Echinomycin

Manufactured by Merck Group

Sourced in United States, United Kingdom

Echinomycin is a laboratory product manufactured by Merck Group. It is a cyclic peptide compound with antimicrobial and antitumor properties. The core function of Echinomycin is to serve as a research tool for scientific investigations.

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Lab products found in correlation

Mg132, by Merck Group (2 mentions) Rapamycin, by Merck Group (2 mentions) Ampicillin sodium salt, by Avantor (1 mentions) Lb agar, by Thermo Fisher Scientific (1 mentions) L arabinose, by Thermo Fisher Scientific (1 mentions) Kanamycin sulfate, by IBI Scientific (1 mentions) Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Primocin, by Thermo Fisher Scientific (1 mentions) Deferoxamine, by Merck Group (1 mentions) Mirneasy micro kit, by Qiagen (1 mentions) Qiazol, by Qiagen (1 mentions) Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Gapdh, by SCBT (1 mentions) Pdgfa, by Abcam (1 mentions) β actin, by SCBT (1 mentions) Vegfa, by Abcam (1 mentions) γh2ax, by Novus Biologicals (1 mentions) Cobalt 2 chloride, by Merck Group (1 mentions) Etoposide, by Merck Group (1 mentions) α tubulin hrp, by Abcam (1 mentions)

24 protocols using echinomycin

E. coli Culture Protocols with Antibiotic Resistance

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E. coli strains were grown in Luria Bertani broth (LB) (Fisher BioReagents, NJ, USA). All cultures grown in liquid media were grown at 37°C with orbital shaking at 200 rpm. Strains cultured on solid media were plated on LB agar (Fisher BioReagents, NJ, USA) and incubated at 37°C. Ampicillin (amp) resistant strains were grown with 50 μg amp/ml liquid media (Ampicillin sodium salt prepared in H₂O, Amresco, Ohio, USA) and 100 μg amp/ml solid media. Kanamycin (kan) resistant strains were treated with 30 μg kan/ml for liquid media (kanamycin sulfate prepared in H₂O, IBI Scientific, NJ. USA), and 50 μg kan/ml for solid media. For induction, 0.2% L‐(+)‐arabinose (Alfa Aesar, G.B.) was added to either liquid or solid media. Strains grown with echinomycin were treated with 1 μM‐20 μM of echinomycin diluted in H₂O from a 908 μM echinomycin (Sigma, USA) solution prepared in methanol (Fisher Chemical, NJ, USA). All strains and primers used for this study are listed in Tables S2 and S3.

Erlandson A., Gade P., Menikpurage I.P., Kim C, & Mera P.E. (2022). The UvrA‐like protein Ecm16 requires ATPase activity to render resistance against echinomycin. Molecular Microbiology, 117(6), 1434-1446.

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Hypoxic Stress Response of Hippocampal Neurons

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Primary neurons isolated from the hippocampus of postnatal 24 h NMRs were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% heat‐inactivated foetal bovine serum (Life Technologies), 2 mM glucose, 0.0025% Glutamax, and 0.2 mg/ml primocin (Thermo Fisher Scientific). Primary neurons isolated from the hippocampus of postnatal 24 h mice were cultured in DMEM containing 10% heat‐inactivated foetal bovine serum (Life Technologies), 4.5 g/L glucose, 0.0025% Glutamax, and 0.2 mg/ml primocin (Thermo Fisher Scientific). Four hours later, the medium was replaced with neurobasal A medium supplemented with B27 (2%), 250 mM Glutamax, and penicillin/streptomycin (1%). NMR neurons were maintained at 33°C in 21% O2/5% CO₂. Half of the medium was renewed every 48–72 h before further experiments. For hypoxic stress, hippocampal neurons from NMRs were maintained at 33°C (5% CO₂, 8% O₂), and hippocampal neurons from NMRs were maintained at 37°C (5% CO₂, 8% O₂), for the indicated periods of time in the absence or presence of 20 mM echinomycin (Sigma–Aldrich).

Yang W., Wu W., Zhao Y., Li Y., Zhang C., Zhang J., Chen C, & Cui S. (2022). Caveolin‐1 suppresses hippocampal neuron apoptosis via the regulation of HIF1α in hypoxia in naked mole‐rats. Cell Biology International, 46(12), 2060-2074.

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Macrophage Transcriptional Response to ICH

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Healthy donor monocyte-derived macrophages were pre-treated with 50 nM echinomycin (Sigma #SML0477) or vehicle control (0.1% DMSO) in fresh culture medium for 1 hour prior to treatment with 100 μg/mL deferoxamine (Sigma #D9533), ICH-DAMP cocktail, or vehicle control (water), with two replicate wells per condition. After 8 hours of treatment, cells were lysed using Qiazol (Qiagen #79306) and RNA was isolated using the miRNeasy micro kit (Qiagen #217084). cDNA was generated using the SuperScript VILO cDNA Synthesis Kit (Thermofisher #11756500). Because GADPH and ACTB were differentially expressed by myeloid cells after ICH (Tables S4, S5, S9), PSMB2 was used as a housekeeping gene, as previously reported (70 (link)). Taqman primers were used to estimate expression of PSMB2 (Thermofisher #Hs01002946_m1), PTGES (#Hs01115610_m1), and HK2 (#Hs00606086_m1). Statistical significance between was determined using two-way ANOVA with Dunnett’s correction for multiple comparisons.

Askenase M.H., Goods B.A., Beatty H.E., Steinschneider A.F., Velazquez S.E., Osherov A., Landreneau M.J., Carroll S.L., Tran T.B., Avram V.S., Drake R.S., Gatter G.J., Massey J.A., Karuppagounder S.S., Ratan R.R., Matouk C.C., Sheth K.N., Ziai W.C., Parry-Jones A.R., Awad I.A., Zuccarello M., Thompson R.E., Dawson J., Hanley D.F., Love J.C., Shalek A.K, & Sansing L.H. (2021). Longitudinal transcriptomics define the stages of myeloid activation in the living human brain after intracerebral hemorrhage. Science immunology, 6(56), eabd6279.

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Hypoxia Signaling Pathway Regulation

Cited 1 time

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Cobalt (II) chloride, DMOG, echinomycin, and etoposide were purchased from Sigma-Aldrich. Daprodustat (GSK1278863) was purchased from DCC Chemicals. VH298 (CAS#2097381-85-4) was purchased from Cayman Chemical. MG was prepared by acid-catalyzed hydrolysis of dimethyl pyruvaldehyde, purified by fractional distillation, and analyzed by NMR as previously described (Tamae et al, 2011 (link)).
Antibodies used include XPA (MA5-13835; Invitrogen), XPC (A301-122A; Bethyl Laboratories), XPD (#11963; CST), XPG (sc12558; SCBT), CSB (24291-AP-1; Proteintech), PHD3 (NB100-139; Novus), HIF-1α (NB100-105 and BD 610959; Novus), HIF-1α-OH P562 (#3434; CST), HIF1AN (MA5-27619; Thermo Fisher Scientific), VEGFA (ab46154; Abcam), PDGFA (ab38562; Abcam), γ-H2AX (NB100-78356; Novus), p-ATR Ser428 (#2853; CST), p-ATM Ser1981 (#13050; CST), ATM (#2873; CST), ATR (#13934; CST), H2AX (#2595; CST), p-AKT (#13038; CST), IDH1 (ab172964; Abcam), α-tubulin-HRP (ab185067; Abcam), GAPDH (sc32233; SCBT), β-actin (#4970, rabbit; CST), β-actin (sc47778, mouse; SCBT), rb-α-ms-HRP (ab6728; Abcam), and gt-α-rb-HRP (ab6721; Abcam).

Ciminera A.K., Shuck S.C, & Termini J. (2021). Elevated glucose increases genomic instability by inhibiting nucleotide excision repair. Life Science Alliance, 4(10), e202101159.

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Cell Culture Conditions and Treatments

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Cell lines were grown at 37°C in a humidified incubator containing 5% CO₂. HeLa cells (ATCC CCL‐2), U2OS (ATCC HTB‐96) and HEK293T (ATCC CRL‐3216) cells were maintained in Dulbecco's modified Eagle's medium/nutrient mixture F‐12 GlutaMAX™ (DMEM/F‐12; Thermo Fisher Scientific) supplemented with 10% foetal bovine serum. Fibroblast cells derived from a patient with homozygous p.Arg435* FBXL4 have been previously published (Bonnen et al, 2013 (link)) (Alsina et al, 2020 (link)) and were cultured in DMEM/F‐12 GlutaMAX™ with 20% FBS and 5 mg/ml penicillin and streptomycin (Thermo Fisher Scientific). All cell lines were regularly screened for mycoplasma contamination. Where indicated, cells were treated with cycloheximide (CHX; 100 μg/ml; 66‐81‐9), deferiprone (DFP; 1 mM; 379409), DMOG (0.5 mM; D3695) and echinomycin (10 nM; SML0477), which were purchased from Sigma. MLN4924 (0.5 μM; 85923S) was obtained from Cell Signaling Technology. MG132 (10 μM; 474787) was purchased from Merck.

Nguyen‐Dien G.T., Kozul K., Cui Y., Townsend B., Kulkarni P.G., Ooi S.S., Marzio A., Carrodus N., Zuryn S., Pagano M., Parton R.G., Lazarou M., Millard S.S., Taylor R.W., Collins B.M., Jones M.J, & Pagan J.K. (2023). FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors. The EMBO Journal, 42(13), e112767.

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Echinomycin Treatment in Heterotopic Ossification Mice

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Mice (cases) were randomly assigned to one of two groups. The treatment (echinomycin [E]) group included eight cases (n = 8, E.1 - E.8) and the control (C) group included another eight cases (n = 8, C.1 - C.8). The treatment group received echinomycin (10 microgram (mcg) [0.3 mg/kg body weight]) (Sigma-Aldrich, Buchs, Switzerland). echinomycin was diluted in dimethyl sulfoxide (DMSO) and administered subcutaneously in the interscapular region. Injections were performed once a week for a total amount of four weeks [1 (link)]. The dosage was chosen due to the following considerations. A previous in vitro study [16 (link)] reported that the lethal dose 50 (LD50) of echinomycin was 12.3 mg/kg. However, further in vitro murine studies [29 (link), 30 (link)] have used higher dosages of 10 mg/kg every two days for 14 days [29 (link)] and up to 40 mg/kg every two days for a total of five administrations [30 (link)] without reporting adverse effects. Since Zimmermann et al. [1 (link)] reported an effect of echinomycin on heterotopic ossification without observing any adverse or lethal effects, the same dosage was used in this study. Euthanasia and harvesting of the limbs were performed at ten weeks.

Jentzsch T., Zimmermann S.M., Nicholls F., Cinelli P., Simmen H.P, & Werner C.M. (2016). Echinomycin did not affect the safety of fracture healing: an experimental pilot study on a murine femur fracture model. Patient Safety in Surgery, 10, 7.

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Tempol, Apigenin, and Echinomycin Modulate HIF-1α

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Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidine 1-oxyl free radical) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Apigenin and echinomycin, HIF-1α inhibitors, were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Anti-HIF-1α antibodies (cat# 3716S), anti-myc-tag antibodies (cat# 2272S) and anti-β-actin antibodies (cat# 4970S) were purchased from Cell Signaling Technology, K. K. (Japan).

KAGIYA G., OGAWA R., CHOUDHURI R., COOK J.A., HATASHITA M., TANAKA Y., KODA K., YAMASHITA K., KUBO M., KAWAKAMI F, & MITCHELL J.B. (2015). Selective enhancement of hypoxic cell killing by tempol-regulated suicide gene expression. Oncology Reports, 34(2), 1065-1073.

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Hypoxia-Induced Neutrophil Conditioned Media Effects on PASMC Proliferation

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Mouse PASMCs were isolated from WT mice with a C57BL/6 J background by enzymatic dissociation of the minced lung with collagenase type II (Worthington)^{58 (link)} and cultured in DMEM (Wako) containing 20% fetal bovine serum. The PASMCs were seeded in 96-well plates or on coverslips in 24-well plates. Conditioned medium from neutrophils in hypoxia incubator chamber (10% O₂, ASTEC) 3 h after incubation was collected. The neutrophils were pretreated with Echinomycin (Sigma) prior to hypoxia for 1 h. Then, the PASMCs were incubated with the conditioned medium for 48 h and then subjected to CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) and immunofluorescent analysis with anti-Ki67 (NB600-1252, Novus Biologicals) and αSMA (19245, Cell Signaling Technology) antibodies.

Kimishima Y., Misaka T., Yokokawa T., Wada K., Ueda K., Sugimoto K., Minakawa K., Nakazato K., Ishida T., Oshima M., Koide S., Shide K., Shimoda K., Iwama A., Ikeda K, & Takeishi Y. (2021). Clonal hematopoiesis with JAK2V617F promotes pulmonary hypertension with ALK1 upregulation in lung neutrophils. Nature Communications, 12, 6177.

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Regulation of Hypoxia Signaling Pathway

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HepG2 cells grown to 75% confluency were serum-starved in 2% FBS overnight prior to treatment of cells with echinomycin (Sigma-Aldrich, St. Louis, MO, USA), dimethyloxaloylglycine (DMOG, Cayman Chemical Company, Ann Arbor, MI, USA) or Compound 3 (Sigma-Aldrich, St. Louis, MO, USA). echinomycin is an inhibitor of HIF-1 activity, by blocking the binding of HIF-1 to DNA and preventing its activity as a transcription factor. DMOG increases the expression of HIF proteins by inhibiting their breakdown. Compound 3 (C3) is an inducer of REDD-1. Dose and time dependency studies were performed to identify the optimal drug concentration (echinomycin 10 nM, DMOG 500 μM, and C3 50 μM) and time of incubation (24 hours) used in this study to maximize effects while minimizing cell death (not shown). Prior to treatment, media was changed to 2% FBS overnight (12–16 hours). Cells were subjected to inhibitor/activator treatments in normoxic or hypoxic conditions for 24 hours. Subsequently, cell media was collected and immediately stored at −80°C and cells were stored at −80°C prior to collection of lysate as described previously^{28 (link)}.

Kakadia J., Biggar K., Jain B., Chen A.W., Nygard K., Li C., Nathanielsz P.W., Jansson T, & Gupta M.B. (2021). Mechanisms linking hypoxia to phosphorylation of insulin-like growth factor binding protein-1 in baboon fetuses with intrauterine growth restriction and in cell culture. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 35(9), e21788.

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Antibody Characterization for EMT Analysis

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We purchased primary antibodies specific for WT1, Snail, N-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA), E-cadherin 24E10, GAPDH (Cell Signaling Technology, Danvers, MA), E-cadherin clone 36, plakoglobin, VHL (BD Biosciences, San Jose, California), histone H3 (Upstate BioTech, Lake Placid, NY), rabbit IgG (Life Technologies), anti-HA (Roche Applied Science, Penzberg, Germany), α-SMA (Sigma-Aldrich, St. Louis, MO), ZO-1 (Thermo Fisher Scientific, Waltham, MA), Na,K-ATPase β₁-subunit (Abcam, Cambridge, United Kingdom). Horseradish peroxidase (HRP)-conjugates secondary antibodies for immunoblot analysis were purchased from Cell Signaling Technology, and FITC- and Texas Red-labeled secondary antibodies for immunofluorescence were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Echinomycin was purchased from Sigma-Aldrich (St. Louis, MO).

Sampson V.B., David J.M., Puig I., Patil P.U., de Herreros A.G., Thomas G.V, & Rajasekaran A.K. (2014). Wilms' Tumor Protein Induces an Epithelial-Mesenchymal Hybrid Differentiation State in Clear Cell Renal Cell Carcinoma. PLoS ONE, 9(7), e102041.

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