Journal: European Journal of Immunology

Article Title: Fever‐Induced Heat Shock Protein‐70 Regulates Macrophage IL‐1β and IL‐10 Secretion During Mycobacterium tuberculosis Infection

doi: 10.1002/eji.202551963

Figure Lengend Snippet: HSP70 antagonism decreases Mtb ‐stimulated IL‐1β and IL‐10 secretion and gene expression from MDM at 37°C and 40°C. (A) IL‐1β and (B) IL‐10 secretion from Mtb ‐stimulated MDM, either pretreated with vehicle (open bars) or with 25 µM HSP70 antagonist Ver155008 (black bars, n = 6 donors) at 37°C or 40°C for 24 h. Fold changes in secretion are relative to vehicle control pretreated Mtb ‐infected MDM at 37°C. (C) il1b and (D) il10 fold change in gene expression from Mtb ‐stimulated MDM, either pretreated with vehicle (open bars) or with 25 µM Ver155008 (black bars, n = 3 donors) at 37°C or 40°C for 24 h. Fold change in gene expression is relative to the vehicle control pretreated control MDM at 37°C. Mean ± SEM are shown. Two‐way ANOVA statistical testing was performed (* p < 0.05, *** p < 0.001, **** p < 0.0001).

Article Snippet: In later experiments, MDM were pretreated with 25 μM Ver155008, a HSP70 antagonist (Santa Cruz Biotechnology, USA), or 500 ng/mL recombinant human HSP70 (rhHSP70, Bio‐Techne, UK) 1 h before infection.

Techniques: Gene Expression, Control, Infection

Journal: European Journal of Immunology

Article Title: Fever‐Induced Heat Shock Protein‐70 Regulates Macrophage IL‐1β and IL‐10 Secretion During Mycobacterium tuberculosis Infection

doi: 10.1002/eji.202551963

Figure Lengend Snippet: Recombinant HSP70 increases IL‐1β secretion at 37°C, and inhibition of HSP70 activity increases Mtb ‐induced HSP70 secretion at 37°C, and to a greater extent at 40°C. (A) IL‐1β and (B) IL‐10 secretion from Mtb ‐stimulated MDM, either pretreated with vehicle (open bars) or with 500 ng/mL recombinant human HSP70 (rhHSP70, black bars, n = 4–6 donors) at 37°C or 40°C for 24 h. Fold changes in secretion are relative to vehicle control pretreated Mtb ‐infected MDM at 37°C. (C) HSP70 secretion from Mtb ‐stimulated MDM, either pretreated with vehicle (open bars) or with 25 µM Ver155008 (black bars, n = 4 donors) at 37°C or 40°C for 24, 48, or 72 h. Mean ± SEM are shown. Two‐way ANOVA statistical testing was performed (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: In later experiments, MDM were pretreated with 25 μM Ver155008, a HSP70 antagonist (Santa Cruz Biotechnology, USA), or 500 ng/mL recombinant human HSP70 (rhHSP70, Bio‐Techne, UK) 1 h before infection.

Techniques: Recombinant, Inhibition, Activity Assay, Control, Infection

Journal: Neuroscience

Article Title: THE NUCLEAR FACTOR ERYTHROID 2-LIKE 2 ACTIVATOR, TERT -BUTYLHYDROQUINONE, IMPROVES COGNITIVE PERFORMANCE IN MICE AFTER MILD TRAUMATIC BRAIN INJURY

doi: 10.1016/j.neuroscience.2012.07.070

Figure Lengend Snippet: Viability of SH-SY5Y model neurons treated with tBHQ and HSP70 inhibitor. Undifferentiated human neuroblastoma SH-SY5Y cells were pretreated with vehicle, 10 μm VER155008, 10 μm tBHQ, or both VER155008 and tBHQ and subjected to multiple mild biaxial stretch injuries. Viability was measured 2 h following the injury by counting Annexin V-positive cells measured by confocal microscopy. A significant increase in Annexin staining was observed in injured cells. A decrease in Annexin staining was observed in injured cells treated with tBHQ compared to injured cells treated with vehicle (column 5 and 6). No significant difference in tBHQ treatment can be observed in the presence of HSP70 inhibitor (column 6 and 8). Data analysis was performed with ImageJ (sham n = 3, injured n = 6, *P < 0.01, **P < 0.001).

Article Snippet: The HSP70 inhibitor, VER155008 (Tocris Bioscience/R&D systems, Minneapolis, MN), was dissolved in DMSO to 100 mM and diluted in media prior to adding to the cells for a final concentration of 10 μM.

Techniques: Confocal Microscopy, Staining

Journal: PLoS ONE

Article Title: Mutation in Integrin-Linked Kinase (ILK R211A ) and Heat-Shock Protein 70 Comprise a Broadly Cardioprotective Complex

doi: 10.1371/journal.pone.0077331

Figure Lengend Snippet: A , (top panels) Phase contrast and fluorescent microscopy images of human IPSC-derived cardiomyocytes (Hu iPS-CMs) (on the left) and primary rabbit cardiomyocytes (on the right) transduced with GFP-linked vector (ad-GFP) versus non transduced cells (C). The Hu iPS-CM cells express monomeric red fluorescent protein and blasticidin resistance under the control of the α-myosin heavy chain promoter that allows simultaneous cardiomyocyte purification and identification. Green fluorescence indicates efficient transduction of ILK constructs. Scale bar, 80 µm. (bottom panels) Western blots showing expression levels of ILK, Hsp70 and Hsc70 in Hu iPS-CMs (on the left) and in primary rabbit cardiomyocytes (on the right) transduced with ILK WT and ILK R211A as compared to non-transduced cells and cells transduced with the vector bearing GFP only. GAPDH was used as a loading control. B , Immunoblot for ILK expression levels in Hu IPS-CMs transduced with ad-ILK WT and ad-ILK R211A as compared to non-transduced cells following treatment with cycloheximide (CHX) at 80 µM for 0, 3, 6 and 18 hours. C , Western blot analysis showing the protein levels of ILK, Hsp70 and Hsp-90 in Hu iPS-CMs transduced with ad-GFP, ad-ILK WT , ad-ILK R211A and in non-transduced cells (C) following treatment with indicated doses of Hsc/p70-ATPase inhibitor (Ver-155008) for 24 hours. GAPDH was used as a loading control. Each experiment was performed at least three times on independent samples and one representative blot is shown.

Article Snippet: To determine the effects of Hsp70 inhibition, cardiomyocytes were transduced with ad-GFP, ad-ILK WT and ad-ILK R211A and incubated for 48h before exposure to different concentrations of the specific Hsc/p70-ATPase inhibitor Ver-155008 (catalogue number: 3803, Tocris Bioscience).

Techniques: Microscopy, Derivative Assay, Transduction, Plasmid Preparation, Control, Purification, Fluorescence, Construct, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Mutation in Integrin-Linked Kinase (ILK R211A ) and Heat-Shock Protein 70 Comprise a Broadly Cardioprotective Complex

doi: 10.1371/journal.pone.0077331

Figure Lengend Snippet: A , Percentage of TUNEL-labelled apoptotic nuclei in Hu iPS-CMs transduced with ad-GFP, ad-ILK WT and ad-ILK R211A and then treated for 24 hours with 40 µM of Hsc/p70-ATPase inhibitor Ver-155008 in the presence or absence of 1 µM DOX. Ver-155008 was added one hour prior to exposure to DOX. Bar graphs represent mean values ±SD, n=10 (random fields). The apoptotic rates following exposure to Ver-155008 plus DOX are not statistically significantly different from one to another. B , Western blot analysis of human iPS-CMs transduced with ad-GFP, ad-ILK WT and ad-ILK R211A and then treated for 24 hours with 40 µM of Ver-155008 versus untreated cells in the presence of 1µM DOX. The blot was probed for ILK, Hsp70, Hsc70, Hsp90, pGsk-3β, total Gsk-3β, pAkt, total Akt, total PLN, and GAPDH was used as a loading control.

Article Snippet: To determine the effects of Hsp70 inhibition, cardiomyocytes were transduced with ad-GFP, ad-ILK WT and ad-ILK R211A and incubated for 48h before exposure to different concentrations of the specific Hsc/p70-ATPase inhibitor Ver-155008 (catalogue number: 3803, Tocris Bioscience).

Techniques: TUNEL Assay, Transduction, Western Blot, Control

Journal: International Journal of Oncology

Article Title: Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells

doi: 10.3892/ijo.2014.2804

Figure Lengend Snippet: Increase of HSP70/72 by SHK and synergistic cytotoxic effects of SHK in combination with HSP70/72 inhibitor. (A) Western blot analyses of HSP70. SHK at a concentration of 2.5 μM transiently increased HSP70 in KMS-12-PE cells in a time-dependent manner, while this was less evident at 5 μM. (B) Western blot analyses of HSP70, HSP72, and HSP90. U266, KMS-12-PE and KMM1 were treated with 2.5 and 5 μM SHK for 7 h. Induction of HSP70 and HSP72 by SHK was observed in all cell lines and maximized at 2.5 μM. There was no change in the expression of HSP90. (C) Cytotoxic effect of the HSP70/72 inhibitor VER-155008 in KMS-12-PE cells. Cells were cultured with various concentrations of VER-155008 for 24 h and evaluated by WST-8 analysis. VER-155008 alone induced cytotoxic effects in MM cells. Note that VER-155008 at ~3 μM showed 55% growth inhibition (dotted line). (D) Combination effects of VER-155008 and SHK. KMS-12-PE cells were treated with SHK at concentrations varying from 0.19 to 0.5 μM either with 3 μM VER-155008 (solid bars) or SHK alone (blank bars) for 24 h. Combinations of SHK and VER-155008 showed significant synergistic effects in induction of cytotoxicity (CI=0.72). (E) The populations of dead cells induced by the combination of SHK and VER-155008 (VER) were partly inhibited by Z-VAD-FMK (P<0.0001). (F) Combination of SHK and VER-155008 did not show toxic effects in normal PBMCs. PBMCs from a normal donor were cultured with SHK and VER-155008 (VER) at 0.5 and 3 μM, respectively, for 24 h and evaluated by trypan blue dye exclusion analysis. No cytotoxic effect was observed.

Article Snippet: VER-155008, an ATP-derivative inhibitor of HSP70, was purchased from Enzo Life Sciences (Farmingdale, NY, USA).

Techniques: Western Blot, Concentration Assay, Expressing, Cell Culture, Inhibition