The GSC-ECLs used in this study were previously isolated from human biopsies following relevant guidelines and national regulations. These cell lines have been characterized as described in previous reports9 (link),10 (link),43 (link),44 (link). Prior to their isolation, the use of these cell lines for research purposes have been approved by the Biomedical Research Ethics Committee “Comité de Ética en Investigaciones Biomédicas de la Fundación para la Lucha contra Enfermedades Neurológicas de la Infancia (FLENI)”. Written informed consent was received from each patient whose tumor tissue was used. NP cells were derived from human embryonic stem cells (WA09, provided by University of Wisconsin—Dr. J. Thomson. hPSCReg ID: WAe009-A)51 (link). Both GSC-ECLs and NPs were grown in a serum-free medium consisting of Neurobasal supplemented with N2, B27, 20 ng/ml epidermal growth factor (EGF), 20 ng/ml basic fibroblast growth factor (bFGF) and plated onto Geltrex-coated plates (10 μg/ml) (all from Thermo Scientific, Rockford, IL, USA). BH3-mimetics and chemotherapeutic agents: ABT-263 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), WEHI-539 (Genentech USA Inc, San Francisco, CA, USA), S63845 (Cayman Chemical, Ann Arbor, MI, USA), TMZ, VCR, and CCNU (Sigma, St. Louis, MO, USA).
S63845
Manufactured by Cayman Chemical
Sourced in United States
S63845 is a laboratory equipment product manufactured by Cayman Chemical. It is designed for use in research and scientific applications. The core function of this product is to provide a controlled environment for various experimental or analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Tunicamycin, by Merck Group (2 mentions)
Etoposide, by Thermo Fisher Scientific (2 mentions)
Oligomycin, by Merck Group (2 mentions)
Paclitaxel, by Merck Group (2 mentions)
Thapsigargin, by Merck Group (2 mentions)
N acetylcysteine, by Merck Group (2 mentions)
Bortezomib, by Thermo Fisher Scientific (2 mentions)
2 deoxyglucose, by Merck Group (2 mentions)
Jnk inhibitor 8, by Merck Group (2 mentions)
Q vd oph, by Thermo Fisher Scientific (2 mentions)
Trametinib, by Selleck Chemicals (2 mentions)
Nutlin 3, by Selleck Chemicals (2 mentions)
Vinblastine, by Selleck Chemicals (2 mentions)
Camptothecin, by Selleck Chemicals (2 mentions)
A 1155463, by Selleck Chemicals (2 mentions)
Pimasertib, by Selleck Chemicals (2 mentions)
Staurosporine, by Apexbio (2 mentions)
Abt 737, by Selleck Chemicals (2 mentions)
7 protocols using s63845
1
Glioblastoma Stem Cell Characterization
2
Prostate Cancer Cell Line Culturing
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Human prostate cancer cell lines (DU 145 and PC-3) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured according to ATCC's instructions. Human recombinant TGF-β1 was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). DTX and SB-505124 were purchased from MilliporeSigma (St. Louis, MO, USA). ABT-199 and S63845 were purchased from Cayman Chemical (Ann Arbor, MI, USA). The jetPRIME transfection reagent (Polyplus transfection, New York, NY, USA) was used for plasmid transfection according to the manufacturer's protocol.
3
Evaluation of Bioactive Compounds in Cell Screening
4
Tumor Growth Inhibition Assay
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To investigate tumor growth difference, each flank of NSG mouse was subcutaneously implanted with 1 million engineered LS174T cells in 100 µL cold phosphate-buffered saline (PBS)−Matrigel (Corning) mixture (1:1 ratio). Implanted mice were randomly assigned to each treatment group. Steap4 expression was continuously induced by Doxycycline in drinking water (2 mg/mL with 1% sucrose). TTM (10 mg/kg, Sigma), S63845 (Mcl-1 inhibitor, 12.5 mg/kg, Cayman, Cat# 21131) and BMS-34554133 (link),34 (link) (IKKα/β inhibitor,125 mg/kg, Sigma, Cat# B9935) were administered through intraperitoneal injection every other day after implantation. Normal saline was injected as the control group. Tumor size was measured using caliber and was initiated 3 days after implantation. Tumor volume was calculated as length/2 × (width)2.
5
Anticancer Combination Therapy Evaluation
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The starting cell seeding density was 0.5–0.7 × 106 viable cells/mL. The cell line was incubated with cytarabine (Cayman Chemical Company, Ann Arbor, MI, USA), idarubicin (MilliporeSigma, Burlington, MA, USA), metformin (Cayman Chemical Company, Ann Arbor, MI, USA), venetoclax (Cayman Chemical Company, Ann Arbor, MI, USA), and S63845 (Cayman Chemical Company, Ann Arbor, MI, USA)—alone or in different combinations for the duration of 96 h.
During the 96 h of treatment period, cell growth characteristics were monitored using Trypan blue staining. Cell suspension was mixed in equal portions with 0.2% Trypan blue (MilliporeSigma, Burlington, MA, USA). The cells were then counted in a hemocytometer (Neubauer-improved, 0.1 mm depth of the chamber; Paul Marienfeld GmbH & Co. KG, Lau-da-Königshofen, Germany).
The viability of the cell cultures was estimated using 7AAD staining (Miltenyi Biotec, Inc., Auburn, CA, USA) following the manufacturer’s instructions.
Antiproliferative IC50 values and metabolic activity were evaluated using the XTT Cell Proliferation Assay Kit (ATTC, Manassas, VA, USA) following the manufacturer’s instructions.
During the 96 h of treatment period, cell growth characteristics were monitored using Trypan blue staining. Cell suspension was mixed in equal portions with 0.2% Trypan blue (MilliporeSigma, Burlington, MA, USA). The cells were then counted in a hemocytometer (Neubauer-improved, 0.1 mm depth of the chamber; Paul Marienfeld GmbH & Co. KG, Lau-da-Königshofen, Germany).
The viability of the cell cultures was estimated using 7AAD staining (Miltenyi Biotec, Inc., Auburn, CA, USA) following the manufacturer’s instructions.
Antiproliferative IC50 values and metabolic activity were evaluated using the XTT Cell Proliferation Assay Kit (ATTC, Manassas, VA, USA) following the manufacturer’s instructions.
6
Evaluation of Bioactive Compounds in Cell Screening
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Camptothecin (Cat# S1288), vinblastine (Cat# S1248), pimasertib (Cat# S1457), trametinib (Cat# S2673), ABT-737 (Cat# S1002), A-1155463 (Cat# S7800), and nutlin-3 (Cat# S1061) were purchased from Selleck Chemicals (Houston, TX). Bortezomib (Cat# NC0587961), etoposide (Cat# ICN19391825), MG-132 (Cat# 17-485), and Q-VD-OPh (Cat# OPH00101M) were purchased from Thermo Fisher Scientific. N-acetylcysteine (Cat# A8199), thapsigargin (Cat# T9033), tunicamycin (Cat# T7765), paclitaxel (Cat# T7191), JNK Inhibitor VIII (Cat# 420135), 2-deoxyglucose (Cat# D8375), oligomycin (Cat# O4876), and cycloheximide (Cat# C7698) were obtained from Sigma-Aldrich (St. Louis, MO). S63845 (Cat#21131) was obtained from Cayman Chemical (Ann Arbor, MI). Staurosporine (Cat# A8192) was obtained from ApexBio (Houston, TX). Erastin was the kind gift of Brent Stockwell (Columbia University). Erastin2 (compound 35MEW28 in Dixon et al., [2014 (link)]) and ML162 (CAS: 1035072-16-2) were synthesized by Acme Bioscience (Palo Alto, CA). Chemical screening was conducted as described below; the library of 261 bioactive compounds was obtained from Selleck Chemicals (Cat# L2000).
7
Antibody and Inhibitor Protocols
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Antibodies used in this study are those for IL1RAP (73070S); IκBα, NF-κB1, and IKBKG (9936T); cleaved PARP (5625S); PARP (cat. 9542S); cleaved caspase-3 (9661S); and BCL-xL and BAX (9941T; all from Cell Signaling Technology); vinculin (V9264; Sigma-Aldrich); and GAPDH (sc-32233; Santa Cruz Biotechnology).
Inhibitor A-1331852 (HY-19741) was purchased from MedChemExpress. S63845 (21131) was purchased from Cayman Chemical. ABT-199 (S8048) was from Selleck Chemicals. IL-1α human recombinant (SRP3310) and IL-1β human recombinant (SRP3083) were purchased from Sigma-Aldrich.
Inhibitor A-1331852 (HY-19741) was purchased from MedChemExpress. S63845 (21131) was purchased from Cayman Chemical. ABT-199 (S8048) was from Selleck Chemicals. IL-1α human recombinant (SRP3310) and IL-1β human recombinant (SRP3083) were purchased from Sigma-Aldrich.
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