Journal: PLoS Pathogens

Article Title: Hsp70 Isoforms Are Essential for the Formation of Kaposi’s Sarcoma-Associated Herpesvirus Replication and Transcription Compartments

doi: 10.1371/journal.ppat.1005274

Figure Lengend Snippet: SILAC results from NE fractions highlighted chaperones from the HSP70 family and their associated co-chaperones

Article Snippet: The inhibitor for Hsp70 isoforms (VER-155008) was obtained from Tocris Bioscience.

Techniques: Multiplex sample analysis

Journal: PLoS Pathogens

Article Title: Hsp70 Isoforms Are Essential for the Formation of Kaposi’s Sarcoma-Associated Herpesvirus Replication and Transcription Compartments

doi: 10.1371/journal.ppat.1005274

Figure Lengend Snippet: (A) Cytotoxicity of VER-155008 was assessed in unreactivated cells exposed to increasing inhibitor concentrations for 24 h. (i) Cell metabolic activity was reduced in a dose-dependent manner as quantified using an MTS assay. (ii) A dose-dependent cleavage of full length (FL) PARP1 protein into cleaved (CL) PARP1 was observed in the presence of VER-155008. (B) Unreactivated cells were exposed for 24 h to increasing inhibitor concentrations. Concentrations higher than 3 μM resulted in increased cytotoxicity and activation of effector caspases-3/7 as demonstrated by quantification with ApoTox-Glo Triplex Assay. (C) Immunoblot analysis showing that reactivated cells treated with non-cytotoxic inhibitor concentrations (1 to 2.5 μM) for 24 h revealed a decrease in viral proteins compared with DMSO-treated samples while cellular proteins remained unaffected. RNAPIIo denotes hypophosphorylated RNAPII. Ser5 and Ser2 RNAPII denote serine 5- and serine 2- hyperphosphorylated RNAPII forms respectively. (D) Cells were reactivated for 24 h to allow robust viral protein production. Then, DMSO control (0.1%) or VER-155008 was added in conjunction with cycloheximide (CHX) at 50 μg/ml to block de novo protein synthesis. Protein lysates were collected at several times post-CHX addition (0, 4, 8, 16 and 24 h) and analysed by Western blotting. VER-155008 did not alter the half-life of RTA or ORF57 protein, thus these proteins were not clients of Hsp70 isoforms.

Article Snippet: The inhibitor for Hsp70 isoforms (VER-155008) was obtained from Tocris Bioscience.

Techniques: Activity Assay, MTS Assay, Activation Assay, Western Blot, Control, Blocking Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Hsp70s regulate circadian rhythm by interacting with PERIOD

doi: 10.1186/s12964-026-02805-3

Figure Lengend Snippet: Hsp70s Regulate Circadian Rhythm under various conditions. A Immunoprecipitation assays were performed to compare the interaction strength between wild-type PER and Hsp70Ba under various temperature conditions. Western blot analysis was performed with mouse anti-V5 (ABclonal, Cat#AE017), mouse anti-HA (ABclonal, Cat#AE008) antibodies and rabbit anti-Actin (ABclonal, #AC026). B Statistics of ( A ). N = 3. Statistical differences were assessed using one-way ANOVAs and post-hoc Tukey tests (P < 0.05). C Representative double plot actograms showing average locomotor activity for the respective genotypes under heat shock conditions. White background indicates light, and black indicates darkness. D Analysis of circadian rhythm persistence in Hsp70 mutants and w 1118 controls following heat shock. P-values from two-sided Fisher’s exact test are indicated (***P < 0.001)

Article Snippet: For drug treatment, 100 μM of the Hsp70 inhibitor VER-155,008 (Target Mol, #T7010) was dissolved in the media.

Techniques: Immunoprecipitation, Western Blot, Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Hsp70s regulate circadian rhythm by interacting with PERIOD

doi: 10.1186/s12964-026-02805-3

Figure Lengend Snippet: Hsp70 Protein Interacts with PER, Affecting PER Phosphorylation and Cellular Localization. A Relative per expression at different time points in Hsp70 mutants and the control group. B Antibody specificity evaluation in western blot analysis for PER protein. C PER protein detection by western blots at ZT3, ZT9, ZT15, and ZT21. B - C Western blot analysis was performed with anti-PER (a gift from Dr. Jeffrey Price’s laboratory at the University of Missouri-Kansas City, U. S.) and mouse anti-β-Tubulin (ABclonal, Cat#AC021). D Quantification of results in ( C ). N = 5. Statistical differences were measured using an unpaired Student’s t-test. **P < 0.01, ***P < 0.001. E Statistics of the cellular localization of PER in s-LNvs and l-LNvs clusters of clock neurons in indicated genotypes. C represents cytoplasm, and N represents nucleus. F Immunoprecipitation experiments to detect the interaction between PER, TIM, and Hsp70s. Western blot analysis was performed with mouse anti-V5 (ABclonal, Cat#AE017), mouse anti-HA (ABclonal, Cat#AE008) antibodies and mouse anti-β-Tubulin (ABclonal, Cat#AC021). Samples were prepared in S2 cells, with β-tubulin used as an internal control. G Calculation of average TIM binding by PER molecule. N = 3. Significance of differences was determined using one-way ANOVAs and post-hoc Tukey tests. H Immunoprecipitation experiments to detect the interaction between PER and Hsp70s in tim 01 and w 1118 controls. Western blot analysis was performed with anti-PER (a gift from Dr. Jeffrey Price’s laboratory at the University of Missouri-Kansas City, U. S.) and anti-β-Tubulin (ABclonal, Cat#AC021). β-tubulin was used as an internal control. ( I ) Calculation of average Hsp70 binding by PER molecule. N = 5. The t-test was used to calculate the significance of differences. **P < 0.01

Article Snippet: For drug treatment, 100 μM of the Hsp70 inhibitor VER-155,008 (Target Mol, #T7010) was dissolved in the media.

Techniques: Phospho-proteomics, Expressing, Control, Western Blot, Immunoprecipitation, Binding Assay

Journal: bioRxiv

Article Title: Trophoblast survival signaling during human placentation requires HIF-induced transcription of HSP70

doi: 10.1101/043851

Figure Lengend Snippet: HSP70 was inhibited using the pharmacological inhibitor VER 155008 during culture of HTR-8/SVneo cells for 4 h at 20% or 2% O 2 . At 2% O 2 , HSP70 inhibition caused a dose-dependent decrease in both MMP2 (A) and HBEGF (B), measured by immunohistochemistry, and concomitantly increased cell death (C), measured by TUNEL. Cells cultured at 20% O 2 were unaffected. D. ELISA for HBEGF in cellular extracts of TB cells cultured for 4 h at either 20% or 2% O 2 in the presence of recombinant MMP2 (rMMP2) and HSP70 inhibitor (VER 155008), as indicated. *, p<0.05, compared to vehicle/20% O 2 .

Article Snippet: Cells were treated by adding to the culture medium 1-10 μg/ml α-amanitin (Sigma-Aldrich), 250 μM CoCl 2 (Sigma-Aldrich), inhibitors (EDM Chemicals, Inc., Gibbstown, NJ) specific for MMP2 and MMP9 ((2R)-[(4-Biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide, BiPS; 100 nM), MMP2 only ((2-((isopropoxy)-(1,1′-biphenyl-4-ylsulfonyl)-amino))-N-hydroxyacetamide; 250 nM) or MMP9 only (MMP9 Inhibitor I; 100 nM), specific inhibitors for ERK, JNK, and p38 , HSP70 inhibitor (VER-155008, Santa-Cruz Biotech; 0.05-100 μM), and 10 nM recombinant MMP2 (R&D Systems; rMMP2;).

Techniques: Inhibition, Immunohistochemistry, TUNEL Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: bioRxiv

Article Title: Trophoblast survival signaling during human placentation requires HIF-induced transcription of HSP70

doi: 10.1101/043851

Figure Lengend Snippet: First trimester villous explants cultured for 6 h at either 20% or 2% O 2 in the presence of recombinant MMP2 (rMMP2) and VER 155008 (HSP70 inh.), as indicated, were dually stained for DAPI and MMP2 (A), HBEGF (C), or TUNEL (E), using immunofluorescence microscopy. Size bars indicating 100 μm are shown in A, C and E. Cell extracts were assayed by ELISA for MMP2 (B) and HBEGF (D), as well as for cell death by TUNEL (F). *, p<0.0001 in B and D, p<0.05 in F, compared to vehicle/20% O 2 .

Article Snippet: Cells were treated by adding to the culture medium 1-10 μg/ml α-amanitin (Sigma-Aldrich), 250 μM CoCl 2 (Sigma-Aldrich), inhibitors (EDM Chemicals, Inc., Gibbstown, NJ) specific for MMP2 and MMP9 ((2R)-[(4-Biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide, BiPS; 100 nM), MMP2 only ((2-((isopropoxy)-(1,1′-biphenyl-4-ylsulfonyl)-amino))-N-hydroxyacetamide; 250 nM) or MMP9 only (MMP9 Inhibitor I; 100 nM), specific inhibitors for ERK, JNK, and p38 , HSP70 inhibitor (VER-155008, Santa-Cruz Biotech; 0.05-100 μM), and 10 nM recombinant MMP2 (R&D Systems; rMMP2;).

Techniques: Cell Culture, Recombinant, Staining, TUNEL Assay, Immunofluorescence, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Trophoblast survival signaling during human placentation requires HIF-induced transcription of HSP70

doi: 10.1101/043851

Figure Lengend Snippet: Low (2%) O 2 stabilizes cytoplasmic HIF1A and HIF2A (1) that accumulate and translocate to the nucleus (2) where they dimerize to HIF1B and bind to hypoxia response elements (HRE) in the regulatory regions of target genes, including HSPA6/HSP70B’ (3). HSP70 regulates the accumulation of activated MMP2 at the cell surface (4), which is required for shedding (5) of the extracellular domain of proHBEGF. The released sHBEGF binds (6) to its receptors, ERBB1 and ERBB4, through its EGF-like domain and to heparan sulfate proteoglycans (HSPG) through its heparin-binding domain. ERBB downstream signaling activates MAPKs (7) required for biosynthesis of proHBEGF from the pool of HBEGF mRNA (8). HBEGF autocrine signaling thereby increases HBEGF secretion to achieve extracellular HBEGF concentrations (>1 nM) sufficient to inhibit apoptosis at 2% O 2 .

Article Snippet: Cells were treated by adding to the culture medium 1-10 μg/ml α-amanitin (Sigma-Aldrich), 250 μM CoCl 2 (Sigma-Aldrich), inhibitors (EDM Chemicals, Inc., Gibbstown, NJ) specific for MMP2 and MMP9 ((2R)-[(4-Biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide, BiPS; 100 nM), MMP2 only ((2-((isopropoxy)-(1,1′-biphenyl-4-ylsulfonyl)-amino))-N-hydroxyacetamide; 250 nM) or MMP9 only (MMP9 Inhibitor I; 100 nM), specific inhibitors for ERK, JNK, and p38 , HSP70 inhibitor (VER-155008, Santa-Cruz Biotech; 0.05-100 μM), and 10 nM recombinant MMP2 (R&D Systems; rMMP2;).

Techniques: Binding Assay