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17 dmag

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17-DMAG is a synthetic, water-soluble benzoquinone ansamycin that functions as a heat shock protein 90 (Hsp90) inhibitor. It is used in research applications to study the role of Hsp90 in various cellular processes.

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Lab products found in correlation

Mg132, by Merck Group (5 mentions) 17 aag, by Merck Group (4 mentions) Ver 155008, by Merck Group (4 mentions) Resveratrol, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Tranit lt1, by Mirus Bio (1 mentions) Ici 182 780, by Merck Group (1 mentions) Phospho ampkα, by Cell Signaling Technology (1 mentions) Control rabbit igg, by Santa Cruz Biotechnology (1 mentions) Ikkα β, by Abcam (1 mentions) Monoclonal antibodies against, by Cell Signaling Technology (1 mentions) P p70s6k, by Abcam (1 mentions) P ikkα β, by Abcam (1 mentions) Secondary antibodies coupled to irdye800 fluorophore, by Rockland Immunochemicals (1 mentions) Odyssey infrared imaging system, by Rockland Immunochemicals (1 mentions) Ampkα, by Cell Signaling Technology (1 mentions) Lamin b1, by Abcam (1 mentions) β actin, by Abcam (1 mentions) P iκbα, by Abcam (1 mentions) Hif 1α, by Abcam (1 mentions)

15 protocols using 17 dmag

1

Evaluating RNA Stability Regulators

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Cells were plated in six-well plates at ∼200 000 cells/well. A media change was performed 1 day after plating. Actinomycin D (ActD; Sigma) and DRB (Sigma) treatment were started around 2 days after plating for 0, 2, 4, 6, 12 and 24 h. To understand the roles of HSP90 inhibition, HDMYZ cells were treated with 1 μM of 17-DMAG (Sigma) for 0, 4, 8, 12 and 24 h. In addition, for concentration titration, HDMYZ cells were treated with 0.008, 0.04, 0.2, 1, 5 and 25 μM of either 17-DMAG or 17-AAG (Sigma) for 4 h. Experiments for each time point were performed with biological duplicate (ActD treatment) or triplicate (DRB and 17-DMAG treatment). The concentration of ActD and DRB for different cell lines was titrated so that cells were mostly alive after 24 h of treatment. Effectiveness of treatment was verified by analyzing c-myc RNA; see Supplementary Table S1 for ActD and DRB concentrations. After treatment at indicated time points, cells were washed with phosphate buffered saline (PBS) once and lysed in TriZol reagent immediately (Invitrogen). RNA extraction was performed following the manufacturer's protocol.
Guo Y., Liu J., Elfenbein S.J., Ma Y., Zhong M., Qiu C., Ding Y, & Lu J. (2015). Characterization of the mammalian miRNA turnover landscape. Nucleic Acids Research, 43(4), 2326-2341.
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2

Comparative Evaluation of Cancer Cell Lines

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Hep3B, Huh7, 293 T and HEK-293 cells were bought from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). DMEM (Dulbecco’s modified Eagle’s medium; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), penicillin (100 units/ml, Sigma, St. Louis, MO, USA) and streptomycin (100 μg/ml, Sigma) was used for cell culture. All these cells were kept in a humidified 5% CO2 incubator at 37 °C. For 17-AAG (Sigma) and 17-DMAG (Sigma) treatment, the cells were cultured in serum-free DMEM for 12 h, and then were cultured in serum-free DMEM containing 17-AAG or 17-DMAG for 24 h.
Xu Q., Tu J., Dou C., Zhang J., Yang L., Liu X., Lei K., Liu Z., Wang Y., Li L., Bao H., Wang J, & Tu K. (2017). HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma. Molecular Cancer, 16, 178.
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3

Pharmacological Inhibition Assay

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E2, ICI 182,780, MG132, Resveratrol, and 17-DMAG were purchased from Sigma. Dip G was isolated as reported previously (Ge et al., 2010 ). Dip G analogues (Dipto-Y-01 to Dipto-Y-06) were synthesized as previously described (Kim and Kim, 2010 (link)). Transfection reagent TranIT-LT1 was purchased from Mirus (Madison, WI).
Zhao Z., Wang L., Jung Y., Kim I., Tan R, & Xu W. (2015). Reciprocal regulation of ERα and ERβ stability and activity by Diptoindonesin G. Chemistry & biology, 22(12), 1608-1621.
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4

Profiling HSP90 and HSP70 Complexes

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HSP90 inhibitors used in this study including PU-H71, PU-DZ13, NVP-AUY922, and SNX-2112 were synthesized as previously reported7 (link),19 (link). 17-DMAG was purchased from Sigma. HSP90 bait (PU-H71 beads)21 (link), HSP70 bait (YK beads)22 (link), biotinylated YK (YK-biotin)22 (link), fluorescently labelled PU-H71 (PU-FITC)23 (link), the control derivatives PU-TEG and PU-FITC9 (ref. 24 ), and the radiolabelled PU-H71-derivative 124I-PU-H71 (ref. 25 (link)) were generated as previously described. The specificity of PU-H71 for HSP90 and over other proteins was extensively analysed7 (link). Thus binding of PU-H71 in cell homogenates, live cells and organisms denotes binding to HSP90 species characteristic of each analysed tumour or tissue. Combined with the findings that PU-H71 binds more tightly to HSP90 in type 1 than in type 2 cells, an observation true for cell homogenates, live cells, and in vivo, at the organismal level, we propose that labelled versions of PU-H71 are reliable tools to perturb, identify and measure the expression of the high-molecular-weight, multimeric HSP90 complexes in tumours. The specificity of YK probes for HSP70 was previously reported22 (link),26 (link)–28 (link).
Rodina A., Wang T., Yan P., Gomes E.D., Dunphy M.P., Pillarsetty N., Koren J I.I.I., Gerecitano J.F., Taldone T., Zong H., Caldas-Lopes E., Alpaugh M., Corben A., Riolo M., Beattie B., Pressl C., Peter R.I., Xu C., Trondl R., Patel H.J., Shimizu F., Bolaender A., Yang C., Panchal P., Farooq M.F., Kishinevsky S., Modi S., Lin O., Chu F., Patil S., Erdjument-Bromage H., Zanzonico P., Hudis C., Studer L., Roboz G.J., Cesarman E., Cerchietti L., Levine R., Melnick A., Larson S.M., Lewis J.S., Guzman M.L, & Chiosis G. (2016). The epichaperome is an integrated chaperome network that facilitates tumour survival. Nature, 538(7625), 397-401.
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5

Signaling Pathway Antibody Panel

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Primary antibodies against Hsp90β, TAK1, p-TAK1 (phospho-T187), TAB-1, ERK1/2, p-ERK1/2 (ERK1: phospho-Y204, ERK2: phospho-Y187), JNK1/2, p-JNK1/2 (phospho-T183/Y185), p38, p-p38 (phospho-T180/Y182), IκBα, p-IκBα (phospho-S32/S36), NF-κB p65, Bcl-2, cytochrome c, p-Bcl-2 (phospho-S87), p-p70S6 K (phospho-T389), p-mTOR (phospho-S2448), IKKα/β, p-IKKα/β (phsopho-S176/S177), HIF-1α, Lamin B1 (internal standard in nuclear protein fractions), β-actin (internal standard in cytosolic protein fractions) were from Abcam. Monoclonal antibodies against MKK3/6, p-MKK3/6 (MKK3: phospho-S189, MKK6: phospho-S207), AMPKα and phospho-AMPKα (phosphor-T172) were from Cell Signaling Technology and R&D Systems, respectively. Control rabbit IgG was from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated secondary antibodies were obtained from Calbiochem. Secondary antibodies coupled to IRDye800 fluorophore for use with the Odyssey Infrared Imaging System were purchased from Rockland. Hsp90 inhibitor, the water-soluble 17-DMAG, was purchased from Sigma Aldrich.
Yan S.H., Zhao N.W., Jiang W.M., Wang X.T., Zhang S.Q., Zhu X.X., Zhang C.B., Gao Y.H., Gao F., Liu F.M, & Fang Z.Y. (2016). Hsp90β is involved in the development of high salt-diet-induced nephropathy via interaction with various signalling proteins. Open Biology, 6(4), 150159.
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6

Transient Transfection of HEK 293 Cells

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HEK 293 cells were grown in DMEM containing 10% FBS and 100 U/ml penicillin/streptomycin (Gibco). Transient transfection was performed using LipoTransfectin (Attendbio) in cells after reaching approximately 80% confluence, and experiments were performed 24 h after transfection. All drugs were dissolved in dimethyl sulfoxide (DMSO) and diluted in DMEM to a final DMSO concentration of <0.5%. Ver-155008 and 17-DMAG were purchased from Sigma.
Capera J., Navarro-Pérez M., Moen A.S., Szabó I, & Felipe A. (2022). The Mitochondrial Routing of the Kv1.3 Channel. Frontiers in Oncology, 12, 865686.
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7

Evaluating Benzoquinone Ansamycin HSP90 Inhibitors

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17-AAG and 17-DMAG, two benzoquinone ansamycin HSP90 inhibitors were purchased from Sigma-Aldrich and dissolved in DMSO. Cells were seeded into 96-well plates at a density of 1500-3000 cells per well. After overnight incubation, cells were treated for 48h with vehicle alone (0.1% DMSO) or with various concentrations of 17-AAG or 17-DMAG (0.001, 0.01, 0.1, 1 and 10 uM, in 0.1% DMSO) in 100 ul of culture medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin. Each concentration was tested in duplicate; experiments were repeated two to three times for each cell. Cell viability was measured by MTS assays (Promega) according to the manufacturer’s recommendations. The concentration of drug inhibiting cells growth by 50% relative to the untreated control (GI50) was calculated after curve fitting with GraphPad Prism 5.0 software.
Schulze K., Imbeaud S., Letouzé E., Alexandrov L.B., Calderaro J., Rebouissou S., Couchy G., Meiller C., Shinde J., Soysouvanh F., Calatayud A.L., Pinyol R., Pelletier L., Balabaud C., Laurent A., Blanc J.F., Mazzaferro V., Calvo F., Villanueva A., Nault J.C., Bioulac-Sage P., Stratton M.R., Llovet J.M, & Zucman-Rossi J. (2015). Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nature genetics, 47(5), 505-511.
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8

Identifying Conditions for K11/K48-linked Chain Formation

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To identify conditions of K11/K48-linked chain formation HEK 293T or HeLa cells were treated with different drugs: Epoxomycin (1µM, 6h, Sigma-Aldrich), MG132 (10µM, 6h, Sigma-Aldrich), Pifithrin µ (10µM, 6h, Sigma-Aldrich), VER155008 (40µM, 6h, Sigma-Aldrich), 17 DMAG (1µM, 6h, Sigma-Aldrich), Chloroquine (100µM, 6h, Abcam), DBEQ (10µM, 6h, Sigma-Aldrich), Oligomycin/Antimycin (each 10µM, 1h, Sigma-Aldrich), CCCP (10µM, 2h, Abcam), Cycloheximide (100µg/mL, 6h, Sigma-Aldrich), Puromycin (25µM, 1h, Sigma-Aldrich), DTT (2mM, 6h), Tunicamycin (10µg/mL, 2h, Sigma-Aldrich), Doxorubicin (5µM, 6h, Sigma-Aldrich). Translation and transcription inhibitors were used at the following concentrations: Cycloheximide (100µg/ml), Harringtonine (2µg/ml), Emetine (20µg/ml), Puromycin (1µg/ml), α- Amanitin (5µg/ml). Accumulation of K11/K48-linked chains was monitored by Western blot or immunofluorescence microscopy.
Yau R.G., Doerner K., Castellanos E.R., Haakonsen D.L., Werner A., Wang N., Yang X.W., Martinez-Martin N., Matsumoto M.L., Dixit V.M, & Rape M. (2017). Assembly and Function of Heterotypic Ubiquitin Chains in Cell Cycle and Protein Quality Control. Cell, 171(4), 918-933.e20.
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9

Screening for Pyroptosis Inhibitors

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To screen for inhibitors of pyroptosis, 155 chemicals from microbial natural product library (Target Molecule, Wellesley Hills, MA) were added into individual wells of 96-well-plates at 50 μM 1 h before the induction of pyroptosis. The effect of chemicals on pyroptosis was measured with supernatant LDH activity and cell viability MTT assays.
To test the effect of HSP90 inhibitors on pyroptosis, Geldanamycin, 17-DMAG, 17-AAG and Radicicol (all from Sigma) were added to the culture system 1 h before the induction of pyroptosis with LPS and Nigericin. MG132 (Sigma) was added to the culture 1 h before HSP90 inhibitors if indicated. Ver-155008 (Selleckchem, Huston, TX) was added to the cell culture system simultaneously with HSP90 inhibitors. To test protein expression after HSP90 inhibition, cells were collected and lysed without the induction of pyroptosis at the indicated time after HSP90 inhibitors were added.
Zhou Z., Li X., Qian Y., Liu C., Huang X, & Fu M. (2020). Heat shock protein 90 inhibitors suppress pyroptosis in THP-1 cells. The Biochemical journal, 477(20), 3923-3934.
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10

DNMT3A Mutant Protein Stability Assay

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Bicistronic DNMT3A mutant reporter plasmids generated as previously described were transfected into HEK293T cells with four biological replicates in 96-well plates. Four controls, including untransfected, bicistronic DNMT3AWT, bicistronic DNMT3AW297Del, and bicistronic control (Addgene 92194), were always transfected in the same plate. Mean fluorescence intensity (MFI) of GFP and DsRed in the bicistronic DNMT3A mutant–transfected cells was measured 24 hours after transfection using flow cytometry. The stability ratio of DNMT3A protein was measured through MFI of GFP divided by MFI of DsRed and normalized with the bicistronic control. For experiments with various inhibitor treatments, including 10 μmol/L MG132 (a proteasome inhibitor; Sigma-Aldrich), 10 μmol/L MLN 4924 (a Cullin-RING E3 ligase inhibitor; Medchem), 50 μmol/L chloroquine (an autophagy inhibitor; Cell Signaling), 5 mmol/L 3-methyladenine (an autophagy inhibitor; Sigma-Aldrich), 50 μmol/L IREi (an unfolded protein response inhibitor; Sigma-Aldrich), 1 μmol/L 17-DMAG (HSP90i; Sigma-Aldrich), and 40 μmol/L VER155008 (HSP70i; Sigma-Aldrich), the inhibitors were added 24 hours after transfection, and MFI of fluorescence protein was measured 48 hours after transfection.
Huang Y.H., Chen C.W., Sundaramurthy V., Słabicki M., Hao D., Watson C.J., Tovy A., Reyes J.M., Dakhova O., Crovetti B.R., Galonska C., Lee M., Brunetti L., Zhou Y., Tatton-Brown K., Huang Y., Cheng X., Meissner A., Valk P.J., Van Maldergem L., Sanders M.A., Blundell J.R., Li W., Ebert B.L, & Goodell M.A. (2021). Systematic Profiling of DNMT3A Variants Reveals Protein Instability Mediated by the DCAF8 E3 Ubiquitin Ligase Adaptor. Cancer Discovery, 12(1), 220-235.
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