Journal: The Journal of Cell Biology

Article Title: Hsc70 chaperone activity is required for the cytosolic slow axonal transport of synapsin

doi: 10.1083/jcb.201604028

Figure Lengend Snippet: Inactivation of Hsc70 ATPase activity blocks slow axonal transport and presynaptic accumulation of synapsin. (A) Schematic of assay to determine slow axonal transport of synapsin using photoactivatable synapsin (PAGFP:synapsin; ; ). In brief, PAGFP:synapsin was photoactivated in a discrete axonal ROI, and the fluorescence was tracked over time by live imaging. (B) Kymographs from photoactivation experiments in A. Note the anterogradely biased dispersion of PAGFP:synapsin fluorescence in control axons, thought to represent slow transport (midpoint of photoactivated zone marked by red arrowhead and dashed line; top). Also note that the Hsc70 inhibitor (100 µM VER155008; bottom) eliminated the anterograde bias. (C) Quantification of the transport experiments. In brief, the centroid of the photoactivated zone was quantified in each image of a given time-lapse video, and the displacement of the centroid was quantified over time (intensity center shift; ). Note that although the population of photoactivated synapsin was transported anterogradely, pharmacologic (VER155008) or genetic (DN-Hsc70) inactivation of Hsc70 ATPase activity prevented the biased transit. (D) Attenuation of endogenous synapsin levels at presynaptic boutons upon selective inactivation of Hsc70 in axons. Axons and presynaptic boutons were isolated from somatodendritic compartments using a triple-chamber microfluidic device (see Fig. S1 for design of device). Thereafter, the Hsc70 inhibitor VER155008 (or DMSO control) was selectively added to the axonal/presynaptic chamber, and the neurons were fixed and immunostained with anti-VAMP2 (to detect all presynapses; green) and anti-synapsin (red) antibodies. Representative images from axonal/presynaptic chambers are shown. (E) Quantification of colocalization between synapsin and VAMP2. Note that Hsc70 inactivation significantly attenuated the presynaptic localization of synapsin. Error bars show means ± SEM. **, P < 0.01.

Article Snippet: VER155008 was used as the Hsc70 inhibitor (3803; Tocris Bioscience).

Techniques: Activity Assay, Fluorescence, Imaging, Dispersion, Control, Isolation

Journal: The Journal of Cell Biology

Article Title: Hsc70 chaperone activity is required for the cytosolic slow axonal transport of synapsin

doi: 10.1083/jcb.201604028

Figure Lengend Snippet: Inactivation of Hsc70 ATPase activity leads to disruption of axonal synapsin. (A) Strategy to analyze the fluorescence decay of PAGFP:synapsin in an axon ROI over time. PAGFP:synapsin was photoactivated in a discrete axonal ROI, and the dispersion of fluorescence was quantified over time. (B) Fluorescence decay of PAGFP:synapsin in axon ROIs. Note faster decay of fluorescence upon Hsc70 inactivation. (C) Representative images showing endogenous distribution of synapsin in axons upon pharmacologic and genetic Hsc70 inactivation (VER155008 and Hsc70-DN). Note that the punctate distribution is disrupted upon Hsc70 inhibition. (D) Quantification of the periodicity of axonal synapsin particles with or without Hsc70 inactivation. Periodicity of endogenous synapsin particles (i.e., distances between adjacent puncta) was determined from axonal line scans, as shown in the example (top left; red boxed region from C). Cumulative data from control axons is shown on the top right, represented by FFTs (see the Colocalization analysis of widefield images… section of Materials and methods for details). Note the major peak at ∼2.77 µm, representing the typical spacing of synapsin puncta in control axons (dashed line over minor peaks). Also note loss of the major peak upon Hsc70 inhibition, reflecting disruption of the periodic synapsin distribution in axons. (E) HEK293 cells were transfected with GFP:synapsin, immunoprecipitated with an anti-GFP antibody, and blotted with anti-GFP and anti-Hsc70 antibodies (with or without VER155008). Note that addition of the Hsc70 inhibitor led to a significant reduction in the amount of immunoprecipitated Hsc70. Molecular mass is shown in kilodaltons. (F) Quantification of blots in E. n = 3 separate experiments. Error bars show means ± SEM. **, P < 0.01.

Article Snippet: VER155008 was used as the Hsc70 inhibitor (3803; Tocris Bioscience).

Techniques: Activity Assay, Disruption, Fluorescence, Dispersion, Inhibition, Control, Transfection, Immunoprecipitation

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