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).
Wehi 539
Manufactured by Apexbio
Sourced in United States
WEHI-539 is a laboratory equipment product manufactured by Apexbio. It is a small molecule that functions as a selective inhibitor of the BCL-2 family of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Abt 737, by Selleck Chemicals (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Umi 77, by Selleck Chemicals (1 mentions)
Doxorubicin, by Selleck Chemicals (1 mentions)
Zvad fmk, by Bachem (1 mentions)
Abt 199, by Apexbio (1 mentions)
Abt 199, by Selleck Chemicals (1 mentions)
Powerplex 18d kit, by Promega (1 mentions)
Gsk2606414, by Bio-Techne (1 mentions)
A1210477, by Active Motif (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Glutamate, by Merck Group (1 mentions)
Q vd oph, by Cayman Chemical (1 mentions)
Thymidine, by Merck Group (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Taxol, by Merck Group (1 mentions)
Zm447439, by Bio-Techne (1 mentions)
8 protocols using wehi 539
1
Evaluating Bcl-xL Inhibitors in Cancer Cells
2
Tumor Sphere Formation Assay
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1 × 103 cells were plated in six-well ultralow attachment surface plate and cultured as previously described48 (link). After 10 days, spheres were photographed and counted. In the case of silencing experiments, cells were plated after 48 h of silencing and tumor sphere-forming capacity was evaluated after 10 days. The capacity to form tumor sphere was also evaluated after 4 days in presence of WEHI-539 (20 μM), zVAD (50 μM), or ABT-199 (1 μM), a specific BCL-2 inhibitor (Apexbio, Houston, USA).
3
Cell Line Preparation and BH3 Mimetic Assay
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Refer to Supplementary Table 1 for a full list of all cell lines used in this study and their growth media. All commercially available cell lines were obtained from ATCC, except OVSAHO which was obtained from the JRCB, tested for mycoplasma contamination, and authenticated with the Promega PowerPlex 18D kit for STR profiling. A subset of patient-derived cell lines were either generated at Duke University12 (link) or at John Hopkins University31 . All cell lines were grown at optimal confluency (no lower than 30% or higher than 80%) and were given fresh media every 2–3 days. For BH3 mimetics, ABT-199 (obtained from Selleckchem), WEHI-539 (obtained from ApexBio), and A-1210477 (obtained from Active Biochem) were used as BCL-2, BCL-XL, and MCL-1 inhibitors, respectively. The PERK inhibitor GSK2606414 was purchased from Tocris Bioscience and used at the indicated concentrations.
4
Isolation and Culture of Primary Rat Hippocampal Neurons
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Primary rat hippocampal neurons were prepared from rat feti (Sprague-Dawley, day 18 of gestation; Harlan, Indianapolis, IN, USA) as described previously13 (link), 52 (link), 53 (link) with modifications specific for this study. After isolation of hippocampi from prenatal brains, neurons were dissociated and seeded (0.2 × 106 cells/35 mm plate) onto plates containing medium with 5% FBS. After 2 h incubation, cells were maintained in neurobasal medium supplemented with B-27, glutamine and antibiotics (Invitrogen GIBCO Life Technologies, Carlsbad, CA, USA). Neurons were grown at 37 °C in 5% CO2 and 20% O2 in a humidified incubator, and assayed at DIV 20-22. Glutamate treatment: 20 μM Glutamate (Sigma-Aldrich, St. Louis, MO, USA) was freshly made in sterile PBS as an aqueous solution then added to the cell culture medium as described in relevant figure legends. Bcl-xL inhibitor treatment: a stock solution of ABT-737 (Selleckbio, Houston, TX, USA), or WEHI-539 (Apex Bio, Houston, TX, USA) were prepared in dimethyl sulfoxide (DMSO). ABT-737 (1 μM or 10 nM), WEHI-539 (5 μM or 10 nM) or the same volume of DMSO was added into the culture dishes 20 min prior to Glutamate treatment. Neurons were transfected at days in vitro (DIV) 7 using lipofectamin LTX with Plus Reagent (Invitrogen).
5
BCL-xL Inhibition in H9 Differentiation
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H9 cells were differentiated using 17D differentiation protocol to D7 and treated with 10 μM of WEHI-539 (ApexBio), an inhibitor of BCL-xL. On D8, the cells were harvested for QPCR, western blot and immunostaining. 10 µM broad-spectrum caspase inhibitor QVD-OPh (Cayman Chemical) was used to inhibit apoptosis.
6
Preparation and Storage of Pharmacological Compounds
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The following drugs were dissolved in DMSO and stored at −20°C: WEHI-539 [32 (link)] (Apexbio); taxol (Sigma); nocodazole (Sigma); AZ138 [8 (link)] (AstraZeneca)); AZ3146 [43 (link)] (AstraZeneca); BI 2536 [38 (link)] (Boehringer Ingelheim); GSK923295 [39 (link),70 ]; MLN8054 [41 (link)] (Millennium Pharmaceuticals); ZM447439 [42 (link)] (Tocris); A-1210477 [31 (link)] (Medchemexpress). Tetracycline (Sigma) was dissolved in water, stored at −20°C, and used at concentrations indicated in the figure legends. Thymidine (Sigma) was dissolved in PBS at a concentration of 200 mM, and stored short-term at 4°C.
7
Evaluation of Bcl-2 Inhibitors in Vitro and In Vivo
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The selective Bcl-2 inhibitor S55746 was kindly provided and developed by Servier (Suresnes, France). For in vitro experiments S55746 was dissolved in phosphate buffered saline (PBS). ABT-737 (S1002, Selleckchem, Houston, TX, USA), WEHI-539 (ApexBio Technology, Houston, TX, USA) and Z-vad-FMK22 (link) (550377, BD biosciences, San Jose, CA, USA) were dissolved in DMSO and stored at −20 °C. The animal experiments were performed with freshly dissolved S55746 according to the manufacturer’s indications at 30 mg/mL in a solution of 40% polyethylene glycol (Sigma-Aldrich, St Louis, MO, USA), 10% ethanol (Sigma-Aldrich) and 50% sterile water for injection (B. Braun, Melsungen, Germany). A-1155463 (S7800, Selleckchem, Houston TX USA) was dissolved in 5% DMSO, 10% ethanol, 20% PEG 35 and 65% sterile water as recommended by the manufacturers. Doxorubicin (2 mg/ml in a 0.9% NaCl solution) and cisplatin (1 mg/ml in a 0.9% NaCl solution) were obtained from the in house hospital pharmacy from the Leiden University Medical Centre for in vitro experiments or Centre Léon Bérard for in vivo experiments.
8
Glycolysis and OXPHOS Assessment in hESCs
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hESCs were differentiated to D7 using the 17D differentiation protocol and treated with DMSO or WEHI-539 (Apexbio) for 24 h. D8 cells were then trypsinized and 150,000 cells plated per well on Seahorse XF96 cell culture microplates (Agilent). Growth media was changed to Seahorse XF Base Medium (Agilent) supplemented with l -Glutamine (Sigma) and placed in a non-CO2 incubator 1 h prior to assay. Glycolysis was measured via the Seahorse XF Glycolysis Stress Test Kit (Agilent) with a Seahorse XF 96 analyzer (Agilent) following the manufacturer’s protocol. Using the same setup, oxidative phosphorylation was measured using Seahorse XF Mito Stress Kit (Agilent).
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