Journal: Journal of Biological Chemistry

Article Title: Muscle-specific MicroRNA1 (miR1) Targets Heat Shock Protein 70 (HSP70) during Dexamethasone-mediated Atrophy*

doi: 10.1074/jbc.m112.390369

Figure Lengend Snippet: FIGURE 5. Overexpression of HSP70 during Dex-induced atrophy resulted in rescue of enhanced levels of atrophy-related proteins. A, real time qPCR analysis of miR1 expression in mRNA extracted from empty vector or HSP70 expression plasmid-transfected C2C12 myotubes after ethanol or Dex treatments. Values are mean S.E. (n 3) expressed as fold-difference relative to empty vector-transfected controls, *, p 0.05. B, Western blot analysis of pFOXO3, FOXO3, pAKT, AKT, MuRF1, and Atrogin-1 protein levels in empty vector and HSP70 expression plasmid-transfected C2C12 myotubes treated with ethanol or Dex. The level of GAPDH was measured as a loading control. C and D, corresponding densitometric analysis of the levels of phosphorylated FOXO3 and AKT, expressed as a percentage of total FOXO3 and AKT, respectively, in empty vector or HSP70 expression plasmid-transfected C2C12 myotubes following treatment with ethanol or Dex. E, corresponding densitometric analysis of the levels of MuRF1 and Atrogin-1 in empty vector or HSP70 expression plasmid- transfected C2C12 myotubes, following treatment with ethanol or Dex. Values represent mean S.E. (n 3); *, p 0.05.

Article Snippet: Dex and RU486 were from Sigma and the HSP70 inhibitor (VER155008) (26) from Tocris Biosciences.

Techniques: Over Expression, Expressing, Plasmid Preparation, Transfection, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Muscle-specific MicroRNA1 (miR1) Targets Heat Shock Protein 70 (HSP70) during Dexamethasone-mediated Atrophy*

doi: 10.1074/jbc.m112.390369

Figure Lengend Snippet: FIGURE6.MstnregulatesmiR1duringDex-mediatedmuscleatrophy.A,WesternblotanalysisofpFOXO3,FOXO3,HSP70,pAKT,AKT,MuRF1,andAtrogin-1 levelsinwholecelllysatesfromC2C12myotubestreatedwithDex(lane2)andDexAnt1(lane3),ascomparedwithethanol-treatedcontrols(lane1).Thelevel of GAPDH was measured as a loading control. B and C, corresponding densitometric analysis of the levels of phosphorylated FOXO3 and AKT, expressed as a percentage of total FOXO3 and AKT, respectively. Error bars represent mean S.E. (n 3); **, p 0.01, and *, p 0.05. D, representative graph showing densitometric analysis of HSP70, MuRF1, and Atrogin-1 protein levels (normalized to GAPDH) in ethanol-, Dex-, and Dex Ant1-treated C2C12 myotubes. Error bars represent mean S.E. (n 3); **, p 0.01. E, Western blot analysis of pFOXO3, FOXO3, HSP70, pAKT, and AKT protein levels in BF muscle collected from saline- or Dex-injected WT (lanes 1 and 2) and Mstn/ (lanes 3 and 4) mice. The level of GAPDH was measured as a loading control. F and G, corresponding densitometric analysis of the levels of phosphorylated FOXO3 and AKT, expressed as a percentage of total FOXO3 and AKT, respectively, in WT and Mstn/ mice after injection with saline or Dex. H, densitometric analysis of HSP70 protein levels in WT and Mstn/ muscle from saline or Dex-injected mice; *, p 0.05 and **, p 0.01. Error bars represent mean S.E. (n 3).

Article Snippet: Dex and RU486 were from Sigma and the HSP70 inhibitor (VER155008) (26) from Tocris Biosciences.

Techniques: Control, Western Blot, Saline, Injection

Journal: Journal of Biological Chemistry

Article Title: Muscle-specific MicroRNA1 (miR1) Targets Heat Shock Protein 70 (HSP70) during Dexamethasone-mediated Atrophy*

doi: 10.1074/jbc.m112.390369

Figure Lengend Snippet: FIGURE 7. Proposed mechanism behind Dex-induced miR1-mediated skeletal muscle atrophy. Dex activates GR, which translocates to the nucleus and up-regulates miR1. Dex also up-regulates Mstn, which increases nuclear translocation of GR and miR1 expression. miR-1-mediated loss of HSP70 and enhanced activation of GR leads to further up-regulation of miR1 expression. Thus, increased miR1 expression may feedforward to further enhance its own expression. The reduced levels of HSP70 observed following Dex and Mstn treatment would further exacerbate skeletal muscle atrophy by decreasing phosphorylation of AKT, which results in activation of downstream proteosomal signaling components, such as FOXO3, MuRF1, and Atrogin-1. Arrows represent stimulation and blunt-ended lines represent inhibition.

Article Snippet: Dex and RU486 were from Sigma and the HSP70 inhibitor (VER155008) (26) from Tocris Biosciences.

Techniques: Translocation Assay, Expressing, Activation Assay, Phospho-proteomics, Inhibition

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: 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: Scientific Reports

Article Title: Heat shock protein A2 is a novel extracellular vesicle-associated protein

doi: 10.1038/s41598-023-31962-5

Figure Lengend Snippet: Autophagy inhibition does not prevent proteasome inhibition-related reduction in intracellular levels of HSPA2. ( a ) Effects of the single or combined treatment with bafilomycin A (BAF) and bortezomib (BTZ) on the HSPA1, HSPA2, and p62 protein levels. ( b ) Effects of a single or combined treatment (16 h) with VER-155008 (VER), BTZ, or chloroquine (ChQ) on the protein levels of HSPA1, HSPA2 and p62. In either experiment, HSPA1 and p62 were used as a BTZ or ChQ/BAF treatment control, respectively. β-actin was used as a protein loading control. Prior to incubation with primary antibodies membranes were cut according to the molecular ladder band (55 kDa). For chemiluminescent signal detection X-ray film was used. Original autoradiograms/immunoblots are presented in Fig. . Graphs on the right side show results of densitometric analysis of immunoblots representative for HSPA2 expression (mean ± SD; n ≥ 4). The protein level is presented relative to β-actin. Statistical significance was determined by the two-tailed t-test performed in regard to cells exposed to DMSO solvent only. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The following stock solutions were used in the incubation experiments: manumycin A (MA; 10 mM in DMSO; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), MG132 (10 mM in DMSO; Selleck Chemicals, Houston, TX, USA), VER-155008 (VER; 20 mM in DMSO; Merck KGaA, Darmstadt, Germany), bortezomib (BTZ; 1.6 mM in DMSO; Selleckchem, Houston, TX, USA), bafilomycin A1 (BAF; 0.1 mM in DMSO; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), chloroquine (ChQ; 50 mM in DMSO; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany).

Techniques: Inhibition, Incubation, Western Blot, Expressing, Two Tailed Test

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