CB-839, mitomycin C, cryptotanshinone, altretamine, GSK583, liproxstatin-1, spautin-1, PD-1/PD-L1 inhibitor 2, RVX-208, selinexor, ferrostatin-1, diacerein, LY2603618, and patupilone were purchased from Selleckchem (Houston, TX). Cisplatin, carboplatin and oxaliplatin were purchased from Sigma-Aldrich (St. Louis, MO). Olaprib, niraparib, veliparib were purchased from Cayman Chemical (Ann Arbor, MI). As2O3 was purchased from BioTang (Lexington, MA). C1–27 is a GSTO1 inhibitor previously identified and synthesized by our laboratory (5 (link)).
Spautin 1
Manufactured by Selleck Chemicals
Sourced in United States
Spautin-1 is a small molecule inhibitor that targets the autophagy-related proteins ULK1 and ULK2. It is a tool compound used in research for studying the role of autophagy in biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Ferrostatin 1, by Selleck Chemicals (2 mentions)
Liproxstatin 1, by Selleck Chemicals (2 mentions)
Cisplatin, by Merck Group (2 mentions)
Bafilomycin a1, by Selleck Chemicals (2 mentions)
3 methyladenine, by Selleck Chemicals (2 mentions)
Cisplatin, by Selleck Chemicals (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Selinexor, by Selleck Chemicals (1 mentions)
Cryptotanshinone, by Selleck Chemicals (1 mentions)
Veliparib, by Cayman Chemical (1 mentions)
Oxaliplatin, by Merck Group (1 mentions)
Cb 839, by Selleck Chemicals (1 mentions)
Mitomycin c, by Selleck Chemicals (1 mentions)
Rvx 208, by Selleck Chemicals (1 mentions)
Ly2603618, by Selleck Chemicals (1 mentions)
Carboplatin, by Merck Group (1 mentions)
Lox 1, by R&D Systems (1 mentions)
Control sc 37007, by Santa Cruz Biotechnology (1 mentions)
Oil red o, by Merck Group (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
11 protocols using spautin 1
1
Comprehensive Anticancer Compound Evaluation
2
Cholesterol Efflux Regulation Mechanisms
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Spautin-1 and MG132 were purchased from Selleckchem (Houston, TX, USA). Control (SC-37007) and USP10 (SC-76811) siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human dil-labeled oxLDL (YB-0010) oxidized low density lipoprotein (oxLDL) (YB-002) were from Yiyuan Biotechnologies (Guangzhou, China). Antibodies are as follows: anti-GAPDH (#5174), anti-USP10 (#8501), anti-ubiquitin (#3936), anti-K48-linkage Specific Polyubiquitin (#12805), anti-CD36 (ab13365), ABCA1 (#96292), ABCG1 (ab52617), SR-B1 (ab217318), SR-A (ab123946). Lox-1 was from R&D system. Oil Red O was from Sigma-Aldrich. CO-IP were obtained from Novus biologicals (USA).
3
Investigating Cellular Signaling Pathways
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Spautin-1 (S7888) and cycloheximide (S7418) were obtained from Selleckchem. Antibodies, anti-USP10 (#8501), anti-Skp2 (#2652), anti-p-Rb (#8516), anti-Rb (#9309), anti-CDK4 (#12790), anti-Cyclin D1 (#55506), anti-p27 (#3686), anti-MMP2 (#40994), anti-ubiquitin (#3936), anti-α-SMA (#19245), and anti-GAPDH (#5174) were purchased from Cell Signaling Technology (CST). Human PDGF-BB was obtained from Peprotech.
4
Antibody and Inhibitor Sources for Cell Signaling
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The antibodies to MAP1LC3B (#3868), actin (#3700), c-CASP8 (#8592 and #9496), C-CASP9 (#9505 and #9509), BAD (#9268), BBC3 (#4976), BAX (#2772), BCL2L1 (#2764), BCL2 (#2872), C-CASP3 (#9664), JNK (#9252), p-JNK (#9255), ERK (#4695), p-ERK (#4370), p38 (#8690), p-p38 (#4511), JUN (#9165), and p-JUN (#3270) were obtained from Cell Signaling Technology (Danvers, MA, USA). Monoclonal anti-HMGB1 neutralizing antibody (clone 2G7) was a gift from Dr. Kevin Tracey. Monoclonal anti-TFAM neutralizing antibody (clone F-6; #sc-166965) was obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). Monoclonal anti-AGER neutralizing antibody (clone 697023; #MAB11795) was obtained from R&D System (Minneapolis, MN, USA). Spautin-1 (#S7888), 3-methyladenine (#S2767), LY294002 (#S1105), chloroquine (#S4157), bafilomycin A1 (#S1413), oxaliplatin (#S1224), Z-VAD-FMK (#S7023), necrostatin-1 (#S8037), necrosulfonamide (#S8251), ferrostatin-1 (#S7234), liproxstatin-1 (#S7699), SP600125 (#S1460), SB203580 (#S1076), SB239063 (#S7741), SCH772984 (#S7101), and LY3214996 (#S8534) were obtained from Selleck Chemicals (Houston, TX, USA). 5-fluorouracil (#F6627), TRAIL (#T5694), Mito-TEMPO (#SML0737), and CC-401 (#SML1613) were obtained from Sigma (St. Louis, MO, USA).
5
Cell Culture and Inhibitor Assay
6
Cell Viability Assay with Inhibitors
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The HEK293T and tumor cell lines were originally obtained from ATCC or JCRB in 2014, where mycoplasma contamination was tested and cell characterization was performed using polymorphic short tandem repeat (STR) profiling. Cells were cultured in RPIM 1640 (Life Technologies) supplemented with 10% fetal bovine serum (Millipore). ABT-263, spautin-1 and other inhibitors were purchased from Selleck Chemicals. All inhibitors were reconstituted in DMSO (Sigma-Aldrich) at a stock concentration of 10 mM. For cell viability assays, cells were seeded at 100,000 cells per well in growth media supplemented with 10% fetal bovine serum in 6-well plates, allowed to adhere overnight, and treated with a serial dilution of inhibitors for 5 days. Cells were fixed with formalin and stained with crystal violet.
7
Glioblastoma Spheroid Cultivation and Metabolic Modulation
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Patient-derived GBM cells were maintained in NeuroCultTM NS-A Proliferation Medium supplemented with 20 ng/mL EGF, 10 ng/mL bFGF, 2 µg/mL of Heparin, and 1X Anti-Anti (NeuroCult complete media) and propagated as non-adherent neurospheres. Patient-derived cells were grown in 6 cm tissue culture plates at 37˚C in a humidified incubator containing 5% CO2 . Patient-derived GBM neurospheres were passaged by manual dissociation of neurospheres into single cells through gentle trituration by pipetting.
Cells were treated with 2-DG (Sigma Aldrich) at the indicated doses for 24 h prior to analysis unless otherwise specified. Cells were treated with 10 µM of Spautin-1 (Selleckchem) for 24 h prior to analysis. For flux assays, cells were treated with 2-DG (1 mM) for 4 h and then treated with 12 µM of CQ 24 h prior to sample collection and analysis.
Cells were treated with 2-DG (Sigma Aldrich) at the indicated doses for 24 h prior to analysis unless otherwise specified. Cells were treated with 10 µM of Spautin-1 (Selleckchem) for 24 h prior to analysis. For flux assays, cells were treated with 2-DG (1 mM) for 4 h and then treated with 12 µM of CQ 24 h prior to sample collection and analysis.
8
Solubilization of Pharmacological Agents
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The following chemical reagents were used in this study: AMG9810 (Sigma-Aldrich, St. Louis, MO, USA, A2731), capsaicin (Sigma-Aldrich, M2028), gefitinib (Selleckchem, Houston, TX, USA, S1025), cisplatin (Selleckchem, S1166), spautin-1 (Selleckchem, S78880), and bafilomycin A1 (Sigma-Aldrich, B1793-2UG). The chemical reagents we used were dissolved in below solvents. AMG9810, gefitinib, spautin-1, or bafilomycin A1: Dimethyl sulfoxide (DMSO); cisplatin: Dimethyl formamide (DMF); capsaicin: ethanol.
9
Combinatorial Anticancer Drug Screening
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Cisplatin, Paclitaxel, Spautin-1, and MK-2206 were obtained from Selleck Chemicals (Selleckchem, Houston, TX). PT and PTU cells were seeded, and 24 h later, the cells were treated with Cisplatin, Paclitaxel, MK-2206, or combination treatments of Spautin-1 and MK-2206. After 24 h (for Spautin-1, MK-2206) and 48 h (for Cisplatin, Paclitaxel), cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma), and absorbance was measured by a plate reader at 570 nm. Data are represented as the mean ± standard deviation. Student t-tests were performed to compare cell viability across cell lines.
10
Culturing HT1080 and JFCR39 Cell Lines
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HT1080 human fibrosarcoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The JFCR39 human cancer cell line panel was developed and cultured as described previously31 (link). HT1080 cells and the JFCR39 cell lines were maintained in RPMI1640 (FUJIFILM Wako Pure Chemical Industry, Osaka, Japan) supplemented with 10% or 5% fetal bovine serum (FBS) and 100 μg/mL kanamycin, unless otherwise stated. Spautin-1, bafilomycin A1, HCQ, SAR405, and rotenone were purchased from Selleck Chemicals (Houston, TX, USA) and dissolved in DMSO. We obtained 2DG from Sigma-Aldrich (St Louis, MO, USA) and 2DG was dissolved in sterilized distilled water. Tunicamycin (Nacalai Tesque, Kyoto, Japan) and thapsigargin (FUJIFILM Wako Pure Chemical Industry) were dissolved in DMSO.
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