Journal: Cells

Article Title: Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

doi: 10.3390/cells10071701

Figure Lengend Snippet: Determination of occupancy of CL phosphosites in human Hsp90β (HSP90AB1). ( A ) Amino acid sequence of Hsp90β’s CL with S226, S255 and S261 indicated by red arrows. ( B ) Typical peptides spanning S226 or S255 resulting from a tryptic digestion of the CL. The peptides marked in red were selected for targeted MS. ( C ) The complete list of peptides selected for the targeted MS method and their charge state (z number). Normalization peptides are required to determine the H/L ratio for the whole protein. Both phosphopeptides and non-phosphopeptides from the CL were measured. ( D ) Typical chromatogram for a normalization peptide (here SIYYITGESK, left pane), a non-phosphopeptide from the CL (IEDVGSDEEDDSGK, middle pane), and its corresponding phosphopeptide (IEDVGpSDEEDDSGK, right pane). The chromatogram for the heavy (H) peptide is the blue curve, and the light (L) chromatogram the red curve. ( E ) Average and standard deviation of phosphorylation occupancy for 5 human cell lines ( n = 3).

Article Snippet: Plasmids were generated using standard procedures and verified by sequencing. pCMV3-HA-Hsp90β WT (“WT plasmid”) was ordered from SinoBiological Inc. (Beijing, China; HG11381-NY), which served as template to produce pCMV3-Hsp90β S226A/S255A (“AA plasmid”) and no tag control plasmids.

Techniques: Sequencing, Standard Deviation

Journal: Cells

Article Title: Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

doi: 10.3390/cells10071701

Figure Lengend Snippet: Quantitative exploration of the interactome of WT vs. AA. ( A ) Volcano plot of the inverse log 10 of the adjusted p -value as a function of the normalized H/L ratio in log 2 scale for all 83 quantified interactors (SILAC experiment). Points represent individual proteins. Point color code: co-chaperones in green, Hsp40 proteins in pink, Hsp70 in orange, Hsp90 in red, other already known interactors in blue, and putative interactors in grey. Classification of co-chaperones and known interactors is based on the “Hsp90 interactors” table by Didier Picard ( www.picard.ch , accessed on 17 May 2021). Horizontal dashed line marks the 0.05 threshold for the adjusted p -value, vertical dashed lines mark absolute fold-changes greater than 0.4, and vertical red line marks Hsp90β. ( B ) Gene Ontology (GO) term analysis of the proteins enriched in the AA co-IP. Vertical axis: enriched GO terms. Horizontal axis: the GeneRatio is the ratio between the number of proteins enriched with the corresponding GO term and the total number of proteins identified in the input assigned to the corresponding GO term. Spot size is proportional to the count of proteins enriched (legend on the right of the plot). Spot color corresponds to the adjusted p -value as described in the color scale on the right of the plot.

Article Snippet: Plasmids were generated using standard procedures and verified by sequencing. pCMV3-HA-Hsp90β WT (“WT plasmid”) was ordered from SinoBiological Inc. (Beijing, China; HG11381-NY), which served as template to produce pCMV3-Hsp90β S226A/S255A (“AA plasmid”) and no tag control plasmids.

Techniques: Co-Immunoprecipitation Assay

Journal: Cells

Article Title: Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

doi: 10.3390/cells10071701

Figure Lengend Snippet: Average occupancy and standard deviation (s.d.) determined for Hsp90β immunopurified from conditioned medium of K562 suspension cultures ( n = 3). p -values from a t -test comparing the present occupancy values with K562 intracellular Hsp90β occupancy values.

Article Snippet: Plasmids were generated using standard procedures and verified by sequencing. pCMV3-HA-Hsp90β WT (“WT plasmid”) was ordered from SinoBiological Inc. (Beijing, China; HG11381-NY), which served as template to produce pCMV3-Hsp90β S226A/S255A (“AA plasmid”) and no tag control plasmids.

Techniques: Standard Deviation

Journal: Blood

Article Title: Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias

doi: 10.1182/blood-2009-10-247239

Figure Lengend Snippet: Hsp90 inhibitors decrease tumor growth and WT1 expression in leukemia tumor models. (A-B) 17-AAG and STA-9090 treatment at their respective HNSTDs significantly inhibited tumor growth in the MV4-11 (A) and the Kasumi-1 (B) xenograft models. In both studies, intravenous dosing was conducted 5× per week (arrowheads). STA-9090 treatment significantly inhibited tumor growth relative to vehicle in both models (*P < .05), and there was a strong trend toward STA-9090 being superior to 17-AAG in the MV4-11 model, although this difference or the difference between the 17-AAG and vehicle-treated groups did not reach statistical significance. Error bars represent ± SEM (n = 8/group). (C-D) Both compounds were well tolerated, as indicated by minimal affects on body weight loss in the MV4-11 (C) and Kasumi-1 (D) xenograft models over the course of the study. The differences in body weight changes between the vehicle- and Hsp90 inhibitor–treated groups were largely due to increasing tumor mass in the vehicle-treated animals over the course of these studies. (E) MV4-11 tumor-bearing animals were given single doses of vehicle, STA-9090 and 17-AAG, and after 6 hours, tumors were removed for Western blot analysis using anti-WT1, anti–Bcl-2, anti–c-Myc, and anti–β-actin. The expression levels of WT1, c-Myc, and Bcl-2 were significantly decreased after treatment with both Hsp90 inhibitors.

Article Snippet: An Hsp90 IMAGE cDNA clone was obtained from OriGene Technologies, and a full-length Hsp90 sequence was polymerase chain reaction (PCR) amplified and subcloned into pCR3.1 at the Not I and Xba I sites.

Techniques: Expressing, Western Blot

Journal: Blood

Article Title: Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias

doi: 10.1182/blood-2009-10-247239

Figure Lengend Snippet: Direct interaction between WT1 and Hsp90. (A) Equal amounts of K562 protein extracts were immunoprecipitated (IP) with agarose-conjugated mouse immunoglobulin G (IgG) or anti-Hsp90 antibodies, and immunoprecipitates were subjected to SDS-PAGE and immunoblotted (IB) for WT1 (top panel) and Hsp90 (bottom panel). Input represents ∼5% of the total protein extract used for immunoprecipitation. (B) Subcellular colocalization of WT1 and Hsp90. K562 cells were stained with DAPI (blue, nuclear stain) and antibodies to WT1 (green) or Hsp90 (red), and confocal images were acquired at 100× magnification. (C) GST pull-down assay. In vitro–translated and 35S-methionine–labeled full-length Hsp90 was incubated with GST or GST-WT1 protein immobilized on glutathione-sepharose beads, and bound WT1 was detected by fluorography (top panel). 20% of the in vitro–translated protein was used for pull-downs. The bottom panel shows the purity of GST-fused proteins on a Coomassie blue-stained SDS-PAGE gel. (D) Dose-dependent binding of Hsp90 to WT1. Increasing amounts of 35S-methionine–labeled Hsp90 were added to GST-WT1, and binding was analyzed by autoradiography.

Article Snippet: An Hsp90 IMAGE cDNA clone was obtained from OriGene Technologies, and a full-length Hsp90 sequence was polymerase chain reaction (PCR) amplified and subcloned into pCR3.1 at the Not I and Xba I sites.

Techniques: Immunoprecipitation, SDS Page, Staining, Pull Down Assay, In Vitro, Labeling, Incubation, Binding Assay, Autoradiography

Journal: Blood

Article Title: Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias

doi: 10.1182/blood-2009-10-247239

Figure Lengend Snippet: Mapping of Hsp90–WT1 interacting domains by GST pull-down assay. (A) Schematic diagram of Hsp90 domains on the left, with the expression constructs and their binding to WT1 on the right. GST pull-down assays were performed using bacterially expressed GST-WT1 (1-517 aa) and GST (negative control) and in vitro–translated and 35S-methionine–labeled full-length and deletion mutants of Hsp90. Bound proteins were separated by SDS-PAGE and visualized by fluorography. Bottom panel shows the in vitro–translated Hsp90 proteins. (B) Schematic diagram of WT1 domains on the left, with the expression constructs and their binding to Hsp90 on the right. GST pull-down assays were performed using bacterially expressed GST-Hsp90 (1-282 aa), GST (negative control), and in vitro–translated and 35S-methionine–labeled full-length and deletion mutants of WT1 and analyzed in panel A. A vertical line has been inserted to indicate a repositioned gel lane in the bottom panel.

Article Snippet: An Hsp90 IMAGE cDNA clone was obtained from OriGene Technologies, and a full-length Hsp90 sequence was polymerase chain reaction (PCR) amplified and subcloned into pCR3.1 at the Not I and Xba I sites.

Techniques: Pull Down Assay, Expressing, Construct, Binding Assay, Negative Control, In Vitro, Labeling, SDS Page

Journal: Blood

Article Title: Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias

doi: 10.1182/blood-2009-10-247239

Figure Lengend Snippet: Pharmacologic inhibition of Hsp90 down-regulates WT1 protein. (A) K562 and (B) KG-1 leukemia cells were treated with the Hsp90 inhibitor 17-AAG for 24 hours. The cells were lysed, and protein extracts were subjected to SDS-PAGE and analyzed by Western blotting for WT1, Hsp90, and/or the WT1-regulated protein c-Myc. β-actin was used as loading control. (C) K562, (D) KG1, (E) Kasumi-1, and (F) MV4-11 leukemia cells were treated with the Hsp90 inhibitor STA-9090 for 24 hours and analyzed for WT1 expression by Western blotting. (G) Primary myeloid leukemia blasts from 5 AML patients (PS#1-5) were isolated, treated with STA-9090 for 24 hours, and analyzed for WT1 and β-actin by Western blotting.

Article Snippet: An Hsp90 IMAGE cDNA clone was obtained from OriGene Technologies, and a full-length Hsp90 sequence was polymerase chain reaction (PCR) amplified and subcloned into pCR3.1 at the Not I and Xba I sites.

Techniques: Inhibition, SDS Page, Western Blot, Control, Expressing, Isolation

Journal: PLoS ONE

Article Title: Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase

doi: 10.1371/journal.pone.0165227

Figure Lengend Snippet: NIH 3T3 cells grown in 24-well plate with cover slips were transfected with expression constructs of DDK tagged TFR1, DDK tagged Na + /K + ATPase, or untagged HSP90. Cells were fixed with acid alcohol followed by staining with AF20, TFR1, DDK, and HSP90 antibodies, respectively. Note that AF20 mAb only revealed signals in TFR1 transfected cells, although all three proteins were successfully expressed in NIH 3T3 cells. In addition, cell death or unusual morphology (elongated) was observed in cells expressing the exogenous proteins (dead cells are indicated by arrows). All images were taken at 20x magnification.

Article Snippet: Expression constructs for TFR1 (Myc-DDK-tagged, RC200980, NM_003234.1), HSP90 (untagged, SC108085, NM_007355.2), and Na + /K + ATPase (Myc-DDK-tagged, RC201009, NM_000701) were purchased from Origene.

Techniques: Transfection, Expressing, Construct, Staining

Journal: Emerging Microbes & Infections

Article Title: Human coronavirus dependency on host heat shock protein 90 reveals an antiviral target

doi: 10.1080/22221751.2020.1850183

Figure Lengend Snippet: Hsp90 is required for MERS-CoV replication. (A) Huh7 cells treated with 10 µM 17-AAG or DMSO in triplicate were inoculated with MERS-CoV at a MOI of 0.01. At the indicated hours post infection, cell lysate and supernatant were collected for the viral gene copy detection. (B) Huh7 cells infected at a MOI of 0.01 were treated with various concentrations of 17-AAG. Cell-free culture media were harvested at 24 hpi for viral load quantification and plaque assay. (C) Huh7 cells treated with 10 µM 17-AAG were inoculated with MERS-CoV at the indicated MOIs. Cell-free culture media were harvested at 24 hpi for viral load quantification. (D) The indicated cells treated with 10 µM 17-AAG were inoculated with MERS-CoV at a MOI of 0.01. Cell-free culture media were harvested at 24 hpi for viral load quantification. Results present mean and SD of one representative experiment independently repeated three times. Student’s t test was used for data analysis. *** p ≤ 0.001.

Article Snippet: pCMV3-flag-Hsp90β expressing an N-terminal FLAG-tagged Hsp90β was purchased from Sino Biological (HG11381-NF).

Techniques: Infection, Plaque Assay

Journal: Emerging Microbes & Infections

Article Title: Human coronavirus dependency on host heat shock protein 90 reveals an antiviral target

doi: 10.1080/22221751.2020.1850183

Figure Lengend Snippet: Hsp90 inhibition reduced MERS-CoV replication in intestinal organoids. The differentiated intestinal organoids treated with 10 µM 17-AAG or DMSO in triplicate were inoculated with MERS-CoV at an estimated MOI of 0.1. At indicated hours post infection, the organoids were applied to viral load detection, cell-free Matrigel and culture medium were applied to viral load detection (A) and viral titration (B). Results present mean and SD of one representative experiment independently repeated three times. Student’s t test was used for data analysis. *** p ≤ 0.001. (C) The infected or mock-infected organoids were fixed at 24 hpi, followed by immunofluorescence staining of MERS-CoV NP (green) and confocal imaging. Nuclei and cellular actin filaments are counterstained with DAPI (blue) and Phalloidin-647 (purple) respectively. Scale bar, 20 μm.

Article Snippet: pCMV3-flag-Hsp90β expressing an N-terminal FLAG-tagged Hsp90β was purchased from Sino Biological (HG11381-NF).

Techniques: Inhibition, Infection, Titration, Immunofluorescence, Staining, Imaging

Journal: Emerging Microbes & Infections

Article Title: Human coronavirus dependency on host heat shock protein 90 reveals an antiviral target

doi: 10.1080/22221751.2020.1850183

Figure Lengend Snippet: Genetic depletion of Hsp90β suppressed MERS-CoV replication and abolished virus spread. At 24 h after transfection of Hsp90α, Hsp90β or scrambled siRNA in triplicate, A549 cells were infected with MERS-CoV at a MOI of 0.1. (A) The expression levels of Hsp90α and Hsp90β in both infected and mock-infected cells were detected by Western blot. (B) At the indicated hours post infection, cell-free culture media were applied to viral load detection and viral titration. (C, D) The infected or mock-infected cells were fixed at the indicated hours post infection and labelled for flow cytometry. (C) Histograms show the infection rates of one representative experiment. (D) Results present mean and SD of infection rate and MFI of one representative experiment independently repeated three times. Student’s t test was used for data analysis. *** p ≤ 0.001, * p ≤ 0.05.

Article Snippet: pCMV3-flag-Hsp90β expressing an N-terminal FLAG-tagged Hsp90β was purchased from Sino Biological (HG11381-NF).

Techniques: Virus, Transfection, Infection, Expressing, Western Blot, Titration, Flow Cytometry

Journal: Emerging Microbes & Infections

Article Title: Human coronavirus dependency on host heat shock protein 90 reveals an antiviral target

doi: 10.1080/22221751.2020.1850183

Figure Lengend Snippet: Co-IP assay identified MERS-CoV NP as an Hsp90β client protein. 293T cells were transfected with Hsp90β expression plasmid and His-tag plasmid expressing the indicated MERS-CoV structural proteins or blank vector. Cell lysates of the transfectants were applied to verify the expression of His-tagged viral proteins and Hsp90β. The cell lysates (input) were used for immunoprecipitation with an α-Hsp90β antibody; the co-precipitated partner (output) was detected by Western blot using an α-His antibody.

Article Snippet: pCMV3-flag-Hsp90β expressing an N-terminal FLAG-tagged Hsp90β was purchased from Sino Biological (HG11381-NF).

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Emerging Microbes & Infections

Article Title: Human coronavirus dependency on host heat shock protein 90 reveals an antiviral target

doi: 10.1080/22221751.2020.1850183

Figure Lengend Snippet: Hsp90β is required to maintain NP stability. (A & B) At 24 h post twice transfection of Hsp90α, Hsp90β or scrambled siRNA, NP plasmid was co-transfected with the respective siRNA. (A) The transfected cells were harvested at 48 h after co-transfection and applied to Western blot to detect the NP expression. (B) At 5 h post co-transfection of NP plasmid and Hsp90β or scrambled siRNA, the cells were incubated in the presence of absence or 1.25 µM MG132 for 43 h and then applied to NP detection by Western blot.

Article Snippet: pCMV3-flag-Hsp90β expressing an N-terminal FLAG-tagged Hsp90β was purchased from Sino Biological (HG11381-NF).

Techniques: Transfection, Plasmid Preparation, Cotransfection, Western Blot, Expressing, Incubation

Journal: Emerging Microbes & Infections

Article Title: Human coronavirus dependency on host heat shock protein 90 reveals an antiviral target

doi: 10.1080/22221751.2020.1850183

Figure Lengend Snippet: Hsp90 inhibitor suppressed SARS-CoV and SARS-CoV-2 replication. (A & B) Huh7 cells treated with 10 µM 17-AAG or DMSO in triplicate were inoculated with SARS-CoV or SARS-CoV-2. At the indicated hours post infection, cell-free culture media were collected for viral gene copy quantification and viral titration by plaque assay. Student’s t test was used for data analysis. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Article Snippet: pCMV3-flag-Hsp90β expressing an N-terminal FLAG-tagged Hsp90β was purchased from Sino Biological (HG11381-NF).

Techniques: Infection, Titration, Plaque Assay