Journal: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet:

Article Snippet: LaminA/C (Cat#: sc-6215, WB 1/2000) and Hsp90β (Cat#: sc-1057, WB 1/2000) were from Santa Cruz.

Techniques: Control, Membrane, Virus, Recombinant, Sample Prep, Flow Cytometry, Western Blot, Microscopy, Plasmid Preparation, Phospho-proteomics, Variant Assay, shRNA, Software, CRISPR

Journal: Theranostics

Article Title: Jujuboside A promotes Aβ clearance and ameliorates cognitive deficiency in Alzheimer's disease through activating Axl/HSP90/PPARγ pathway

doi: 10.7150/thno.26164

Figure Lengend Snippet: Aβ induces depletion of HSP90 and PPARγ. (A) Brain tissue from 8-month-old Wt or APP/PS1 transgenic mice was collected and subjected to Western blot analyses. BV2 cells (B) or primary microglia (C) were administrated Aβ42 (5 μM) for the indicated times. The detergent-soluble lysates from cells were extracted for Western blot assay. BV2 cells (D) or primary microglia (E) were administrated Aβ42 (5 μM) for 12 h. Total protein was extracted and subjected to IP study with indicated antibodies. (F) BV2 cells were incubated with Aβ42 (5 μM) for the indicated times and protein levels of HSP90α, HSP90β and PPARγ were detected. (G) BV2 cells were transfected with indicated siRNA for 48 h. (H) BV2 cells were transfected with p-HSP90β and administrated Aβ42 (5 μM) for 12 h. Whole cell proteins were collected for western blot assay. All Experiments were repeated three times. * p < 0.05, *** p < 0.001 vs. indicated control.

Article Snippet: Mouse non-target control siRNA (sc-37007), HSP90β siRNA (sc-35607), HSP90α siRNA (sc-35609), Axl siRNA (sc-29770) were purchased from Santa Cruz (Dallas, USA).

Techniques: Transgenic Assay, Western Blot, Incubation, Transfection, Control

Journal: Theranostics

Article Title: Jujuboside A promotes Aβ clearance and ameliorates cognitive deficiency in Alzheimer's disease through activating Axl/HSP90/PPARγ pathway

doi: 10.7150/thno.26164

Figure Lengend Snippet: HSP90β is essential for maintaining PPARγ function in microglia. BV2 cells were pretreated with JuA at 1 μM, 5 μM or 25 μM for 30 min, followed by administration of Aβ42 (5 μM) for 12 h. Total protein was extracted and subjected to western blot (A) and IP study (B) with indicated antibodies. (C) BV2 cells were transfect with HSP90β siRNA for 48 h, and then pretreated with JuA for 30 min followed by administration of Aβ42 (5 μM) for 12 h. DSMO at 0.1% was used as Ctrl. Whole cell proteins were collected for western blot assay. All experiments were repeated three times. ### p < 0.001, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse non-target control siRNA (sc-37007), HSP90β siRNA (sc-35607), HSP90α siRNA (sc-35609), Axl siRNA (sc-29770) were purchased from Santa Cruz (Dallas, USA).

Techniques: Western Blot

Journal: Theranostics

Article Title: Jujuboside A promotes Aβ clearance and ameliorates cognitive deficiency in Alzheimer's disease through activating Axl/HSP90/PPARγ pathway

doi: 10.7150/thno.26164

Figure Lengend Snippet: JuA up-regulates HSP90β in an Axl/ERK-dependent manner. BV2 cells were treated with JuA (25 μM) for indicated periods of time (A) , or treated with JuA at indicated concentrations for 0.5 h (B) . (C) BV2 cells were pretreated with 0.1% DMSO (Ctrl), JuA (25 μM) or JuA (25 μM) + SCH772984 (10 μM) for 30 min, followed by administration of Aβ42 (5 μM) for 6 h. (D) BV2 cells were pretreated with 0.1% DMSO (Ctrl), JuA (25 μM) or JuA (25 μM) with the indicated antagonist of RTKs (Dovitinib at 1 μM, Gefinitib at 2.5 μM, Sunitinib at 2.5 μM and LDC1267 at 1 μM) for 30 min, followed by administration of Aβ42 (5 μM) for 12 h. (E) BV2 cells were pretreated with 0.1% DMSO (Ctrl), JuA (25 μM) or JuA (25 μM) with the indicated antagonist of TAM receptor (LDC1267 at 1 μM, UNC2250 at 5 μM, R428 at 5 μM) for 30 min, followed by administration of Aβ42 (5 μM) for 12 h. (F) BV2 cells were transiently transfected with non-target control siRNA or siAxl for 48 h. (G) BV2 cells were transfected with siAxl for 48 h, then pretreated with JuA (25 μM) followed by administration of Aβ42 (5 μM) for 12 h. The whole-cell proteins were subjected to western blot with the antibodies indicated. All experiments were repeated three times. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse non-target control siRNA (sc-37007), HSP90β siRNA (sc-35607), HSP90α siRNA (sc-35609), Axl siRNA (sc-29770) were purchased from Santa Cruz (Dallas, USA).

Techniques: Transfection, Control, Western Blot

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Heat shock protein 90-β over-expression is associated with poor survival in stage I lung adenocarcinoma patients

doi:

Figure Lengend Snippet: Hsp90-β protein expression in lung ADC tissues as observed by immunohistochemical staining with a rabbit polyclonal antibody against Hsp90-β applied to formalin-fixed and paraffin-embedded tissues in the manner of tissue microarrays. A. The expression intensity of Hsp90-β immunostaining in normal alveoli was IRS0; B-D. The expression intensities of Hsp90-β immunostaining in lung ADCs were IRS01, IRS2, and IRS3, respectively.

Article Snippet: TMA sections (5 m thick) were incubated at 4°C in a humid chamber overnight with a rabbit polyclonal antibody against human Hsp90-β (1:100, ABGENT, catalog #AP7867d, San Diego, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunostaining

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Heat shock protein 90-β over-expression is associated with poor survival in stage I lung adenocarcinoma patients

doi:

Figure Lengend Snippet: Correlation between clinicopathological features and Hsp90-β expression in lung adenocarcinomas

Article Snippet: TMA sections (5 m thick) were incubated at 4°C in a humid chamber overnight with a rabbit polyclonal antibody against human Hsp90-β (1:100, ABGENT, catalog #AP7867d, San Diego, CA, USA).

Techniques: Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Heat shock protein 90-β over-expression is associated with poor survival in stage I lung adenocarcinoma patients

doi:

Figure Lengend Snippet: Protein expression of Hsp90-β in lung ADC tissues was detected by Western blotting. A. Hsp90-β expression in paired normal (N) and tumor (T) tissues of 24 ADC patients. B. The Western blot results indicated high expression levels of Hsp90-beta in the cancer tissues than the adjacent normal tissues (P<0.001).

Article Snippet: TMA sections (5 m thick) were incubated at 4°C in a humid chamber overnight with a rabbit polyclonal antibody against human Hsp90-β (1:100, ABGENT, catalog #AP7867d, San Diego, CA, USA).

Techniques: Expressing, Western Blot

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Heat shock protein 90-β over-expression is associated with poor survival in stage I lung adenocarcinoma patients

doi:

Figure Lengend Snippet: The Kaplan-Meier survival analysis revealed that the patients with high (n=16) Hsp90-β expression had a significantly poorer outcome than those with moderate (n=20) or low (n=8) Hsp90-β expression (P=0.034).

Article Snippet: TMA sections (5 m thick) were incubated at 4°C in a humid chamber overnight with a rabbit polyclonal antibody against human Hsp90-β (1:100, ABGENT, catalog #AP7867d, San Diego, CA, USA).

Techniques: Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Heat shock protein 90-β over-expression is associated with poor survival in stage I lung adenocarcinoma patients

doi:

Figure Lengend Snippet: Multivariate cox regression analysis for overall survival in early stage patients with lung ADC cancer

Article Snippet: TMA sections (5 m thick) were incubated at 4°C in a humid chamber overnight with a rabbit polyclonal antibody against human Hsp90-β (1:100, ABGENT, catalog #AP7867d, San Diego, CA, USA).

Techniques:

Journal: Cell Adhesion & Migration

Article Title: Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

doi: 10.1080/19336918.2015.1103421

Figure Lengend Snippet: Undersulfation of HSPGs leads to a severe loss of surface-bound Hsp90α and Hsp90β in A-172 and HT1080 cells. Cells were grown in DMEM-FBS containing 30 mM sodium chlorate for 24 h at 37°C, stained with anti-Hsp90α, anti-Hsp90β, and anti-heparan sulfate antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and chlorate-treated (red lines) cells stained with Hsp90-specific antibodies, as well as for cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β expression after chlorate treatment. The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P < 0.05) from untreated cells. The representative results from 3 independent experiments are shown. ( D ) Western blot analyses of total (intracellular and cell-surface) levels of Hsp90α, Hsp90β, and β-actin (loading control) in chlorate-treated and control A-172 and HT1080 cells. The representative results from 3 independent experiments are shown.

Article Snippet: Rabbit Hsp90α- and Hsp90β-specific antibodies directed to N-terminal fragments of human Hsp90α (PEETQTQDQPM) and Hsp90β (EEVHHGEEEVE) were used (USBiological H1834–55A and H1832–98H).

Techniques: Staining, Confocal Microscopy, Flow Cytometry, Control, Negative Control, Membrane, Expressing, Western Blot

Journal: Cell Adhesion & Migration

Article Title: Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

doi: 10.1080/19336918.2015.1103421

Figure Lengend Snippet: Digestion of cell surface HSPGs with heparinase considerably reduces the level of surface-bound Hsp90α and Hsp90β in A-172 and HT1080 cells. Cells were incubated for 1 h at 37°C with a heparinase I/III blend, stained with anti-Hsp90α, anti-Hsp90β, and anti-heparan sulfate antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and heparinase-treated (red lines) cells stained with Hsp90-specific antibodies, as well as for cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β expression after heparinase treatment. The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P < 0.05) from untreated cells. The representative results from 3 independent experiments are shown. ( D ) Western blot analyses of total (intracellular and cell-surface) amounts of Hsp90α, Hsp90β, and β-actin (loading control) in heparinase-treated and control A-172 and HT1080 cells. The representative results from 3 independent experiments are presented.

Article Snippet: Rabbit Hsp90α- and Hsp90β-specific antibodies directed to N-terminal fragments of human Hsp90α (PEETQTQDQPM) and Hsp90β (EEVHHGEEEVE) were used (USBiological H1834–55A and H1832–98H).

Techniques: Incubation, Staining, Confocal Microscopy, Flow Cytometry, Control, Negative Control, Membrane, Expressing, Western Blot

Journal: Cell Adhesion & Migration

Article Title: Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

doi: 10.1080/19336918.2015.1103421

Figure Lengend Snippet: Treatment of A-172 and HT1080 cells with heparin results in a significant loss of membrane-bound Hsp90β and Hsp90α. Cells were treated for 1 h at 37°C ( A, B, D, F ) or at 37°C and 4°C ( E ) with heparin at concentrations of 50 μg/ml ( A, B, E, F ) or 0–100 μg/ml ( D ), stained with anti-Hsp90α and anti-Hsp90β antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B, D, E, F ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and heparin-treated (red lines) cells stained with Hsp90-specific antibodies, as well as cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Western blot analyses of total (intracellular and cell-surface) levels of Hsp90α, Hsp90β, and β-actin (loading control) in A-172 and HT1080 cells treated with heparin at a concentration of 50 μg/ml for 1 h at 37°C. The representative results from 3 independent experiments are shown. ( D, E ) The flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β after treatment with heparin at different concentrations ( D ) and different temperatures ( E ). The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. ( F ) The percentage of restoration of membrane-bound Hsp90α and Hsp90β is presented. The levels of membrane-bound Hsp90α and Hsp90β were estimated by the respective specific MFI. The levels of membrane-associated Hsp90 in heparin-treated cells and control cells were taken as a 0% restoration and a 100% restoration, respectively. Each bar ( D, E ) and point ( F ) represent the mean ± SD (n = 5–6). *Statistically significantly different ( P < 0.05) from untreated cells. The representative results from 3 independent experiments are shown.

Article Snippet: Rabbit Hsp90α- and Hsp90β-specific antibodies directed to N-terminal fragments of human Hsp90α (PEETQTQDQPM) and Hsp90β (EEVHHGEEEVE) were used (USBiological H1834–55A and H1832–98H).

Techniques: Membrane, Staining, Confocal Microscopy, Flow Cytometry, Control, Negative Control, Western Blot, Concentration Assay

Journal: Cell Adhesion & Migration

Article Title: Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

doi: 10.1080/19336918.2015.1103421

Figure Lengend Snippet: Binding of Hsp90 to cells ( A-C ) and colocalization of cell surface Hsp90 with cellular HSPGs ( D ). ( A, B ) A-172 and HT1080 cells were treated with sodium chlorate or heparinase. A suspension of cells was incubated for 1 h at 4°C with Hsp90-FITC. Untreated cells were incubated with Hsp90-FITC in the presence and absence of heparin (20 μg/ml). Then, the cells were washed, fixed with formaldehyde, and analyzed by flow cytometry. Representative flow cytometry histograms for A-172 ( A ) and HT1080 ( B ) cells are presented. Red lines – chlorate- and heparinase-treated cells and cells incubated with heparin; green lines - untreated cells; black lines – control cells (autofluorescence). ( C ) Flow cytometry-based quantification of Hsp90 binding to cells. The data are presented as the FITC-Hsp90-specific MFI of treated and control cells, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P < 0.05) from untreated cells. ( D ) Live untreated cells and cells treated with heparin at a concentration of 50 μg/ml for 1 h at 37°C were stained simultaneously with antibodies specific to Hsp90α or Hsp90β (red fluorescence) and HSPGs (green fluorescence), counterstained with Hoechst 33258, and analyzed by confocal microscopy. Representative confocal microscopy images showing surface staining with antibodies are presented. Inserts are enlarged images of regions of interest. Yellow fluorescence indicates the colocalization of cell-surface Hsp90 and HSPGs. Scale bar: 20 μm.

Article Snippet: Rabbit Hsp90α- and Hsp90β-specific antibodies directed to N-terminal fragments of human Hsp90α (PEETQTQDQPM) and Hsp90β (EEVHHGEEEVE) were used (USBiological H1834–55A and H1832–98H).

Techniques: Binding Assay, Suspension, Incubation, Flow Cytometry, Control, Concentration Assay, Staining, Fluorescence, Confocal Microscopy

Journal: British Journal of Cancer

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation

doi: 10.1038/bjc.2013.710

Figure Lengend Snippet: STCA destabilises the HSP90 client proteins HER2, RAF1, and mutant p53, and stabilises WT p53. ( A ) Domain structure of human HSP90 β . The N-terminal domain (NTD) is the site of binding of ATP and some co-chaperones. The middle domain (MD) is where many of the HSP90 client proteins and co-chaperones interact, and the C-terminal domain (CTD) contains a dimerisation motif. The white bars indicate the distribution and positions of the cysteine residues. ( B – D ) MCF7 cells (2.5 × 10 5 per well) in six-well plates were exposed to vehicle (0.1% acetonitrile) or STCA for 16 h. Cells were then lysed in RIPA buffer, proteins in aliquots from cell lysates were resolved by SDS–PAGE, transferred to immobilon-P, and probed with antibodies against HER2, HSP70 ( B ), RAF1( C ), and glycogen synthase kinase 3 (GSK3) α /GSK3 β ( D ). ( E ) MDA-MB-231 cells (5 × 10 5 per well) in six-well plates were treated with vehicle (0.1% acetonitrile) or STCA for 24 h. The levels of mutant p53 and HSP70 were detected by western blot analyses. ( F ) MCF7 cells (2.5 × 10 5 per well) in six-well plates were treated with vehicle (0.1% acetonitrile) or STCA for 24 h. The levels of WT p53 were detected by western blot analyses. ( G ) MCF7 cells were treated with vehicle (0.1% acetonitrile) or 75 μ M STCA for 2, 4, 8 or 24 h. For all panels, the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were also determined as a loading control. The data represent results from three independent experiments.

Article Snippet: Human HSP90β clone was kindly provided by Dr Anutosh Chakraborty and Dr Solomon H Snyder (Johns Hopkins University).

Techniques: Mutagenesis, Binding Assay, SDS Page, Western Blot, Control

Journal: British Journal of Cancer

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation

doi: 10.1038/bjc.2013.710

Figure Lengend Snippet: STCA does not affect the ability of HSP90 to bind ATP. MCF7 cells (5 × 10 5 per dish) in 6-cm dishes were exposed to vehicle (0.1% acetonitrile) or STCA for 24 h. Control treatments were also performed with vehicle (0.1% DMSO), 1 μ M GA and 0.8 μ M celastrol (CL). Cells were lysed and subjected to ATP pulldown using ATP-agarose beads. For the ATP pulldown and input samples, the levels HSP90 and GAPDH were determined by western blot analyses. The data represent results from three independent experiments.

Article Snippet: Human HSP90β clone was kindly provided by Dr Anutosh Chakraborty and Dr Solomon H Snyder (Johns Hopkins University).

Techniques: Control, Western Blot

Journal: British Journal of Cancer

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation

doi: 10.1038/bjc.2013.710

Figure Lengend Snippet: STCA modifies cysteine residues of HSP90. Purified recombinant HSP90 β was incubated with STCA for 2 h, dialysed to remove STCA, subject to click reaction with biotin azide, and digested by trypsin. The tryptic digest was incubated with avidin-coated beads. The captured peptides were eluted by 0.5 M acetic acid and analysed by MALDI-mass spectroscopy.

Article Snippet: Human HSP90β clone was kindly provided by Dr Anutosh Chakraborty and Dr Solomon H Snyder (Johns Hopkins University).

Techniques: Purification, Recombinant, Incubation, Avidin-Biotin Assay, Mass Spectrometry

Journal: British Journal of Cancer

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation

doi: 10.1038/bjc.2013.710

Figure Lengend Snippet: S-4 induces transcription of hsp70a1a and hsp70a1b, causes destabilisation of HSP90 client proteins, and inhibits cell proliferation. ( A ) WT MEFs (2.1 × 10 5 per well) in six-well plates were exposed to vehicle (0.1% acetonitrile) or increasing concentrations of each sulphoxythiocarbamate for 8 h. Cells were then lysed and total RNA were extracted. The amount of mRNA for hsp70a1a and hsp70a1b was analysed by quantitative RT–PCR, using β -actin mRNA as an internal control. The mRNA from each sample was measured separately, in triplicate. Data represent means±s.d. and are expressed as ratio of the relative transcription units in treated over control samples. ( B – F ) Cells seeded in six-well plates at a density of 2.5 × 10 5 per well ( B , D and E ) or in 6-cm dishes at a density of 10 6 per dish ( C ) were exposed to vehicle (0.1% acetonitrile) or sulphoxythiocarbamates for either 24 h ( B and F ) or 16 h ( C – E ). Cells were lysed in RIPA buffer, proteins in aliquots from cell lysates were resolved by electrophoresis, transferred to immobilon-P, and probed with the specified antibodies. The data represent results from two independent experiments. ( G ) MCF7 cells (5 × 10 3 per well) in 96-well plates were treated with vehicle (0.1% acetonitrile, ○), 2.5 μ M (♦), 5 μ M (▴), or 10 μ M S-4 (◊) for 24 or 48 h. Cell proliferation was assessed using the Alamar Blue fluorometric assay. * P <0.001 relative to vehicle-treated cells.

Article Snippet: Human HSP90β clone was kindly provided by Dr Anutosh Chakraborty and Dr Solomon H Snyder (Johns Hopkins University).

Techniques: Quantitative RT-PCR, Control, Electrophoresis