Fibroblasts were treated with ACM for 24 h, washed once with MEM, and treated with the specified concentration of duramycin (Sigma D3168) or duramycin and PtdEtn diluted in MEM (1:3 duramycin:PtdEtn mol/mol) for 20 min at 37°C. Cells were washed twice with PBS and stained with the LIVE/DEAD® Viability/Cytotoxicity Kit (Life Technologies L3224) according to manufacturer's instructions. Cells were visualized by fluorescent microscopy and hand‐scored. Percent death was calculated as a ratio of ethidium‐stained red cells to total cells.
Duramycin
Manufactured by Merck Group
Sourced in United States, United Kingdom
Duramycin is a laboratory equipment product from the Merck Group. It is designed for specific laboratory applications. No further details can be provided while maintaining an unbiased and factual approach.
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Lab products found in correlation
Yeast extract, by BD (3 mentions)
Nitd008, by Merck Group (2 mentions)
7 deaza 2 c methyl d adenosine, by Biosynth (2 mentions)
Sncl2, by Merck Group (2 mentions)
Tricine, by Merck Group (2 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
Stearylamine, by Merck Group (1 mentions)
Ezview flag beads, by Merck Group (1 mentions)
Monoclonal flag antibody, by Merck Group (1 mentions)
Irdye 800cw goat anti rabbit igg h l, by LI COR (1 mentions)
Azithromycin, by Merck Group (1 mentions)
Rabbit anti active caspase 3, by Cell Signaling Technology (1 mentions)
Mouse igg1 isotype control antibody, by R&D Systems (1 mentions)
Rabbit anti gata4 antibody, by Abcam (1 mentions)
Goat anti axl antibody, by R&D Systems (1 mentions)
Human bfgf, by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Biotin, by Merck Group (1 mentions)
Zeba desalt spin column, by Thermo Fisher Scientific (1 mentions)
Phalloidin tetramethyl rhodamine, by Merck Group (1 mentions)
20 protocols using duramycin
1
Duramycin and PtdEtn-mediated Cell Death Assay
2
Axl Kinase and Duramycin Inhibition of Zika Virus Infection
3
Yeast Growth Sensitivity Analysis
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The yeast cells were grown at 28 °C in liquid SGA-Ura medium to an A600 of 0.6–0.8 and diluted with sterile water to an A600 of 0.1. Ten-fold serial dilutions (10–1, 10–2, 10–3, 10–4) were made in sterile water. A 10-μl aliquot of each of the diluted cell suspensions was then spotted on a plate with SGA-Ura or SDA-Ura medium. The plates were then placed at 28 °C under different light conditions for 3 days. For growth sensitivity to duramycin, the yeast cells grown in liquid SDA-Ura were diluted as above, and 2 μl of each of the diluted cell suspensions were examined on YPDA plates containing 20 μM duramycin [Sigma-Aldrich, St. Louis, MO] at 28 °C.
4
Quantitative Analysis of RhoA Dynamics
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Human breast cancer cell line MDA-MB-231 was purchased from ATCC. RhoA antibody (sc-418) and donkey anti-mouse IgG-FITC (sc-2099) were purchased from Santa Cruz. Duramycin, biotin and phalloidin tetramethylrhodamine (Cat#P1951) were from Sigma; biotinylated Duramycin was synthesised as described earlier (8 (link)); avidin-Texas red conjugate (A820) and avidin-FITC (Cat#43-4411) were from Life Technologies; Zeba desalt spin column (Cat#89890) was from Thermo Scientific. GFP-tagged wild-type RhoA (GFP-RhoA), constitutively active RhoA (GFP-CA-RhoA), wild-type RhoA with positive charges in the PBR mutated to Q (GFP-RhoA-PBRQ) and constitutively active RhoA with positive charges in the PBR mutated to Q (GFP-CA-RhoA-PBRQ) were prepared and used as previously described (9 (link)). Lact-C2-GFP (Cat#22852), 2PH-PLCdelta-GFP (Cat#35142) and PM-GFP (Cat#21213) were purchased from Addgene. LifeAct–TagGFP2 (Cat#60101) was purchased from Ibidi.
5
Radiolabeling of HYNIC-Duramycin with Technetium-99m
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A modified single-step kit protocol based on the method described by Zhao was used [23 (link)]. Briefly, duramycin (Sigma) was conjugated with hydrazinonicotinic acid (HYNIC; Solulink, CA, USA) at a molar ratio of 20:1 in anhydrous dimethylformamide. HYNIC-conjugated duramycin was purified by C18 reversed-phase ultra-performance liquid chromatography (UPLC) at a flow rate of 4 mL/min at room temperature. For radiolabeling with 99mTc, 100 μg HYNIC-conjugated duramycin was added to 100 mg tricine (Sigma), 32 mg trisodium triphenylphosphine-3,3,3-trisulfonate (Lianshuo, Shanghai, China), and 21 μg SnCl2 (Sigma) in a volume of 2 mL. Aliquots of 500 μL were freeze-dried overnight. The reaction vial was mixed with approximately 740 MBq of 99mTc-sodium pertechnetate (Atom High Tech, Beijing, China) in 500 μL of saline and then heated to 80°C for 20 min. The radiochemical purity was greater than 98%, as determined by instant thin-layer chromatography.
6
Duramycin-Mediated Cell Staining Assay
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Cells (2 x 10 5 ) were re-suspended in 50µl PBS and added to a 5ml polypropylene tube (Sarstedt, UK). Then either 10µl of duramycin (duramycin from Streptoverticillium cinnamoneus, Sigma-Aldrich, UK) at appropriate concentration or 10µl normal rabbit serum (1:5000 dilution) as negative control was added to the tube and incubated at room temperature for 30 minutes. Cells were washed with PBS, centrifuged at 320g for 3 minutes and re-suspended in 300µl PBS and 10µl of anti-duramycin antibody (Abcam, UK) (final dilution 1:600) was added and incubated in the dark at room temperature for 30 minutes. The cells were then washed and re-suspended as before and 50µl of sheep anti-rabbit IgG:FITC antibody (AbD Serotec ® ) (final dilution 1:600) added and incubated in the dark at room temperature for 30 minutes. Cells were washed, centrifuged and resuspended as before and then analysed using a BD FACScalibur (BD Biosciences, UK), which was the flow cytometer, used for all flow cytometric analysis in this study. Data is expressed as median fluorescence intensity (MFI) ratio which is calculated by division of the MFI value for the positive sample by the MFI value of the negative control sample giving the level of shift in fluorescence intensity of a population of cells expressed as a ratio.
7
Yeast Cell Culture and Lipid Treatment
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The yeast strains used in this study are listed in Table S3. Mutant strains and isogenic control strains were maintained on synthetic defined (SD) plates (Giaever et al. 2002 (link)). The conditions for cell culture were as reported previously (Kobayashi et al. 2005 (link)). Briefly, fresh colonies were inoculated into YPD medium and incubated overnight, unless otherwise stated. Overnight cultures of yeast cells were diluted to an optical density at 600 nm (OD600) of 0.2 in YPD medium and cultured. Cell growth was monitored by OD600 measurements. Cells in the logarithmic phase of growth were used. SR medium (6.7 g of yeast nitrogen base without amino acids [Difco, Detroit, MI] plus 20 g of raffinose per liter) was used for induction of the GAL1 promoter. Duramycin (Sigma Chemical Co., St. Louis, MO) was dissolved in water. ISP-1/myriocin (Sigma) was dissolved in methanol, and PHS (Sigma) and stearylamine (Sigma) were dissolved in ethanol. Aureobasidin A and dihydrosphingosines were dissolved in ethanol and methanol, respectively. Cells were treated with PHS (20 μM) and incubated for 15 or 30 min prior to harvesting. Treatment with ISP-1 (500–750 ng mL−1) was performed as described previously (Sun et al. 2000 (link)).
8
Yeast Strain Growth and Plasmid Maintenance
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General chemicals were purchased from Wako Pure Chemicals Industry (Osaka, Japan) unless otherwise stated. Papuamide B was from the collection of R. Andersen (University of British Columbia, Canada). Duramycin was purchased from Sigma-Aldrich (St. Louis, MO). Yeast strains were grown in YPDA-rich medium (1% yeast extract [Difco Laboratories, Detroit, MI], 2% Bacto-peptone [Difco], 2% glucose, and 0.01% adenine). Strains carrying plasmids were grown in SD synthetic medium (0.67% yeast nitrogen base without amino acids [Difco] and 2% glucose) that contained the required nutritional supplements (Rose, 1990 ). The SDA medium was SD medium that contained 0.5% casamino acid (Difco). For the induction of the GAL1 promoter, 3% galactose and 0.2% sucrose were used as carbon sources (YPGA and SGA-Ura media).
9
Yeast Mutants Sensitivity Assay
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S. cerevisiae mutant strain ZHY709 (MATα his3 leu2 ura3 met15 dnf1Δ dnf2Δ drs2::LEU2)16 (link) was used and transformed by the lithium acetate method. Transformants were cultured at 30 °C in standard synthetic glucose (SD) or galactose (SG) medium lacking the appropriate amino acids. SG gradient plates were stored at 4 °C for 2 days before use. Gradients contained the following maximum concentrations: 0.3 µg/mL papuamide A (Flintbox, Lynsey Huxham), 3 µM duramycin (Sigma-Aldrich), or 5 µg/mL miltefosine (hexadecylphosphocholine, Calbiochem, La Jolla, CA).
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S. cerevisiae mutant strain ZHY709 (MATα his3 leu2 ura3 met15 dnf1Δ dnf2Δ drs2::LEU2)16 (link) was used and transformed by the lithium acetate method. Transformants were cultured at 30 °C in standard synthetic glucose (SD) or galactose (SG) medium lacking the appropriate amino acids. SG gradient plates were stored at 4 °C for 2 days before use. Gradients contained the following maximum concentrations: 0.3 µg/mL papuamide A (Flintbox, Lynsey Huxham), 3 µM duramycin (Sigma-Aldrich), or 5 µg/mL miltefosine (hexadecylphosphocholine, Calbiochem, La Jolla, CA).
10
Papuamide A Purification and Antibody Detection
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