Journal: Veterinary Research

Article Title: HSP70 positively regulates translation by interacting with the IRES and stabilizes the viral structural proteins VP1 and VP3 to facilitate duck hepatitis A virus type 1 replication

doi: 10.1186/s13567-024-01315-9

Figure Lengend Snippet: HSP70 is crucial for DHAV-1 replication in DEFs. A The chemical formula for VER155008. B VER15508 blocks ATP binding to HSP70. C Sequence alignment of human HSP70 with HSP70 of other species. The key sites involved in the interaction of HSP70 with VER155008 are marked in red. D Molecular docking of HSP70 and VER155008. E DEFs were treated with different concentrations of VER155008, and cell viability was analyzed via a CCK-8 kit after 36 h. F Different concentrations of the VER155008 inhibitor were applied, and DEFs were infected with DHAV-1 at the same time. The cells were harvested at 36 h, and the viral copy number was quantified via one-step TaqMan fluorescent quantitative RT‒PCR. G , H Time course of the drug treatment experiment. VER155008 was applied at different times. The viral copy number was quantified. **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: An Alexa FluorTM 568-conjugated goat anti-mouse IgG antibody (Cat: A11004) and an Alexa FluorTM 488-conjugated goat anti-rabbit IgG antibody (Cat: A11008) were purchased from Thermo Fisher Scientific Co., Ltd. VER155008 (Cat: S7751), MG132 (Cat: S2619), and CHX (Cat: S7418) were purchased from Selleckchem.

Techniques: Binding Assay, Sequencing, CCK-8 Assay, Infection

Journal: Veterinary Research

Article Title: HSP70 positively regulates translation by interacting with the IRES and stabilizes the viral structural proteins VP1 and VP3 to facilitate duck hepatitis A virus type 1 replication

doi: 10.1186/s13567-024-01315-9

Figure Lengend Snippet: HSP70 affects multiple steps of the DHAV-1 life cycle. A , B Attachment assay. DEFs were precooled at 4 °C for 30 min, after which DMED was replaced with VER155008, DMSO or DHAV-1 (0.1 MOI, 1 MOI, or 10 MOI). After incubation at 4 °C for 4 h, the cells were washed three times with cold PBS. For the internalization assay, After the attachment of DHAV-1, the medium was replaced with VER155008 or DMSO, and the cells were incubated at 37 °C for 2 h and then washed three times. One-step TaqMan fluorescent quantitative RT‒PCR was used to measure the viral copy number. C DEFs were incubated with VER155008, DMSO, or DHAV-1, and the cells were harvested at 6 h, 12 h, 24 h, or 36 h. The total vial negative viral copy number of DHAV-1 were analyzed via RT‒PCR. Viral production was analyzed by TCID 50 . D , E Schematic diagram of the dual-luciferase plasmids. DEFs were transfected with Rluc-IRES-Fluc and incubated with VER155008 or DMSO. After 24 h, the cells were harvested for dual-luciferase assays. F , G , H DEFs were infected with DHAV-1. After 36 h, VER155008 or DMSO was added to the cells, and the cells and supernatants were harvested at 6 h, 12 h, and 24 h. The extracellular RNA/intracellular RNA ratio represents viral release efficiency. Supernatants and cells were harvested at 12 h. The viral TCID 50 /viral RNA ratio represents viral assembly efficiency. **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: An Alexa FluorTM 568-conjugated goat anti-mouse IgG antibody (Cat: A11004) and an Alexa FluorTM 488-conjugated goat anti-rabbit IgG antibody (Cat: A11008) were purchased from Thermo Fisher Scientific Co., Ltd. VER155008 (Cat: S7751), MG132 (Cat: S2619), and CHX (Cat: S7418) were purchased from Selleckchem.

Techniques: Incubation, Luciferase, Transfection, Infection

Journal: Veterinary Research

Article Title: HSP70 positively regulates translation by interacting with the IRES and stabilizes the viral structural proteins VP1 and VP3 to facilitate duck hepatitis A virus type 1 replication

doi: 10.1186/s13567-024-01315-9

Figure Lengend Snippet: HSP70 regulates the proteasomal degradation of the structural proteins VP1 and VP3. A, B Cells were co-transfected with different concentrations of HSP70-HA, VP1-Flag, and VP3-Flag, and protein expression was analyzed by Western blotting at 24 h post-transfection. C, D DEFs were transfected with VP1-Flag or VP3-Flag and incubated with VER155008 or DMSO. After 24 h, MG132 was added to the medium, and after 12 h, protein expression in the cell lysates was analyzed by Western blotting. E, F DEFs were co-transfected with HSP70-HA, VP1-Flag, and VP3-Flag, and the cells were treated with CHX (100 mg/μL) after 24 h. The cell lysates were harvested at 0, 3, 6, and 9 h, after which protein expression was measured via Western blotting. G, H DEFs were co-transfected with HSP70(1-384)-HA or HSP70(384-634)-HA and with VP1-Flag or VP3-Flag and treated with CHX. VP1 and VP3 protein expression levels were analyzed via Western blotting.

Article Snippet: An Alexa FluorTM 568-conjugated goat anti-mouse IgG antibody (Cat: A11004) and an Alexa FluorTM 488-conjugated goat anti-rabbit IgG antibody (Cat: A11008) were purchased from Thermo Fisher Scientific Co., Ltd. VER155008 (Cat: S7751), MG132 (Cat: S2619), and CHX (Cat: S7418) were purchased from Selleckchem.

Techniques: Transfection, Expressing, Western Blot, Incubation

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