Journal: The Journal of Cell Biology

Article Title: The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex

doi: 10.1083/jcb.200210028

Figure Lengend Snippet: Hsp 90 is essential for mediating DAB 389 IL-2 C-domain translocation from the lumen of early endosomes to the external milieu. The Hsp 90-specific inhibitors, geldanamycin and radicicol, were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro. Excess partially purified HUT 102/6TG CTF complex and hrHsp 90 were added to geldanamycin/radicicol treated CTF complexes as indicated and assayed for translocation activity in vitro.

Article Snippet: The inhibitors geldanamycin (Alomone Labs), radicicol (Sigma-Aldrich), cis-13-retinoic acid (Sigma-Aldrich), and trans-13-retinoic acid (Sigma-Aldrich) were added as indicated. hrHsp 90 (StressGen Biotechnologies), brTrR-1 (American Diagnostica, Inc.), and hrTrx (American Diagnostica, Inc.) were added as indicated.

Techniques: Translocation Assay, Purification, Activity Assay, In Vitro

Journal: Biochimica et biophysica acta

Article Title: Lysosomal membrane stability plays a major role in the cytotoxic activity of the anti-proliferative agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT).

doi: 10.1016/j.bbamcr.2016.04.017

Figure Lengend Snippet: Fig. 2. The geldanamycin (GA)-induced increase of HSP70 expression or silencing of HSP70 expression in MCF7 cells does not affect Dp44mT-induced lysosomal destabilization. (A, B) Cells were preincubated with Control medium or medium containing GA (200 nM) for 24 h/37 °C followed by a 24 h/37 °C incubation with either Control medium, Dp44mT (5 μM), or DFO (250 μM). (Ai) Western blotting was performed to investigate the alteration of HSP70 expression. Blots shown are representative of 3 experiments. (Aii) Densitometric analysis (arbitrary units) of HSP70 western blots in (Ai). Densitometric analysis is mean ± SD (3 experiments) normalized to β-actin. (Bi) Lysosomal integrity was assessed by staining with ac- ridine orange (20 nM) using fluorescence microscopy. Scale bar: 10 μm. (Bii) Quantification of green and red fluorescence was performed using ImageJ. Fold change of green:red fluores- cence is mean ± SD (3 experiments) normalized to the Control. **p b 0.01, ***p b 0.001 versus the Control or between treatments as indicated. (C, D) MCF7 cells transfected with Negative Control siRNA or HSPA1A siRNA were incubated with Control medium, Dp44mT (5 μM), or DFO (250 μM) for 24 h/37 °C. (Ci) Western blotting was performed examining HSP70 expression. Blots shown are representative of 3 experiments. (Cii) Densitometric analysis (arbitrary units) of the results in (Ci). Densitometric analysis is mean ± SD (3 experiments) normalized to β-actin. (Di) Lysosomal integrity was shown by staining with acridine orange (20 nM) using fluorescence microscopy. Scale bar: 10 μm. (Dii) Quantification of red and green fluorescence was performed using ImageJ. Fold increase of green:red fluorescence is mean ± SD (3 experiments) normalized to Control or between treatments as indicated. *p b 0.05, **p b 0.01, and ***p b 0.001 versus the Control.

Article Snippet: Geldanamycin was obtained from Cell Signaling (Danvers, MA, USA) andwasdissolved inDMSO at 1mM,while Dp44mTwasdissolved in this solvent at 10mM.

Techniques: Expressing, Control, Incubation, Western Blot, Staining, Microscopy, Transfection, Negative Control

Journal: The Journal of General Virology

Article Title: The Feline calicivirus capsid protein VP1 is a client of the molecular chaperone Hsp90

doi: 10.1099/jgv.0.002030

Figure Lengend Snippet: Hsp90 inactivation by geldanamycin causes a reduction in VP1 but not in NS6/7 NS3 and VP2 protein levels. Mock-infected and FCV-infected cells at an MOI of 5 were treated with 0.5% DMSO or 0.5 µM GA. Total protein extracts were obtained, and the levels of ( a ) Hsp90, VP1, NS6/7, NS3 and VP2 were analysed at 3, 6 and 9 hpi by Western blotting. A representative blot of three independent experiments is shown. Hsp70 was used as a control of Hsp90 inhibition and annexin A2 (ANXA2) and actin as loading controls. Mock-infected treated and non-treated cells were included as controls. The relative expression of ( b ) Hsp90, ( c ) VP1, ( d ) NS6/7, ( f ) NS3 and ( g ) VP2 from three independent experiments is shown. Densitometric values were obtained using FIJI software, and the statistical analysis was performed using GraphPad Prism 10 software. No significant (ns) differences are indicated. Values of P < 0.01 (**) and P < 0.0001 (****) calculated by two-way ANOVA are indicated.

Article Snippet: Depending on the experimental conditions, cells were treated with geldanamycin (GA) (Santa Cruz Biotechnology), chloroquine (CQ) (Sigma-Aldrich), carbobenzoxy- l -leucyl- l -leucyl- l -leucinal (MG132) (Sigma-Aldrich) or17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) (Merck Millipore), alone or combined at the indicated concentrations, or the drug vehicles as controls, added to the maintenance media.

Techniques: Infection, Western Blot, Control, Inhibition, Expressing, Software

Journal: The Febs Journal

Article Title: The deubiquitinase inhibitor WP1130 drives nuclear aggregation and reactivation of mutant p53 for selective cancer cell targeting

doi: 10.1111/febs.70036

Figure Lengend Snippet: WP1130 treatment alters the interactions of mutp53 with chaperones and E‐3 ubiquitin Ligases MDM2 and CHIP: (A) C‐33A cells were treated with Vehicle control (Vc) (DMSO) or 10 μ m of WP1130 (WP) for 4 h; Post‐treatment, equal concentrations of proteins were subjected to immunoprecipitation using a p53 antibody (DO‐1). The interaction of HSP40, HSP70, and HSP90 with mutp53 was analyzed using western blot analysis ( n = 2). (B) SK‐BR‐3 cells were transfected with si‐control or siHSP40 for 48 h. Post‐transfection, cells were harvested and the levels of p53 and HSP40 were determined using immunoblotting. Vinculin was used as a loading control ( n = 2). The numbers below the blots indicate the protein quantification normalized to vinculin using densitometry. (C) SK‐BR‐3 and C‐33A cells were treated with DMSO or 2.5 μ m geldanamycin (GA) for 6 h; cells were harvested post‐treatment and protein lysates were subjected to western blotting to detect p53 levels. The total protein lane was used as a loading control ( n = 3). Numbers below the blots indicate p53 quantification normalized to the total protein lane using densitometry. (D) SK‐BR‐3 and C‐33A cells were transfected with si‐control or siHSP70 for 48 h. Levels of p53 and HSP70 were determined using western blotting. Vinculin was used as a loading control ( n = 3). The numbers below the blots indicate protein quantification normalized to the vinculin using densitometry. (E) SK‐BR‐3 cells were treated with DMSO (Vc) or 10 μ m WP1130 (WP) for 4 h. Post‐treatment, equal concentrations of proteins were subjected to immunoprecipitation using a p53 antibody (DO‐1). The interaction of CHIP and MDM2 with mutp53 was analyzed by immunoblotting ( n = 2). (F) SK‐BR‐3 and C‐33A cells were transfected with si‐control or siCHIP for 72 h. Post‐transfection, protein lysate was subjected to immunoblotting to detect p53 and CHIP. Vinculin was used as a loading control ( n = 3). The numbers below the blots indicate protein quantification normalized to the vinculin using densitometry. In A and E, the numbers below the blot indicate the quantification of proteins normalized to immunoprecipitated p53 using densitometry.

Article Snippet: WP1130 (Degrasyn), MG132, Chloroquine (CQ), and Geldanamycin (GA) were obtained from Selleck Chemicals, USA. dimethyl sulfoxide (DMSO) was purchased from HiMedia, India.

Techniques: Ubiquitin Proteomics, Control, Immunoprecipitation, Western Blot, Transfection

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