 (#9661, Cell Signaling Technology), Cleaved CASP-9 (#9509, Cell Signaling Technology), PUMA (#ab9645, Abcam), VDAC1(Abcam, ab15895), Rabbit polyclonal antibodies raised against amino acids 19–32 of mBOK (gift from Francine Ke, WEHI^{22 (link)}), BID (BD Biosciences, #559681), BCLXL (#2764, Cell Signaling Technology), BIM, APAF1, BMF, and CASP9 are from in house (L. O’Reilly, WEHI).
BH3-mimetic ABT737 and venetoclax (ABT199) were obtained from Selleck (Houston, TX). The pan-caspase inhibitor Q-VD-OPH was purchased from MP Biomedicals Cat#03OPH109. IDN-6556 was purchased from MedChem Express LLC. Necrostatin-1 (Nec-1) was synthesized by TetraLogic Pharmaceuticals. Doxycycline and dexamethasone were purchased from Sigma-Aldrich.

Tasquinimod was purchased from Sigma-Aldrich (Diegem, Belgium) and dissolved in dimethylsulfoxide at a stock concentration of 5 mM. For the final concentration, we chose the concentrations 5, 10 and 25 µM. Venetoclax (ABT-199) was purchased from Selleck Chemicals (Houston, Texas, USA) and the final concentrations were 100, 250 and 500 nM for AML cells, and 10, 20 and 50 nM for primary AML samples.

Venetoclax (ABT-199) (1257044-40-8) was purchased from Selleck and Metformin hydrochloride (MB1927-S) was purchased from the Dalian Meilun Biotechnology Co. Cycloheximide was purchased from MCE and MG-132 was obtained from Selleck. Cell Counting Kit (CCK8, B34304) was purchased from Bimake (USA). Annexin V-FITC Apoptosis Detection Kit (C1062M) was purchased from Beyotime (Shang Hai, China). The antibodies β-Actin, BCL-2 and PARP were purchased from Cell Signaling Technology, while BAX and BCL-xl were from ABcone, MCL-1 was purchased from Affinity.

Cells were incubated at 0.1 × 10⁶ cells/mL with AZD1775 (WEE1 inhibitor, Selleckchem) for 18 h. After washing with 1% BSA in PBS, cells were incubated with venetoclax/ABT-199 (BCL-2-selective inhibitor, Selleckchem), S63845 (MCL-1 inhibitor, Selleckchem), or navitoclax/ABT-263 (BCL-XL/BCL-2/BCL-W inhibitor Selleckchem) for 48 h at 37 °C. After incubation, cells were washed with 1% BSA in PBS and stained with propidium iodide (Sigma Aldrich) to assess cell viability by flow cytometry (FACSCalibur, BD Biosciences). Data were analysed in Winlist 3D (Verity Software House, Topsham ME, USA).

Artesunate, MG132, EB, and AO were purchased from Sigma-Aldrich Inc. Venetoclax (ABT-199) was purchased from Selleck Chemical. Cytarabine and Q-VD-OPh were purchased from MedChemExpress. Antibodies to PARP and caspase-8 were obtained from BD Biosciences. Antibodies to Bcl-2 (C-2), Actin (C-2), Mcl-1 (S-19), Mcl-1 (G-7), Bax (6A7) and Chk1 (G-4) were obtained from Santa Cruz Biotechnology, Inc. Antibodies to Mcl-1 (D35A5), Bim (C34C5), Bak (D4E4), Bax poly, Noxa (D8L7U), Cleaved Caspase-3 (Asp175), Phospho-Chk1 (Ser345) (133D3), and Phospho-Histone H2A.X (Ser139) (20E3) were obtained from Cell Signaling Technology, Inc. Antibodies to Noxa were obtained from Abcam, Inc. Bak (Ab-1) was obtained from Merck Millipore. NOXA(sc-37305), BIM(sc-29802), MCL1(sc-35877)siRNA, and a control siRNA were purchased from Santa Cruz Biotechnology, Inc. NOXA(s10708), BIM(s195011), MCL1(s8583)siRNA was purchased from Thermo Fisher Scientific.

T98G, A172, LN229, MCF-10A and HMC3 human microglial cells were from ATCC; U251-MG and SF188 cells were a gift from Dr Chris Jones (ICR, London, UK). GBM cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (cat#11995065, Gibco), HMC3 cells in DMEM/F12 (cat#10565018, Gibco), and MCF-10A cells in mammary epithelial cell basal medium (MEBM) (cat#CC-3151, Lonza). Human astrocytes (cat#3P10251; Innoprot) were cultured in AM (cat#1801, ScienCell) with supplements (cat#1852, ScienCell). Mouse GSCs were isolated and cultured as described previously [47 (link)]. For radiation, cells were irradiated with a linear electron accelerator (Varian Medical Systems) using a 6MV photon field at varying doses ranging from 6−12 Grays. Dexamethasone (cat#D4920), temozolomide (cat#T2577), human PDGF-BB (cat#P3201) and LPS (cat# L5293) were from Sigma/Merck (Madrid, Spain). IFNγ (cat#300-02) was from Peprotech (London, UK) and reversine (cat#ab120921) from abcam (Cambridge, UK). FDA approved drug library (cat# L1300), Ponatinib (cat#S1490), Sunitinib (cat#S7781), Dasatinib (cat# S1021), Venetoclax/ABT-199 (cat# S8048), S63845 (cat# S8383), Vincristine (cat#S1241) and Taxol (cat#S1150) were from Selleckchem (Newmarket, UK). Navitoclax/ABT-263 (cat#M1637) was from AbMole. TKIs were used at concentrations in the range of their IC50s (see Figure S5A).