Journal: OncoTargets and therapy

Article Title: Human epidermal growth factor receptor 2-positive breast cancer: which cytotoxic agent best complements trastuzumab’s efficacy in vitro?

doi: 10.2147/OTT.S46883

Figure Lengend Snippet: ( A–F ) Apoptosis/necrosis plots for MCF-7 cells at 72 hours. ( A ) Untreated control; ( B ) cells exposed to trastuzumab at 72 hours; ( C ) cells exposed to doxorubicin at 72 hours; ( D ) cells exposed to the doxorubicin-trastuzumab combination at 72 hours; ( E ) cells exposed to geldanamycin at 48 hours; ( F ) cells exposed to the geldanamycin-trastuzumab combination at 48 hours. Similar trends were observed in the SK-BR-3 graphs. However, higher percentages for necrosis as opposed to early apoptosis were observed. Notes : FL1 log, annexin V fluorescein isothiocyanate; FL3 log, propidium iodide; H1, early apoptosis; H2, late apoptosis; H3, normal cells; H4, necrosis.

Article Snippet: Cell culture-compatible doxorubicin (Sigma-Aldrich) and geldanamycin (Tocris Bioscience, Bristol, UK) were dissolved in dimethyl sulfoxide (Merck, Darmstadt, Germany), stored at -80°C, and reconstituted in the appropriate medium prior to use.

Techniques: Control

Journal: OncoTargets and therapy

Article Title: Human epidermal growth factor receptor 2-positive breast cancer: which cytotoxic agent best complements trastuzumab’s efficacy in vitro?

doi: 10.2147/OTT.S46883

Figure Lengend Snippet: ( A–F ) Flow-cytometry histograms analyzed with deconvolution software dividing phases into G 1 (left), S (center), and G 2 (right). ( A ) Untreated MCF-7 cells in Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum; ( B ) MCF-7 cells exposed to trastuzumab, resulting in G 1 accumulation at 72 hours; ( C ) MCF-7 cells exposed to doxorubicin, resulting in late S-phase accumulation at 48 hours; ( D ) MCF-7 cells exposed to the doxorubicin-trastuzumab combination, resulting in G 1 and late S-phase accumulation at 48 hours; ( E ) MCF-7 cells exposed to geldanamycin, resulting in G 2 accumulation at 48 hours; ( F ) MCF-7 cells exposed to the geldanamycin-trastuzumab combination resulting in G 2 -phase accumulation at 48 hours. Similar trends in phase accumulation were observed in the SK-BR-3 graphs. However, the percentages differed.

Article Snippet: Cell culture-compatible doxorubicin (Sigma-Aldrich) and geldanamycin (Tocris Bioscience, Bristol, UK) were dissolved in dimethyl sulfoxide (Merck, Darmstadt, Germany), stored at -80°C, and reconstituted in the appropriate medium prior to use.

Techniques: Flow Cytometry, Software, Modification

Journal: OncoTargets and therapy

Article Title: Human epidermal growth factor receptor 2-positive breast cancer: which cytotoxic agent best complements trastuzumab’s efficacy in vitro?

doi: 10.2147/OTT.S46883

Figure Lengend Snippet: Cell viability in MCF-7 and SK-Br-3 cells expressed as a percentage of the untreated controls. Statistically significant differences were found between trastuzumab (T; 100 μg/mL) alone versus geldanamycin (GLD; 0.35 μM) alone and the GLD-T combination. Note : Statistical significance is represented by *** P < 0.001.

Article Snippet: Cell culture-compatible doxorubicin (Sigma-Aldrich) and geldanamycin (Tocris Bioscience, Bristol, UK) were dissolved in dimethyl sulfoxide (Merck, Darmstadt, Germany), stored at -80°C, and reconstituted in the appropriate medium prior to use.

Techniques:

Journal: OncoTargets and therapy

Article Title: Human epidermal growth factor receptor 2-positive breast cancer: which cytotoxic agent best complements trastuzumab’s efficacy in vitro?

doi: 10.2147/OTT.S46883

Figure Lengend Snippet: Relative human epidermal growth factor receptor 2 density at 24 hours in SK-BR-3 cells expressed as a percentage of the fluorescence of untreated controls (standardized to 100%). Note : Statistical significance followed the same trend at 12 and 48 hours, and is represented by *** P < 0.001. Abbreviations : GLD, geldanamycin (0.35 μM); T, trastuzumab (100 μg/mL).

Article Snippet: Cell culture-compatible doxorubicin (Sigma-Aldrich) and geldanamycin (Tocris Bioscience, Bristol, UK) were dissolved in dimethyl sulfoxide (Merck, Darmstadt, Germany), stored at -80°C, and reconstituted in the appropriate medium prior to use.

Techniques: Fluorescence

Journal: Genetics

Article Title: Mitochondrial perturbation reduces susceptibility to xenobiotics through altered efflux in Candida albicans

doi: 10.1093/genetics/iyab095

Figure Lengend Snippet: A functional genomic screen reveals that transcriptional repression of mitochondrial genes reduces susceptibility to the Hsp90 inhibitor geldanamycin. (A) Filamentation scores of GRACE strains. Strains were incubated for 6 h at 30°C in the presence of 0.05 µg/mL doxycycline (DOX), to repress target gene expression, and 10 µM geldanamycin (GdA), to induce filamentation. Heatmap summarizes the screening results with representative images showing strains belonging to each degree of filamentation classification. Strains were scored on a scale from 0 to 3, with mutants that grew exclusively as yeast being classified as a “0,” mutants that were mostly yeast with few short filaments classified as “1,” mutants with truncated filaments classified as “2,” and strains with filamentation comparable to wild type classified as “3.” Heatmap was generated using the heatmap.2 function in R. The number of strains that were classified within the different bins is listed in black in the heatmap. Scale bar, 10 µm. (B) Schematic of the prioritization triage. Fifty-four genes were identified for which their depletion reduced susceptibility to geldanamycin. Out of these, 33 strains were involved in mitochondrial function. (C) Percent growth inhibition of GRACE strains in the presence of 0.05 µg/mL DOX for 24 h at 30°C. Growth was determined by optical density at 600 nm (OD600). The data shown are the average of two technical replicates. Percent growth inhibition was calculated relative to the wild-type strain (orange dot). The 201 GRACE strains with at least 50% relative growth are below the red dotted line. (D) Percent growth inhibition of GRACE strains in the presence of 0.05 µg/mL DOX and 10 µM geldanamycin for 24 h at 30°C. Growth was determined by OD600. The data shown are the average of two technical replicates. Percent growth inhibition of strains in the presence of geldanamycin and DOX was calculated relative to the growth in the presence of DOX only. The wild-type strain showed 86% growth inhibition. GRACE strains with threefold reduced susceptibility to geldanamycin relative to the wild-type strain are below the red dotted line. (E) Fifty-four prioritized genes for which transcriptional repression conferred resistance to geldanamycin. Clusters were defined by GO term annotation and gene descriptions from Candida genome database (Skrzypek et al. 2017). Genes with mitochondrial annotations are highlighted in yellow. Asterisks indicate gene name based on S. cerevisiae homolog.

Article Snippet: Stock solutions of geldanamycin (Toronto Research Chemicals Inc), radicicol (AG Scientific Inc), gepinacin (Toronto Research Chemicals), and terbinafine (Sigma-Aldrich) were prepared in DMSO.

Techniques: Functional Assay, Incubation, Expressing, Generated, Inhibition

Journal: Genetics

Article Title: Mitochondrial perturbation reduces susceptibility to xenobiotics through altered efflux in Candida albicans

doi: 10.1093/genetics/iyab095

Figure Lengend Snippet: Repression of some mitochondrial genes reduces susceptibility to Hsp90 inhibitors. (A) The phenotype of GRACE mitochondrial mutants in different concentrations of Hsp90 inhibitors geldanamycin or radicicol, in the presence or absence of 0.05 µg/mL doxycycline, as indicated. Strains were grown for 10 h at 30°C under static conditions. Colors correspond to degree of filamentation (DOF; see color bar). Heatmap was generated using the heatmap.2 function in R. (B) Hsp90 inhibitor susceptibility phenotypes of GRACE strains. Strains were grown overnight in the absence and presence of 20 µg/mL doxycycline to repress gene expression. Strains were subsequently grown in the presence or absence of 10 µM geldanamycin, 20 µM radicicol, and 20 µg/mL DOX, as indicated. The growth was determined by OD600 after 24 h, and values were normalized to the wild-type no-drug controls. Data are presented as mean ± SD of technical triplicates. The growth of each strain in each condition was compared to the growth of the wild type in the absence of doxycycline and the presence of the Hsp90 inhibitor using an unpaired t-test. (C) Deletion of MSU1 or SHY1 reduces susceptibility to Hsp90 inhibitors, enabling filamentous growth upon incubation with higher concentrations of the compound. Strains were grown in the presence of the indicated concentrations of geldanamycin or radicicol for 7 h under shaking conditions prior to imaging. Scale bar, 10 µm. (D) Antifungal susceptibility phenotypes of strains grown in the presence or absence of 10 µM geldanamycin, 20 µM radicicol, 1 µg/mL fluconazole, 10 µg/mL terbinafine, or 20 µM gepinacin. Growth was determined by OD600 after 48 h, and values were normalized to no-drug control for each strain. Data are presented as mean ± SD of technical quadruplicates. The growth of each strain was compared to the growth of the wild-type control under each condition using an unpaired t-tests. *P-value < 0.05; **P-value < 0.01; ****P-value < 0.0001. DOX, doxycycline; RAD, radicicol; GdA, geldanamycin.

Article Snippet: Stock solutions of geldanamycin (Toronto Research Chemicals Inc), radicicol (AG Scientific Inc), gepinacin (Toronto Research Chemicals), and terbinafine (Sigma-Aldrich) were prepared in DMSO.

Techniques: Generated, Expressing, Incubation, Imaging

Journal: Genetics

Article Title: Mitochondrial perturbation reduces susceptibility to xenobiotics through altered efflux in Candida albicans

doi: 10.1093/genetics/iyab095

Figure Lengend Snippet: Mitochondrial mutants display enhanced efflux independent of transcriptional upregulation of efflux transporter genes. (A) Mitochondrial mutants display enhanced efflux of R6G. Cells were treated with R6G, and efflux was initiated by the addition of 1% (wt/vol) glucose. Fluorescence was measured every 15 min by flow cytometry. Mean fluorescence intensity (MFI) values were normalized to the starting MFI for each strain before the addition of glucose. (B) Deletion of TAC1 does not affect the sensitivity of mitochondrial mutants to Hsp90 inhibitors. Cells were inoculated in YPD medium with two-fold dilutions of geldanamycin or radicicol. After incubation at 30°C for 48 h under static conditions, growth was measured by the OD600. Data shown are the average of two technical replicates, normalized relative to the no-drug control for the wild-type strain. Data are quantitatively displayed with color using Java Treeview (see color bar). (C) Deletion of SHY1 or MSU1 does not lead to increased transcript levels of YOR1, CDR1, CDR2, SNQ2, or MDR1. Transcript levels of indicated genes were normalized to ACT1 and are relative to the wild-type control. Data reported are mean fold changes ± standard deviations from three independent experiments in technical triplicates. The significance of differences between the wild type and mitochondrial mutants for each gene was determined by an unpaired t-test. R6G, rhodamine 6G; RAD, radicicol; GdA, geldanamycin.

Article Snippet: Stock solutions of geldanamycin (Toronto Research Chemicals Inc), radicicol (AG Scientific Inc), gepinacin (Toronto Research Chemicals), and terbinafine (Sigma-Aldrich) were prepared in DMSO.

Techniques: Fluorescence, Flow Cytometry, Incubation

Journal: Genetics

Article Title: Mitochondrial perturbation reduces susceptibility to xenobiotics through altered efflux in Candida albicans

doi: 10.1093/genetics/iyab095

Figure Lengend Snippet: Yor1 and Cdr1 efflux Hsp90 inhibitors. (A) Candida albicans efflux pump gene deletion library screen identifies Yor1 and Cdr1 as key Hsp90 inhibitor efflux transporters. Cells were grown in a YPD medium in the absence or presence of 5 µM geldanamycin or 10 µM radicicol. After incubation at 30°C for 24 h under static conditions, growth was measured by the OD600. Data shown are the average of two technical replicates, normalized relative to the no-drug control for the wild-type strain. Data are quantitatively displayed with color using Java Treeview (see color bar). Gray boxes indicate strains not covered in the library. YOR1 and TPO3 are synthetic lethal, and therefore the double mutants are not in this library, as indicated by white crosses. (B) Deletion of YOR1 confers hypersensitivity to geldanamycin and deletion of CDR1 confers hypersensitivity to radicicol. Cells were inoculated in a YPD medium with two-fold dilutions of geldanamycin or radicicol. Data were analyzed as described for Figure 3B. (C) Transcript levels of CDR1 and YOR1 in tetO-YOR1/yor1Δ and tetO-CDR1/tetO-CDR1 strains. Cells were grown overnight in the absence or presence of 20 μg/mL doxycycline to repress target gene expression. Overnights were subcultured into YPD medium with the same doxycycline conditions for 4 h before RNA extraction. Transcript levels were normalized to ACT1 and are relative to the wild-type no-doxycycline control. Data are presented as mean ± SEM of technical triplicates. Expression was compared to the wild-type no-DOX control and significance was determined by an unpaired t-test. Asterisks indicate level of significance observed in both biological replicates. ***P-value < 0.001. (D) Hsp90 inhibitor susceptibility phenotypes of strains grown overnight in YPD medium in the absence or presence of 20 μg/mL doxycycline to repress target gene expression in the tetO strains, as indicated. Cells were inoculated in YPD medium in the absence or presence of 20 μg/mL doxycycline with twofold dilutions of geldanamycin or radicicol. Data were analyzed as described in Figure 3B. (E) Dose-response assays with C. albicans azole-sensitive and azole-resistant clinical strains where CDR1, CDR2, or CDR1 and CDR2 have been deleted. Data were analyzed as described in Figure 3B. (F) Hsp90 inhibitor susceptibility phenotypes of C. auris strains grown in YPD medium with twofold dilutions of geldanamycin or radicicol. Data were analyzed as described in Figure 3B. Roman numerals indicate the clade each C. auris strain belongs to. RAD, radicicol; GdA, geldanamycin; DOX, doxycycline; Flc, fluconazole.

Article Snippet: Stock solutions of geldanamycin (Toronto Research Chemicals Inc), radicicol (AG Scientific Inc), gepinacin (Toronto Research Chemicals), and terbinafine (Sigma-Aldrich) were prepared in DMSO.

Techniques: Incubation, Expressing, RNA Extraction

Journal: Genetics

Article Title: Mitochondrial perturbation reduces susceptibility to xenobiotics through altered efflux in Candida albicans

doi: 10.1093/genetics/iyab095

Figure Lengend Snippet: Mitochondrial perturbation enhances efflux activity in C. albicans. (A, B) Deletion of YOR1 and CDR1 in mitochondrial mutants enhances susceptibility to geldanamycin and radicicol, respectively. Strains were grown in the absence or presence of (A) 10 µM geldanamycin or (B) 20 µM radicicol. The growth was determined by OD600 after 48 h, and values were normalized to no-drug control for each strain. Data are presented as mean ± SD of technical triplicates. Growth of each strain was compared to the growth of the wild-type control under each condition using an unpaired t-test. ***P-value < 0.001; ns, not significant. (C) Deletion of YOR1 and CDR1 in mitochondrial mutants restores the ability of C. albicans to the filament in the presence of geldanamycin and radicicol, respectively. Strains were grown in the absence or presence of 10 µM geldanamycin or 10 µM radicicol for 7 h under shaking conditions prior to imaging. Scale bar, 10 µm. RAD, radicicol; GdA, geldanamycin.

Article Snippet: Stock solutions of geldanamycin (Toronto Research Chemicals Inc), radicicol (AG Scientific Inc), gepinacin (Toronto Research Chemicals), and terbinafine (Sigma-Aldrich) were prepared in DMSO.

Techniques: Activity Assay, Imaging

Journal: PloS one

Article Title: Dynamic impacts of the inhibition of the molecular chaperone Hsp90 on the T-cell proteome have implications for anti-cancer therapy.

doi: 10.1371/journal.pone.0080425

Figure Lengend Snippet: Figure 1. stSILAC experiments and workflow for data analysis. A) Labelling and sample preparation scheme. Geldanamycin or DMSO were added at t = 0. Total protein extracts were collected at t = 6h and 20h, while total mRNA was taken at t = 5h and t = 19h. B) Data analysis and interpretation combined data on protein abundance changes (stSILAC) with protein-protein interactions (PPI) analysed as a network, synthesis and decay values for proteins in the two conditions and transcript levels. Enrichment of Gene Ontology annotation terms was used to extract functional information on protein categories with common behaviours. doi:10.1371/journal.pone.0080425.g001

Article Snippet: Heavy-stSILAC labelled cells were treated with 1 mM Geldanamycin (GA) (Cell Signalling, Danvers, MA) in dimethylsulfoxide (DMSO) for 6 or 20h, and lightstSILAC-labelled cells were treated with the same volume of DMSO and used as a control.

Techniques: Sample Prep, Quantitative Proteomics, Protein-Protein interactions, Functional Assay

Journal: PloS one

Article Title: Dynamic impacts of the inhibition of the molecular chaperone Hsp90 on the T-cell proteome have implications for anti-cancer therapy.

doi: 10.1371/journal.pone.0080425

Figure Lengend Snippet: Figure 6. Changes in decay rate constants, synthesis rates, abundance and half-life for protein categories in response to treatment with geldanamycin. Relative average changes in synthesis (log2 [Vs_GA/Vs_DMSO]), decay (log2 [kd_GA/kd_DMSO]), abundance (log2 [pGA/pDMSO]), and half-life (log2 [T1/2_GA/T1/2_DMSO]) for selected protein categories. Numbers of proteins in each category are indicated in brackets. All values shown are adjusted for global proteome changes in synthesis and decay by subtracting the median of ratios for the whole dataset (911 proteins). doi:10.1371/journal.pone.0080425.g006

Article Snippet: Heavy-stSILAC labelled cells were treated with 1 mM Geldanamycin (GA) (Cell Signalling, Danvers, MA) in dimethylsulfoxide (DMSO) for 6 or 20h, and lightstSILAC-labelled cells were treated with the same volume of DMSO and used as a control.

Techniques: