To extract total RNA or perform the TF-activity assay, yeast cells were cultured for 8 h in 2x complete supplement medium (2x CSM: 0.7% yeast nitrogen base without amino acids, 2% dextrose, 0.2% CSM powder) adjusted to pH 6 with DMSO as control or with 50 μM or 25 μM Geldanamycin (GdA) (Ant-gl-25, InvivoGen, San Diego, CA, USA). Final cell density was controlled to OD600 = 0.2 to 0.8. The cells for promoter activity assays were cultured in cloNAT-containing medium [2x CSM supplied with 9.375 μg/ml cloNAT (Werner BioAgents, Jena, Germany)] and DMSO (D8418, Sigma-Aldrich, St. Louis, MO, USA) or 50 μM GdA. To ensure that the observed phenotypes were not GdA-specific, we also tested a different Hsp90 inhibiter radicicol (#R-1130, AG Scientific, San Diego, CA) and showed that 25 μM radicicol-treated cells exhibited phenotypes similar to GdA-treated cells (Figure S9 ). The cells for growth curve measurement were cultured in rich medium (YPD: 1% yeast extract, 2% bactopeptone, and 2% glucose), or in YPD containing 2 mM H2O2 (#18312, Riedel-deHaen, Seelze, Germany), or in YPD containing 25 nM rapamycin (#1568, BioVision, Milpitas, CA, USA). Hsp90 inhibition was induced by treatment with 50 μM GdA.
Radicicol
Manufactured by AG Scientific
Sourced in United States
Radicicol is a natural product compound that functions as a heat shock protein 90 (Hsp90) inhibitor. It disrupts the chaperone activity of Hsp90, a protein involved in the folding and stabilization of various client proteins.
Automatically generated - may contain errors
Lab products found in correlation
Rapamycin, by Abcam (2 mentions)
D8418, by Merck Group (2 mentions)
Geldanamycin, by Merck Group (2 mentions)
Radicicol, by Merck Group (2 mentions)
Tert butyl peroxide, by Merck Group (2 mentions)
Flat bottom 96 well microtiter plates, by Corning (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Geldanamycin, by AG Scientific (2 mentions)
Hydroxyurea, by Merck Group (1 mentions)
Rifabutin, by Merck Group (1 mentions)
17 aag, by Merck Group (1 mentions)
Nvp auy922, by Selleck Chemicals (1 mentions)
Biib021, by Selleck Chemicals (1 mentions)
Gedunin, by Bio-Techne (1 mentions)
Celastrol, by Bio-Techne (1 mentions)
17 dmag, by LC Laboratories (1 mentions)
Geldanamycin gda, by InvivoGen (1 mentions)
8 protocols using radicicol
1
Transcriptional Profiling of Yeast under Hsp90 Inhibition
2
Hsp90 Inhibitors Impact Yeast Growth
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To extract total RNA or perform the TF-activity assay, yeast cells were cultured for 8 h in 2x complete supplement medium (2x CSM: 0.7% yeast nitrogen base without amino acids, 2% dextrose, 0.2% CSM powder) adjusted to pH 6 with DMSO as control or with 50 µM or 25 µM Geldanamycin (GdA) (InvivoGen, San Diego, CA, USA). Final cell density was controlled to OD600 = 0.2 to 0.8. The cells for promoter activity assays were cultured in cloNAT-containing medium [2x CSM supplied with 9.375 µg/ml cloNAT (Werner BioAgents, Jena, Germany)] and DMSO (D8418, Sigma-Aldrich, St. Louis, MO, USA) or 50 µM GdA. To ensure that the observed phenotypes were not GdA-specific, we also tested a different Hsp90 inhibiter radicicol (#R-1130, AG Scientific, San Diego, CA ) and showed that 25 µM radicicol-treated cells exhibited phenotypes similar to GdA-treated cells (Supplemental Fig. S10A andS10B). The cells for growth curve measurement were cultured in rich medium (YPD: 1% yeast extract, 2% bactopeptone, and 2% glucose), or in YPD containing 2 or 3 mM H2O2 (#18312, Riedel-de Haen, Seelze, Germany), or in YPD containing 25 nM rapamycin (#1568, BioVision, Milpitas, CA, USA). Hsp90 inhibition was induced by treatment with 50 µM GdA.
3
Compound 1 Synthesis and Radicicol Inhibition
4
Isolating Aneuploid Yeast Strains
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We followed the protocol outlined by Chen et al. (2012) (link) to isolate aneuploid derivatives. Haploid strains (derived from S288c (DBY7286), oak strain YPS163 or vineyard strain KCY40) were grown in YPD +20 µg/ml radicicol (AG Scientific, San Diego, CA) for 24 hr to promote chromosome instability and induction of aneuploidy. Cells were then washed 3X with YPD, grown in YPD for 24 hr, and plated on YPD + 8–32 μg/ml fluconazole to select for Chr 8 aneuploidy, since amplification of ERG11 on Chr 8 was shown to confer fluconazole resistance (Chen et al., 2012 (link)). Minimal inhibitory concentrations on fluconazole at which 90% of cells (MIC90) die were determined for each strain: 32, 8, and 16 µg/ml for DBY7286, YPS163 and KCY40, respectively. Cells with Chr 8 amplification were initially screened via qPCR of select Chr 8 genes and confirmed by array comparative genomic hybridization (aCGH).
5
Construction and Maintenance of Yeast Strains
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Supplemental Tables S1 and S2 list yeast strains and plasmids used in this study, respectively. All KO, fluorescent, and epitope-tagged strains were constructed using PCR-based homologous recombination (Gietz and Schiestl, 2007 (link)). For Pif1 and Rrm3 mutants, the ORF was first replaced with a URA3 selectable marker, followed by replacement with the point mutant allele using an alternative selectable marker. All yeast strains were grown at 30°C unless otherwise noted.
qCTF strains were maintained in SD-Leu (Sunrise Biosciences, 1707-500) solid plates or media before the qCTF assay to select for the minichromosome. All other strains were grown in YPD unless otherwise noted. All drug treatments paired with qCTF assays were maintained for a 24-h culture period in SD-Complete medium (Sunrise Biosciences, 1701-500). qCTF assay drug concentrations were as follows: 10 µg/ml Radicicol (AG Scientific, R-1130), 25 mM Hydroxyurea (Sigma, H8627). For spot dilution assays, cells were grown to midlog phase in YPD to an OD of 0.62. A series of 10-fold dilutions was performed on this refreshed culture and 4 µl of each dilution was dropped onto indicated plates.
qCTF strains were maintained in SD-Leu (Sunrise Biosciences, 1707-500) solid plates or media before the qCTF assay to select for the minichromosome. All other strains were grown in YPD unless otherwise noted. All drug treatments paired with qCTF assays were maintained for a 24-h culture period in SD-Complete medium (Sunrise Biosciences, 1701-500). qCTF assay drug concentrations were as follows: 10 µg/ml Radicicol (AG Scientific, R-1130), 25 mM Hydroxyurea (Sigma, H8627). For spot dilution assays, cells were grown to midlog phase in YPD to an OD of 0.62. A series of 10-fold dilutions was performed on this refreshed culture and 4 µl of each dilution was dropped onto indicated plates.
6
Melanoma Cell Culture and Inhibitors
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Culture conditions and the cell lines have been previously described [12] (link). The B16-BL6 murine melanoma cell line was provided by Dr. Andrew Hurwitz [24] (link). Human cell lines were obtained from the ATCC, (Manassas, VA) (including MALME-M3, MM96L, A375, 453A, MM455, Roth, H59-44T, Mel-Juso, JURKAT), or from a patient at the Massashusetts General Hospital (MU89 and MU-X (derived from MU89 as previously described [6] (link), [11] (link)). Construction and culture of the J.RT3-T3.5 cell line expressing a Melan-A/MART-1 specific TCR has been described [15] (link). IFN-β-1a (Avonex) was obtained from Biogen-Idec (Cambridge, MA). The iHsp90s were obtained from the following sources: Radicicol (A.G. Scientific, San Diego, CA); Novobiocin (BioMol, Plymouth Meeting, PA); 17-DMAG (LC laboratories, Woburn, MA); 17-AEP (InVivoGen, San Diego, CA); Rifabutin, PU-H71, and 17-AAG (Sigma, St. Louis, MO); Gedunin, CCT018159, and celastrol (Tocris, Ellisville, MO); and NVP-AUY922 and BIIB021 (Selleck Chemicals, Houston, TX).
7
Antifungal Susceptibility Profiling
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Susceptibility of wild-type and resistant strains to AmB, AmBAU, AmBMU, tert-butyl peroxide (Sigma-Aldrich), geldanamycin and radicicol (A.G. Scientific) was determined in flat bottom, 96-well microtiter plates (Costar) using a broth microdilution protocol adapted from CLSI M27-A3. Overnight cultures (14–20hr) were grown at 30°C in YPD, and approximately 5×103 cells were seeded per well. For AmB, AmBAU, and AmBMU, MIC assays were performed at 37°C in RPMI buffered with MOPS (0.165M) with 10% fetal bovine serum (Sigma-Aldrich) added; for tert-butyl peroxide, geldanamycin, and radicicol, MIC’s were determined in YPD at 30°C. MIC’s were determined after 24h incubation as the concentration of compound resulting in no visible growth in wells. For quantitative display of growth at drug dilutions, OD600 was measured in a spectrophotometer (Tecan) and displayed as heat maps using Java TreeView 1.1.3 (http://jtreeview.sourceforge.net ).
8
Antifungal Susceptibility Profiling
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