Journal: Cancers

Article Title: Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability

doi: 10.3390/cancers12010215

Figure Lengend Snippet: Hakai interacts with heat shock protein 90 (Hsp90): Co-immunoprecipitation of Hakai and Hsp90 was performed in different cell lines: ( a ) HEK293T. ( b ) ACHN. ( c ) HT29. ( d ) HCT116. Hakai immunoprecipitation was performed by employing two different rabbit polyclonal Hakai antibodies indicated in material an methods: one from Bethyl (Bethyl Ab) and one kindly provided by Dr. Fujita (Hakai-2498). GAPDH signal was used as protein loading control.

Article Snippet: Lysosome degradation inhibitor Chloroquine (c-6628, Sigma-Aldrich, St. Louis, MO, USA), proteasome inhibitor MG132 (M8699, Sigma-Aldrich, St. Louis, MO, USA) and HSP90 Geldanamycin inhibitor (T6343, TargetMol, Wellesley Hills, MA, USA) were employed as indicated in figure legends.

Techniques: Immunoprecipitation

Journal: Cancers

Article Title: Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability

doi: 10.3390/cancers12010215

Figure Lengend Snippet: Hakai, Annexin A2, and Hsp90 interact with other: The whole cell extracts of HCT116 cell line cells were prepared and subjected to a co-immunoprecipitation assay. Subsequent western-blotting analysis was performed by using Hakai, Annexin A2, or Hsp90 antibodies ( a ) Hakai is specifically detected in anti-Hsp90 immunoprecipitates. ( b ) Annexin A2 is specifically detected in anti-Hakai immunoprecipitates. ( c ) Hsp90 is specifically detected in anti-Annexin A2 immunoprecipitates. GAPDH signal was used as protein loading control.

Article Snippet: Lysosome degradation inhibitor Chloroquine (c-6628, Sigma-Aldrich, St. Louis, MO, USA), proteasome inhibitor MG132 (M8699, Sigma-Aldrich, St. Louis, MO, USA) and HSP90 Geldanamycin inhibitor (T6343, TargetMol, Wellesley Hills, MA, USA) were employed as indicated in figure legends.

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Cancers

Article Title: Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability

doi: 10.3390/cancers12010215

Figure Lengend Snippet: Hakai regulates Annexin A2 expression levels: ( a ) HEK293T cells were transiently transfected with pcDNA-Flag-Hakai (4 µg), pBSSR-HA-ubiquitin (3 µg) and pSG-v-Src (3 µg) plasmids for 48 h. Cells were harvested and the levels of endogenous Annexin A2 levels and Hsp90 were determined by western blot analysis (left panel) and quantified by densitometry (right panel). The effect of the indicated transfected siRNA-Hakai on Annexin A2 and Hsp90 levels were tested in 293T cells ( b ) and HCT116 ( c ). Whole-cell lysates were subjected to western-blotting 72 h after transfection (top) and protein expression was quantified by densitometry (bottom) using GAPDH as loading control for normalization. Relative quantification of Annexin A2 expression levels was graphically represented as Mean ± SEM for two independent experiments for panel a and three for panel b and c (* p < 0.05, ** p < 0.01).

Article Snippet: Lysosome degradation inhibitor Chloroquine (c-6628, Sigma-Aldrich, St. Louis, MO, USA), proteasome inhibitor MG132 (M8699, Sigma-Aldrich, St. Louis, MO, USA) and HSP90 Geldanamycin inhibitor (T6343, TargetMol, Wellesley Hills, MA, USA) were employed as indicated in figure legends.

Techniques: Expressing, Transfection, Western Blot

Journal: Cancers

Article Title: Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability

doi: 10.3390/cancers12010215

Figure Lengend Snippet: Geldanamycin treatment disrupts Hakai, Hsp90 and Annexin A2 interaction: HCT116 cell line was treated with 10 µM geldanamycin for 24 h. ( a ) Immunoprecipitation was performed by using Hakai antibody. Subsequent western blotting-analysis was performed using Hakai, Annexin A2, or Hsp90 antibodies. ( b ) Immunoprecipitation was performed by using Annexin A2 antibody and western blotting analysis was performed using Hakai and Hsp90 antibodies. GAPDH was used as protein loading control.

Article Snippet: Lysosome degradation inhibitor Chloroquine (c-6628, Sigma-Aldrich, St. Louis, MO, USA), proteasome inhibitor MG132 (M8699, Sigma-Aldrich, St. Louis, MO, USA) and HSP90 Geldanamycin inhibitor (T6343, TargetMol, Wellesley Hills, MA, USA) were employed as indicated in figure legends.

Techniques: Immunoprecipitation, Western Blot

Journal: Cancers

Article Title: Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability

doi: 10.3390/cancers12010215

Figure Lengend Snippet: Hsp90 expression levels in colorectal cancer human samples: ( a ) Representative images of Hsp90 immunoreactivity in normal colonic mucosa, adenoma and colorectal cancer (TNM stages I–IV). Images were obtained with 20× objective. Scale bar 125 µm. ( b ) Statistical quantification of Hsp90 staining intensity in epithelial cancer cells at different colon cancer stages and in adenoma and normal colon tissue. A total of 30 human colon samples were analysed including: different TNM stages (I–IV) from colon adenocarcinomas, colon adenoma compared to human colon healthy tissues (normal colonic mucosa, n = 5; adenoma, n = 5; colorectal cancer, n = 5 of all stages) Scale bar, 200 µm. Black arrows show tumoral tissue and blue arrows show stromal tissue. Five photographs of each tissue were quantified and data are represented as scatter plot. Results are represented as Mean ± SEM for signal intensity scoring per area. Analysis was performed by employing Kruskal-Wallis with Tukey correction test (* p < 0.05).

Article Snippet: Lysosome degradation inhibitor Chloroquine (c-6628, Sigma-Aldrich, St. Louis, MO, USA), proteasome inhibitor MG132 (M8699, Sigma-Aldrich, St. Louis, MO, USA) and HSP90 Geldanamycin inhibitor (T6343, TargetMol, Wellesley Hills, MA, USA) were employed as indicated in figure legends.

Techniques: Expressing, Staining

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: 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:

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: Cancer Cell International

Article Title: The in vitro influences of epidermal growth factor and heregulin-β1 on the efficacy of trastuzumab used in Her-2 positive breast adenocarcinoma

doi: 10.1186/1475-2867-13-97

Figure Lengend Snippet: Cell viability in MCF-7 and SK-Br-3 cells. Mean (± SEM) cell viability determined using a tetrazolium conversion assay. Cells were first exposed to trastuzumab ranging from 1 μg/ml to 500 μg/ml. The result was a reduction in cell viability in SK-Br-3 cells which was not seen in MCF-7 cells. Cells were then exposed to a saturating concentration of trastuzumab combined with either EGF (400 ηg/ml) or HRG-β1 (200 ηg/ml). These endogenous biological molecules significantly abrogated cell viability observed for trastuzumab (Table ).

Article Snippet: Relative Her-2 receptor density was determined after cells were exposed to trastuzumab, growth factors or the positive control [geldanamycin, 0.35 μM (Tocris Bioscience; Ellisville, USA)] for 12, 24 and 48 hours, using an anti-Her-2-FITC affibody molecule (Abcam; Cambridge, UK) which is a protein composed of a three-helix bundled at the unique C-terminal cysteine which does not compete for Her-2 binding with trastuzumab [ ].

Techniques: Concentration Assay

Journal: Cancer Cell International

Article Title: The in vitro influences of epidermal growth factor and heregulin-β1 on the efficacy of trastuzumab used in Her-2 positive breast adenocarcinoma

doi: 10.1186/1475-2867-13-97

Figure Lengend Snippet: Flow cytometry histograms analysed with deconvolution software. Gaussian curves were utilised to divide histograms into G1 phase (left), S phase (centre) and G2 phase (right) A) Untreated MCF-7 cells in DMEM with 10% FCS; B) Trastuzumab treated MCF-7 cells after 72 hours illustrating G1 accumulation; C) Untreated SK-Br-3 cells in RPMI with 10% FCS; D) Trastuzumab treated SK-Br-3 cells after 24 hours illustrating G1 accumulation.

Article Snippet: Relative Her-2 receptor density was determined after cells were exposed to trastuzumab, growth factors or the positive control [geldanamycin, 0.35 μM (Tocris Bioscience; Ellisville, USA)] for 12, 24 and 48 hours, using an anti-Her-2-FITC affibody molecule (Abcam; Cambridge, UK) which is a protein composed of a three-helix bundled at the unique C-terminal cysteine which does not compete for Her-2 binding with trastuzumab [ ].

Techniques: Flow Cytometry, Software

Journal: Cancer Cell International

Article Title: The in vitro influences of epidermal growth factor and heregulin-β1 on the efficacy of trastuzumab used in Her-2 positive breast adenocarcinoma

doi: 10.1186/1475-2867-13-97

Figure Lengend Snippet: Column graphs of cell cycle analysis. Following exposure for 72 hours to trastuzumab, heregulin-β1 or the heregulin-β1-trastuzumab combination histograms were analyzed with deconvolution software and expressed as column graphs. A) Significant alterations within the G1 phase were observed in MCF-7 cells exposed to trastuzumab and the heregulin-β1-trastuzumab combination; B) Significant alterations in the S-phase were observed in SK-Br-3 cells exposed to heregulin-β1 compared to the untreated control. [EGF did not perpetuate noteworthy accumulation or alterations in cell cycle kinetics].

Article Snippet: Relative Her-2 receptor density was determined after cells were exposed to trastuzumab, growth factors or the positive control [geldanamycin, 0.35 μM (Tocris Bioscience; Ellisville, USA)] for 12, 24 and 48 hours, using an anti-Her-2-FITC affibody molecule (Abcam; Cambridge, UK) which is a protein composed of a three-helix bundled at the unique C-terminal cysteine which does not compete for Her-2 binding with trastuzumab [ ].

Techniques: Cell Cycle Assay, Software, Control

Journal: Cancer Cell International

Article Title: The in vitro influences of epidermal growth factor and heregulin-β1 on the efficacy of trastuzumab used in Her-2 positive breast adenocarcinoma

doi: 10.1186/1475-2867-13-97

Figure Lengend Snippet: Overlay plots and column graphs comparing the relative Her-2 receptor density in SK-Br-3 cells. A) Overlay plots of the unstained (black), stained (green) and the positive control, geldanamycin (red) with the associated geometric mean; B) Overlay plots of the unstained (black), stained (green) and cells exposed to trastuzumab (blue) with the associated geometric mean; C) Overlay plots of the unstained (black), stained (green) and cells exposed to heregulin-β1 (pink) or the heregulin-β1-trastuzumab combination (purple) with the associated geometric mean; D) Fluorescence (x-mean) intensity of triplicate runs, expressed as a percentage of untreated control (standardised to 100%). Concurrent heregulin-β1 abrogated relative Her-2 receptor density observed for trastuzumab. Trastuzumab, heregulin-β1 and the heregulin-β1-trastuzumab combination illustrated a progressive decrease in Her-2 receptor density at 24 hours. Similar significance trends were observed in SK-Br-3 cells exposed to EGF [56.82% (± 2.82)] and the EGF-trastuzumab combination [45.01% (± 1.06)].

Article Snippet: Relative Her-2 receptor density was determined after cells were exposed to trastuzumab, growth factors or the positive control [geldanamycin, 0.35 μM (Tocris Bioscience; Ellisville, USA)] for 12, 24 and 48 hours, using an anti-Her-2-FITC affibody molecule (Abcam; Cambridge, UK) which is a protein composed of a three-helix bundled at the unique C-terminal cysteine which does not compete for Her-2 binding with trastuzumab [ ].

Techniques: Staining, Positive Control, Fluorescence, Control

Journal: Cancer Cell International

Article Title: The in vitro influences of epidermal growth factor and heregulin-β1 on the efficacy of trastuzumab used in Her-2 positive breast adenocarcinoma

doi: 10.1186/1475-2867-13-97

Figure Lengend Snippet: Cell viability in MCF-7 and SK-Br-3 cells

Article Snippet: Relative Her-2 receptor density was determined after cells were exposed to trastuzumab, growth factors or the positive control [geldanamycin, 0.35 μM (Tocris Bioscience; Ellisville, USA)] for 12, 24 and 48 hours, using an anti-Her-2-FITC affibody molecule (Abcam; Cambridge, UK) which is a protein composed of a three-helix bundled at the unique C-terminal cysteine which does not compete for Her-2 binding with trastuzumab [ ].

Techniques: