Journal: European journal of immunology

Article Title: Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

doi: 10.1002/eji.201445293

Figure Lengend Snippet: Figure 2. Hsp90 plays a pivotal role in the production of IFN-α by pDCs. (A, B) pDCs derived from HSF-1−/−mice showed decreased Hsp90α expression and impaired ability to produce IFN-α production in response to CpG-A. (A) Western blotting analysis of Hsp90 expres- sion in pDCs isolated from HSF-1−/−and HSF-1+/−mice. One rep- resentative blot out of three independent experiments is shown. (B) IFN-α production from pDCs derived from HSF-1+/−or HSF- 1−/−mice was measured using ELISA. Bars show mean + SEM from three independent experiments. **p < 0.01; Student’s t-test. (C) Cell extracts from 293XL-hTLR7-HA and 293XL-hTLR9-HA cells were immunoprecipitated (IP) with anti-HA or anti-Hsp90 and ana- lyzed by immunoblotting (IB) for coprecipitated HA, Hsp70, or Hsp90. HA and Hsp 90 served as loading controls. One repre- sentative blot out of three independent experiments is shown. (D) Purified human pDCs were stimulated for 15 min with CpG-A in the presence (CpG-A + 17-AAG) or absence (CpG-A) of 17-AAG; cell extracts were IP with anti-Hsp90 or anti-TLR9 and analyzed by immunoblotting (IB) for coprecipitated TLR9 and Hsp90. One representative blot out of three independent experiments is shown.

Article Snippet: A different set of siRNA targeting Hsp90α and Hsp90β (sc 35610) was purchased from Santa Cruz Biotechnology. siRNA was transfected into mouse pDCs twice using Lipofectamine RNAiMAX transfection reagent (Life Technologies) on days 0 and 2.

Techniques: Derivative Assay, Expressing, Western Blot, Isolation, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: European journal of immunology

Article Title: Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

doi: 10.1002/eji.201445293

Figure Lengend Snippet: Figure 5. Hsp90 is essential for CpG-A-mediated TLR9 translocation to early endosomes and production of IFN-α. Mouse pDCs were trans- fected with siRNA against Hsp90α and Hsp90β twice (days 0 and 2) to knock down both Hsp90α and Hsp90β or control siRNA (Cont.). (A) Western blotting analysis of Hsp90 in Hsp90-knocked-down pDCs. β-actin served as a loading control. One representative blot out of three independent experiments is shown. (B) CpG-A-mediated IFN-α produc- tion by mouse pDCs and Hsp90-knocked-down pDCs was measured by ELISA. Bars show mean + SEM from three independent experiments. (C) Translocation of TLR9 in response to CpG-A administration in pDCs and in Hsp90-knocked-down pDCs using laser confocal microscopy. Original magnification: × 630. (D) Analysis of the localization of TLR9 in pDCs and Hsp90-knocked-down pDCs. Bars show mean + SEM. Data are representative of three independent experiments. **p < 0.01; Student’s t-test.

Article Snippet: A different set of siRNA targeting Hsp90α and Hsp90β (sc 35610) was purchased from Santa Cruz Biotechnology. siRNA was transfected into mouse pDCs twice using Lipofectamine RNAiMAX transfection reagent (Life Technologies) on days 0 and 2.

Techniques: Translocation Assay, Knockdown, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Confocal Microscopy

Journal: European journal of immunology

Article Title: Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

doi: 10.1002/eji.201445293

Figure Lengend Snippet: Figure 7. Hsp90 multichaperone complex in pDCs in mice that devel- oped SLE. (Input) Expression of Hsp90, p23, Hop in purified pDCs isolated from ICR (control) mouse or MRL/lpr mouse that developed SLE was compared by Western blot analysis. β-Actin served as a loading control. (IP made with 2D12) Status of Hsp90 was analyzed using immunopre- cipitation made with mAb 2D12, which recognizes both complexed and uncomplexed forms of Hsp90, followed by Western blotting detected with antibodies against p23 or Hop. Note that the protein quantity of Hsp90 in pDC isolated from SLE-developed mouse was increased com- pared to that from ICR mouse. One representative blot out of three independent experiments is shown.

Article Snippet: A different set of siRNA targeting Hsp90α and Hsp90β (sc 35610) was purchased from Santa Cruz Biotechnology. siRNA was transfected into mouse pDCs twice using Lipofectamine RNAiMAX transfection reagent (Life Technologies) on days 0 and 2.

Techniques: Expressing, Isolation, Control, Western Blot

Journal: European journal of immunology

Article Title: Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

doi: 10.1002/eji.201445293

Figure Lengend Snippet: Figure 8. Serum Hsp90α level is increased in SLE patients and is asso- ciated with disease activity. (A) SLE patients were divided into sub- groups according to SLEDAI scores of 0–5, 6–10, and >10. Hsp90 in sera from 17 patients with SLE, six with mixed connective tissue disease, six with autoimmune vasculitis, ten with Sj¨ogren’s syndrome, ten with Micklickz disease, and six healthy controls was measured using ELISA. Each dot shows an individual patient, bars represent mean. *p < 0.05, **p < 0.01; Student’s t-test. (B) Serum Hsp90α levels before treatment and after treatment were compared in SLE patients (n = 17). ***p < 0.001; Student’s t-test.

Article Snippet: A different set of siRNA targeting Hsp90α and Hsp90β (sc 35610) was purchased from Santa Cruz Biotechnology. siRNA was transfected into mouse pDCs twice using Lipofectamine RNAiMAX transfection reagent (Life Technologies) on days 0 and 2.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

doi: 10.1002/eji.201445293

Figure Lengend Snippet: Figure 9. Serum Hsp90 level is dependent on the disease activity of SLE in mouse model. (A) The serum Hs90α level in MRL/lpr mice increased with age in weeks. (B) Comparison of serum Hsp90α level between 17- DMAG-treated mice and PBS-treated mice. Serum Hsp90α levels were determined ELISA. Each dot shows individual mouse, bars represent mean. Statistically significant differences were observed between sub- groups of PBS-treated and 17-DMAG-treated mice. **p < 0.01; Student’s t-test.

Article Snippet: A different set of siRNA targeting Hsp90α and Hsp90β (sc 35610) was purchased from Santa Cruz Biotechnology. siRNA was transfected into mouse pDCs twice using Lipofectamine RNAiMAX transfection reagent (Life Technologies) on days 0 and 2.

Techniques: Activity Assay, Comparison, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

doi: 10.1002/eji.201445293

Figure Lengend Snippet: Figure 10. Extracellular Hsp90 in lupus serum is involved in IFN-α pro- duction by pDCs through binding to self-DNA and anti-DNA Ab. (A) Purified human pDCs (5 × 104/100 μL) were incubated in serum-free RPMI and the serum of an SLE patients (at a final concentration of 50% vol/vol) with or without Hsp90 depletion as shown by Western blotting using anti-Hsp90 antibody. Albumin served as a loading control. One representative blot out of three independent experiments is shown. (B) After 24 h, the concentrations of IFN-α in the culture supernatants were measured by ELISA. (C) Self-DNA or (D) anti-DNA Ab bound to Hsp90 in serum from SLE patients (before treatment and after treatment) was detected using ELISA. (E) Self-DNA purified from active SLE patients serum (10 μg/mL) was mixed with Hsp90 (10 μg/mL) and loaded onto human pDCs for 24 h. IFN-α in culture supernatant was measured. Data show mean + SEM and are representative of three independent exper- iments. *p < 0.01; Student’s t-test.

Article Snippet: A different set of siRNA targeting Hsp90α and Hsp90β (sc 35610) was purchased from Santa Cruz Biotechnology. siRNA was transfected into mouse pDCs twice using Lipofectamine RNAiMAX transfection reagent (Life Technologies) on days 0 and 2.

Techniques: Binding Assay, Incubation, Concentration Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Biochemical Journal

Article Title: The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation

doi: 10.1042/bj20130963

Figure Lengend Snippet: Figure 4 Phosphorylation of Hsp90α Thr115, Thr425 and Thr603 by PKCγ

Article Snippet: For siRNA transfection, HeLa cells were transfected using LipofectamineTM 2000 (Invitrogen). siRNA against human PKCγ and control scrambled siRNA were from Santa Cruz Biotechnology. siRNA against human Hsp70, Hsp90α and Cdc37 were synthesized by GenePharma.

Techniques: Phospho-proteomics

Journal: Biochemical Journal

Article Title: The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation

doi: 10.1042/bj20130963

Figure Lengend Snippet: Figure 5 The effect of threonine phosphorylation of Hsp90α by PKCγ on Hsp90α chaperone function

Article Snippet: For siRNA transfection, HeLa cells were transfected using LipofectamineTM 2000 (Invitrogen). siRNA against human PKCγ and control scrambled siRNA were from Santa Cruz Biotechnology. siRNA against human Hsp70, Hsp90α and Cdc37 were synthesized by GenePharma.

Techniques: Phospho-proteomics

Journal: Biochemical Journal

Article Title: The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation

doi: 10.1042/bj20130963

Figure Lengend Snippet: Figure 6 The effects of threonine set phosphorylation of Hsp90α on its interaction with PKCγ

Article Snippet: For siRNA transfection, HeLa cells were transfected using LipofectamineTM 2000 (Invitrogen). siRNA against human PKCγ and control scrambled siRNA were from Santa Cruz Biotechnology. siRNA against human Hsp70, Hsp90α and Cdc37 were synthesized by GenePharma.

Techniques: Phospho-proteomics

Journal: Biochemical Journal

Article Title: The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation

doi: 10.1042/bj20130963

Figure Lengend Snippet: Figure 7 The effects of PKCγ and Hsp90α on cancer cell migration and survival

Article Snippet: For siRNA transfection, HeLa cells were transfected using LipofectamineTM 2000 (Invitrogen). siRNA against human PKCγ and control scrambled siRNA were from Santa Cruz Biotechnology. siRNA against human Hsp70, Hsp90α and Cdc37 were synthesized by GenePharma.

Techniques: Migration

Journal: Cancer Control : Journal of the Moffitt Cancer Center

Article Title: Secreted Heat Shock Protein 90α Attenuated the Effect of Anticancer Drugs in Small-Cell Lung Cancer Cells Through AKT/GSK3β/β-Catenin Signaling

doi: 10.1177/1073274818804489

Figure Lengend Snippet: Cell viabilities of (A) H69, (B) H82, and (C) H146 cells under the treatment of ABT-737 or doxorubicin. Enzyme-linked immunosorbent assay (ELISA) assay revealed the existence and concentrations of extracellular heat shock protein 90α (HSP 90α) in the medium of (D) H69, (E) H82, and (F) H146 cells under the treatment of ABT-737 or doxorubicin; (G) representative and (H) summary of Western blot assay for the existence and quantities of HSP 90α and β in culture medium of various cell types (as in eHSP90α and eHSP90β), and the amount of intracellular HSP 90α and β. * P < .05, ** P = .01, *** P = .001, and † P > .05, respectively, compared to control. # P < .05, ## P = .01, ### P = .001, and ‡ P > .05, respectively, compared to models or indicated.

Article Snippet: Heat shock protein 90α levels were detected using Hsp90α enzyme-linked immunosorbent assay kit (Cusabio, CSB-E13462 h, Houston, Texas) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Cancer Control : Journal of the Moffitt Cancer Center

Article Title: Secreted Heat Shock Protein 90α Attenuated the Effect of Anticancer Drugs in Small-Cell Lung Cancer Cells Through AKT/GSK3β/β-Catenin Signaling

doi: 10.1177/1073274818804489

Figure Lengend Snippet: Apoptosis of cells after different treatments were tested and presented as (A) representative and (B-G) summaries of cell survival rates. Concentrations of human recombinant heat shock protein 90 (hrHSP90) were 10 and 40 μg/mL. * P < .05, ** P = .01, *** P = .001, and † P > .05, respectively, compared to control. # P < .05, ## P = .01, ### P = .001, and ‡ P > .05, respectively, compared to models or indicated.

Article Snippet: Heat shock protein 90α levels were detected using Hsp90α enzyme-linked immunosorbent assay kit (Cusabio, CSB-E13462 h, Houston, Texas) according to the manufacturer’s instructions.

Techniques: Recombinant, Control

Journal: Cancer Control : Journal of the Moffitt Cancer Center

Article Title: Secreted Heat Shock Protein 90α Attenuated the Effect of Anticancer Drugs in Small-Cell Lung Cancer Cells Through AKT/GSK3β/β-Catenin Signaling

doi: 10.1177/1073274818804489

Figure Lengend Snippet: Extracellular heat shock protein 90α (HSP90α) inhibits glycogen synthase kinase 3β (GSK3β) via the activation of Ak strain transforming (AKT) signaling. A, Western blot assay of phosphorylation of AKT (p-AKT; T308), p-AKT (S473), AKT, phosphorylation of GSK3β (p-GSK3β) and GSK3β in H69, H82, and H146 cells after 40 μg/mL human recombinant heat shock protein 90 (hrHSP90) treatment. B, Summaries of p-AKT (308) and AKT levels in (A). C, Summaries of p-AKT(S473) levels in (A). D, Summaries of p-GSK3β and GSK3β levels in (A). * P < .05, ** P = .01, *** P = .001, and † P > .05, respectively, compared to control. # P < .05, ## P = .01, ### P = .001, and ‡ P > .05, respectively, compared to models or indicated.

Article Snippet: Heat shock protein 90α levels were detected using Hsp90α enzyme-linked immunosorbent assay kit (Cusabio, CSB-E13462 h, Houston, Texas) according to the manufacturer’s instructions.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Recombinant, Control

Journal: Cancer Control : Journal of the Moffitt Cancer Center

Article Title: Secreted Heat Shock Protein 90α Attenuated the Effect of Anticancer Drugs in Small-Cell Lung Cancer Cells Through AKT/GSK3β/β-Catenin Signaling

doi: 10.1177/1073274818804489

Figure Lengend Snippet: Subsequently after the inhibition of glycogen synthase kinase 3β (GSK3β), extracellular heat shock protein 90α (HSP90α) could then activate β-catenin. A, Western blot assay of active β-catenin and β-catenin in H69, H82, and H146 cells after 40 μg/mL human recombinant heat shock protein 90 (hrHSP90) treatment. (B) Summaries of active β-catenin and β-catenin levels in A. (C) Akt inhibitor MK-2206 could partly reverse the phosphorylation states of various proteins after hrHSP90 treatment. * P < .05, ** P = .01, *** P = .001, and † P > .05, respectively, compared to control. # P < .05, ## P = .01, ### P = .001, and ‡ P > .05, respectively, compared to models or indicated.

Article Snippet: Heat shock protein 90α levels were detected using Hsp90α enzyme-linked immunosorbent assay kit (Cusabio, CSB-E13462 h, Houston, Texas) according to the manufacturer’s instructions.

Techniques: Inhibition, Western Blot, Recombinant, Phospho-proteomics, Control

Journal: Cancer Control : Journal of the Moffitt Cancer Center

Article Title: Secreted Heat Shock Protein 90α Attenuated the Effect of Anticancer Drugs in Small-Cell Lung Cancer Cells Through AKT/GSK3β/β-Catenin Signaling

doi: 10.1177/1073274818804489

Figure Lengend Snippet: Extracellular heat shock protein 90α (HSP90α) but not HSP90β increased the phosphorylation levels of Ak strain transforming (AKT) and glycogen synthase kinase 3β (GSK3β) while enhanced the expression of active β-catenin. (A) Tumor volumes of xenograft mouse after various treatments. (B) Representative and (C) summary of the phosphorylation and expression levels in tumor areas. (D) Representative and (E) summary of the transferase dUTP nick end labeling (TUNEL) staining indicating the apoptosis of tumor cells under different treatment. * P < .05, ** P = .01, *** P = .001, and † P > .05, respectively, compared to control. # P < .05, ## P = .01, ### P = .001, and ‡ P > .05, respectively, compared to models or indicated.

Article Snippet: Heat shock protein 90α levels were detected using Hsp90α enzyme-linked immunosorbent assay kit (Cusabio, CSB-E13462 h, Houston, Texas) according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Expressing, End Labeling, TUNEL Assay, Staining, Control