The B16F10 cells were cultured at a density of 4x104 in a 24-well plate. After 24 h, the cells were exposed to one concentration of methanolic extract (0, 0.135 mg/mL) and incubated at different times (24 and 48 h). Etoposide (Enzo Life Sciences, NY, USA) at 1.5 mM was used as a control of apoptosis induction. After treatment, the cells were harvested and washed twice with cold PBS and suspended in 100 μL of binding buffer (0.1 M Hepes/NaOH pH 7.4, 1.4 M NaCl, and 25 mM CaCl2) supplemented with 0.5 μL of APC-Annexin-V and 0.5 μL of propidium iodide (PI). The cell suspension was gently vortexed and incubated for 20 min at 4°C in the dark to be assessed by flow cytometry in a BD AccuriC6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) (total population 10,000 cells). Data was analyzed using FlowJo software (LLC, Ashland, Oregon, USA).
Etoposide
Manufactured by Enzo Life Sciences
Sourced in United States
Etoposide is a laboratory reagent used for research purposes. It is a chemotherapeutic agent that acts as a topoisomerase II inhibitor. Etoposide interferes with the cell's ability to divide and replicate, making it a useful tool for studying cell division and growth processes.
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Lab products found in correlation
Staurosporine, by Enzo Life Sciences (2 mentions)
Nigericin, by Cayman Chemical (2 mentions)
Gapdh clone 14c10, by Cell Signaling Technology (2 mentions)
Xf24 cell culture microplate, by Agilent Technologies (2 mentions)
Cell culture components, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Curcumin, by Enzo Life Sciences (1 mentions)
5 fluorouracil, by Enzo Life Sciences (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Llc1 cells, by American Type Culture Collection (1 mentions)
Tamoxifen, by Enzo Life Sciences (1 mentions)
Cisplatin, by Enzo Life Sciences (1 mentions)
Sitagliptin, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Santa Cruz Biotechnology (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Vildagliptin, by Cayman Chemical (1 mentions)
Saxagliptin, by LGC (1 mentions)
Human il 1β, by R&D Systems (1 mentions)
Ala pro afc, by Bachem (1 mentions)
17 protocols using etoposide
1
Apoptosis Induction in B16F10 Cells
2
Apoptotic Bodies Isolation Protocol
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Lewis lung carcinoma (LLC1) cells were obtained from the American Type Culture Collection (ATCC, Baltimore, U.S.A.). Cultures were maintained in RPMI-1640 supplemented with 10% FCS. All experiments were carried out within 6 months of resuscitation. LLC1 cells were incubated with etoposide (400 μM), curcumin (160 μM), tamoxifen (40 μM), staurosporine (1 μM), cisplatin (400 μM) or 5-fluorouracil (160 μM) (all from Enzo Life Sciences) for 24 hrs to induce apoptosis. Apoptotic bodies were purified as described by Fransen et al., 2009 (28 (link)); briefly, cells were centrifuged at 1500 g for 10 mins at 20°C. The supernatant was centrifuged at 15700 g for 50 mins at 20°C. Apoptotic bodies were resuspended in PBS.
4 x 106 LLC1 cells were incubated with 100 µl RIPA buffer containing a protease inhibitor cocktail (20 µl/ml; Calbiochem), phosphatase inhibitor (Thermo Scientific) and 1% Triton X-100 for 20 mins at 4°C. Supernatants, collected after centrifugation at 18000 g for 30 mins at 4°C, were employed as cell lysate.
4 x 106 LLC1 cells were incubated with 100 µl RIPA buffer containing a protease inhibitor cocktail (20 µl/ml; Calbiochem), phosphatase inhibitor (Thermo Scientific) and 1% Triton X-100 for 20 mins at 4°C. Supernatants, collected after centrifugation at 18000 g for 30 mins at 4°C, were employed as cell lysate.
3
Caspase Activation and Pyroptosis Assays
4
Synthesis and Characterization of Compound 8j
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Compound 8j was synthesized according to previously published protocols29 (link). VbP (Talabostat mesylate) was purchased from R&D Systems and was resuspended in DMSO containing 0.1% TFA to prevent cyclization. Bortezomib was purchased from LC laboratories, zVAD-FMK from Ubpbio, VX-765 from Apexbio Technology LLC, and etoposide from Enzo Life Sciences. LPS (from E. coli O111:B4) was purchased from Invivogen, Nigericin from Cayman Chemicals, Bestatin Methyl Ester from Santa Cruz, and MCC950 from AdipoGen. Antibodies used were: hCASP1 (no. 2225, Cell Signaling Technology), GAPDH (clone 14C10, Cell Signaling Technology), hGSDMD (NBP2-3342, Novus Biologicals), PARP (no. 9542, Cell Signaling Technology), hNLRP1 (AF6788, R&D Systems), hCARD8 (no. ab24186, Abcam), mGSDMD (no. ab209845, Abcam), GSDMD (no. 219800, Abcam), α-Actin (no. A4700, Sigma-Aldrich), mCD45 (no. 103108, Biolegend, FITC conjugate, clone 30-F11), mCD3 (no. 100235, Biolegend, APC conjugate, clone 17A2), rCD3 (no. 201411, Biolegend, PE conjugate, clone 1F4), rCD6 (no. 554904, BD Biosciences, FITC conjugate, clone OX-52), mCaspase-1 (AG20B-0042, Adipogen), mIL-1β (no. D4T2D, Cell Signaling Technologies), NLRC4 (no. ab201792, Abcam), and NLRP3 (no. ab210491, Abcam).
5
Apoptosis Induction in Fibroblasts and Endothelial Cells
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Human Umbilical Vein Endothelial Cells (HUVEC) (Gibco) were maintained in Medium 200 (Gibco) supplemented with Low Serum Growth Supplement (Gibco), 1% penicillin-streptomycin (Lonza), and 1% L-glutamine (Lonza) for no more than 8 passages. Human Forskin Fibroblasts (HFF) (ATCC cell collection) cells were maintained in DMEM (Dulbecco's modified Eagle's medium) (Lonza) supplemented with 10% (v/v) fetal bovine serum (Gibco), 1% penicillin-streptomycin (Lonza), and 1% L-glutamine (Lonza) for no more than 30 passages. Both of the cells were incubated at 37°C in a humidified environmental incubator supplemented with 5% CO2. Etoposide (Enzo) was used to induce apoptosis at 10 μM for 24 h. The cells used in this study are non-transformed cells and represent two different important cell types in diseases such as heart disease and cancer, namely fibroblast and endothelial cells. In addition, these cells would be expected to have different sensitivities to hypoxia exposure.
6
Annexin-V Apoptosis Assay in T Cells
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Isolated T cells were cultured in the presence or absence of dexamethasone (Sigma-Aldrich), etoposide (Enzo Life Sciences), staurosporine (Sigma-Aldrich), plate bound anti-CD3 antibody, or solvent control (dimethyl sulfoxide or ethanol) for 8 hours. Cells were counterstained with anti–CD4-Cy5 and anti–CD8-PE in binding buffer and analyzed for annexin-V positivity, as described (38 (link)).
7
Ruthenium-labeled Protein A/G/L Reagent
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Unlabeled protein A/G/L used for detection in the NHP TAb assay was purchased from BioVision (Milpitas, CA, USA). The ruthenium-labeled protein A/G/L reagent was prepared by reconstituting protein A/G/L according to manufacturer’s recommendation at 10 mg/ml in DPBS without calcium and magnesium. The concentration was adjusted to 1 mg ml−1 in DPBS and labeled with 10 nmol MSD Sulfo-NHS Ruthenium for 1 h at ambient temperature with rotation. The labeled protein A/G/L was purified by three cycles of filtration with Amicon-15 centrifugal filters using DPBS and stored at −80 °C in single-use aliquots. Tris Buffered Saline with 1% (w/v) Casein (TBS-C) was from BioRad (Hercules, CA, USA). The protein A/G columns, protein L columns, and bicinchoninic acid (BCA) protein assay kits were from Thermo Fisher Scientific (South San Francisco, CA, USA). Etoposide was from Enzo Life Sciences (Farmingdale, NY, USA), Steady-Glo Luciferase reagent was from Promega (Sunnyvale, CA, USA), fetal bovine serum (FBS) without heat inactivation was from HyClone (Logan, UT, USA), and Penicillin-Streptomycin-Glutamine (100 ×) was from Gibco (South San Francisco, CA, USA). The HEK293T/17 cell line was purchased from ATCC (Manassas, VA, USA). The GFP quantification kit was purchased from Cell Biolabs (San Diego, CA, USA).
8
Molecular Mechanisms of DNA Damage Response
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SGC8158, doxorubicin, and CHX were obtained from Sigma-Aldrich (St. Louis, MO, USA) and etoposide was obtained from Enzo Life Sciences (Farmingdale, NY, USA). The following antibodies were used for western blotting analysis or immunoprecipitation (IP): β-actin (Santa Cruz Biotechnology, Dallas, TX, USA, sc-47778), p53 (Santa Cruz Biotechnology, sc-126), cyclin B1 (Santa Cruz Biotechnology, sc-752), cyclin D1 (Santa Cruz Biotechnology, sc-8396), cyclin E (Santa Cruz Biotechnology, sc-377100), CDK2 (Santa Cruz Biotechnology, sc-6248), CDK6 (Santa Cruz Biotechnology, sc-7961), PARP1 (Santa Cruz Biotechnology, sc-7150), 53BP1 (Santa Cruz Biotechnology, sc-515841), BRCA2 (Santa Cruz Biotechnology, sc-293185), Hsp70 (Enzo Life Sciences, ADI-SPA-810-F), PRMT7 (Abcam, Cambridge, UK, ab179822, ab181214), MMA (Abcam, ab415), p-Rb (Abcam, ab24), p21 (Cell Signaling Technology, Danvers, MA, USA, #2947), p16 (Cell Signaling Technology, #80772), Rb (Cell Signaling Technology, #9309), CDK (Cell Signaling Technology, #12790), γH2AX (Cell Signaling Technology, #9718), p-chk1 (Cell Signaling Technology, #2348S), p-chk2 (Cell Signaling Technology, #2661S), Ku80 (Cell Signaling Technology, #2180S), p27 (Genetex, Irvine, CA, USA, GTX-100446), and horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch laboratories, Inc., West grove, PA, USA).
9
Rat Cortical Neuron Apoptosis Assay
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Embryonal rat cortical neurons (RCN) were derived as previously described from rat E15-16 cortices35 (link). For each experiment, cortices were obtained from all embryos (mixed, unknown sex) of a single pregnant Sprague-Dawley® dam (Envigo). After dissociation, cells were seeded onto 96-well, 12-well, 60 mm, 100 mm cell culture plates (Corning, Corning, NY) or XF24 cell culture microplates (Agilent, Santa Clara, CA) and maintained in serum-free conditions using the B27 supplement (ThermoFisher, Waltham, MA) as described previously35 (link). We previously showed these cultures are >90% neuronal36 .
Etoposide (Cat. #BML-GR307, Enzo Life Sciences, Farmingdale, NY), staurosporine (Cat. #ALX-380-014, Enzo Life Sciences, Farmingdale, NY), camptothecin (Cat. #ALX-350-015, Enzo Life Sciences, Farmingdale, NY), C2-ceramide (Cat. #BML-SL100, Enzo Life Sciences, Farmingdale, NY), doxorubicin (Cat. #CST-5927, Cell Signaling Technologies, Danvers, MA), and mithramycin (Cat. #11434, Cayman Chemical Company, Ann Arbor, MI) were used to treat 7 days in vitro (DIV) cells at concentrations and for times indicated elsewhere.
Etoposide (Cat. #BML-GR307, Enzo Life Sciences, Farmingdale, NY), staurosporine (Cat. #ALX-380-014, Enzo Life Sciences, Farmingdale, NY), camptothecin (Cat. #ALX-350-015, Enzo Life Sciences, Farmingdale, NY), C2-ceramide (Cat. #BML-SL100, Enzo Life Sciences, Farmingdale, NY), doxorubicin (Cat. #CST-5927, Cell Signaling Technologies, Danvers, MA), and mithramycin (Cat. #11434, Cayman Chemical Company, Ann Arbor, MI) were used to treat 7 days in vitro (DIV) cells at concentrations and for times indicated elsewhere.
10
Preparation of ATO, AT-101, GANT61, Etoposide
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Arsenic trioxide (As2O3, ATO; Sigma-Aldrich) was solved in 1 M NaOH, diluted with PBS (Gibco) to 0.5 M and solved at 80 °C while stirring. The solution was than sterile filtrated and diluted to 1 mM for long-term storage. An intermediate dilution of 15 mM was used for all consecutive dilutions. AT-101 ((−)-Gossypol, Bio-Techne GmbH, Wiesbaden--Nordenstadt, Germany), GANT61 (Sigma-Aldrich) and etoposide (Enzo Life Sciences, Lörrach, Germany) were diluted in DMSO (Carl Roth GmbH, Karlsruhe, Germany).
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