Journal: Pharmaceutical Biology

Article Title: Aspirin relieves the calcification of aortic smooth muscle cells by enhancing the heat shock response

doi: 10.1080/13880209.2021.2007268

Figure Lengend Snippet: Aspirin enhanced the heat shock response in the calcification process of VSMCs. (A) The expression of heat shock proteins in the process of VSMC calcification. Western blot assay was used to detected protein expression of HSF1, HSP70 and HSP90 in VSMCs after different-time calcification induction. β-Actin was used as loading control. (B–D) Grey values analysis of immune bands in western blot assay. n = 3 in each group. ** p < 0.01. (E) The effect of aspirin on expression of heat shock proteins in VSMCs after four-day culture in OIM. Western blot assay was used to detect protein expression of HSF1, HSP70 and HSP90 in VSMCs. β-Actin was used as loading control. (F–H) Grey values analysis of immune bands in western blot assay. n = 3 in each group. ** p < 0.01.

Article Snippet: Aspirin (1 mmol/L or 4 mmol/L; Sangon Biotech, Shanghai, China), HSP70 inhibitor MKT-077 (1 µmol/L; MedChemExpress, Monmouth Junction, NJ) and HSP90 inhibitor 17-DMAG (1 µmol/L; Sigma, St. Louis, MO) were applied to treat the calcification-induced VSMCs.

Techniques: Expressing, Western Blot, Control

Journal: Pharmaceutical Biology

Article Title: Aspirin relieves the calcification of aortic smooth muscle cells by enhancing the heat shock response

doi: 10.1080/13880209.2021.2007268

Figure Lengend Snippet: Inhibition of HSP70 counteracted the anti-calcification effect of aspirin on VSMCs. (A) The effect of HSP70 inhibitor MKT-077 on aspirin alleviating VSMC calcification. Alizarin Red S assay was applied to detect calcium nodules in VSMCs after 10-day culture in OIM. (B) The effect of MKT-077 on aspirin reducing intracellular calcium concentration in VSMCs after 10-day culture in OIM. n = 5 in each group. ** p < 0.01. (C) The effect of MKT-077 on aspirin downregulating osteogenic marker genes expression in VSMCs after four-day culture in OIM. β-Actin was used as loading control. (D, E) Grey values analysis of immune bands in western blot assay. n = 3 in each group. ** p < 0.01. (F) The effect of HSP70/HSP90 siRNA on aspirin alleviating VSMC calcification. The calcium nodules were detected by Alizarin Red S assay in VSMCs after 10-day culture in OIM. (G) The effect of HSP70/HSP90 siRNA on aspirin reducing intracellular calcium concentration in VSMCs after 10-day culture in OIM. n = 5 in each group. * p < 0.05.

Article Snippet: Aspirin (1 mmol/L or 4 mmol/L; Sangon Biotech, Shanghai, China), HSP70 inhibitor MKT-077 (1 µmol/L; MedChemExpress, Monmouth Junction, NJ) and HSP90 inhibitor 17-DMAG (1 µmol/L; Sigma, St. Louis, MO) were applied to treat the calcification-induced VSMCs.

Techniques: Inhibition, Concentration Assay, Marker, Expressing, Control, Western Blot

Journal: BMC Cancer

Article Title: Interaction between the BAG1S isoform and HSP70 mediates the stability of anti-apoptotic proteins and the survival of osteosarcoma cells expressing oncogenic MYC

doi: 10.1186/s12885-019-5454-2

Figure Lengend Snippet: BAG1S mediated cell survival dependent on interaction with HSP70 chaperone. a Schematic demonstrating BAG1S interaction with HSP70 chaperone (top) and a mutant BAG1ΔS that retains BAG1S structure but prevents HSP70 binding via a single amino acid change in the BAG domain (bottom). b U2OS MYC-ER cells transfected with vectors encoding BAG1S, BAG1ΔS or empty vector, followed by endogenous BAG1 depletion or shLUC control. IB demonstrating efficient knockdown of endogenous BAG1 and retained ectopic BAG1S and BAG1ΔS expression. c U2OS MYC-ER cells expressing ectopic vector, BAG1S, or BAG1ΔS depleted of endogenous BAG1 via lentiviral infection. Five days post-infection cells treated with ±100 nM 4-OHT for 48 h. Apoptosis measured by Annexin V-PE plus 7AAD DNA staining and quantified by FACS analysis. d Quantification of three experimental replicates representing average and standard deviation of cumulative early and late apoptosis based on population of Annexin V-PE positive cells. e PARP cleavage and BAG1 knockdown demonstrated by IB. F and C indicate full and cleaved species respectively. f U2OS MYC-ER cells cultured with ±100 nM 4-OHT and 10uM MKT-077 HSP70 inhibitor for 48 h. CAS-3 demonstrated by IB. F and C indicate full and cleaved species respectively. g Model detailing three experimental methods used to interrupt HSP70/BAG1S chaperone complex activity. Limiting HSP70/BAG1S function via silencing BAG1, inhibiting HSP70, and preventing BAG1S binding HSP70 all resulted in increased MYC-dependent death

Article Snippet: The HSP70 inhibitor MKT-077 (Sigma-Aldrich) was used at a final concentration of 10uM for 48 h.

Techniques: Mutagenesis, Binding Assay, Transfection, Plasmid Preparation, Expressing, Infection, Staining, Standard Deviation, Cell Culture, Activity Assay

Journal: BMC Cancer

Article Title: Interaction between the BAG1S isoform and HSP70 mediates the stability of anti-apoptotic proteins and the survival of osteosarcoma cells expressing oncogenic MYC

doi: 10.1186/s12885-019-5454-2

Figure Lengend Snippet: Proteomic analysis identified proteins upregulated in the presence of pro-survival HSP70/BAG1S complex. a U2OS MYC-ER cells expressing ectopic vector, BAG1S, or BAG1ΔS depleted of endogenous BAG1 protein. MYC activity induced for 12 or 24 h with ±100 nM 4-OHT treatment. Lysates analyzed via IB to detect changes in known HSP70 chaperone client proteins GCR, XIAP and RAF1. b Schematic of experimental conditions representing endogenous BAG1 (vector - shLUC), BAG1 knockdown (vector - shBAG1), BAG1S only (BAG1S - shBAG1), or BAG1ΔS only (BAG1ΔS - shBAG1) evaluated for differences in global protein levels. Proteomics analysis outlined with exclusion criteria for significant protein differences between samples. c Efficient knockdown of endogenous BAG1 and rescue of BAG1S and BAG1ΔS shown by IB for samples subjected to proteomics analysis. d Proteomic hits assessed based on schematic of compiled proteins with ≥|1.5| fold change in knockdown compared to control and p ≤ 0.05 across all conditions. Protein expression levels obtained for each sample indexed by specific protein and clustered by UniProt biological process classification. P -values representative of experimental triplicates submitted for proteomic assessment. e Venn diagram showing proteins partially rescued with reintroduction of BAG1S or BAG1ΔS. Increase of ≥10% constitutes a partial rescue. Overlapping proteins with BAG1S or BAG1ΔS indicative of proteins rescued by either ectopic protein. f Verification of proteomics via detection of BAG1S rescued targets SLC7A6 and POLR1D by IB.

Article Snippet: The HSP70 inhibitor MKT-077 (Sigma-Aldrich) was used at a final concentration of 10uM for 48 h.

Techniques: Expressing, Plasmid Preparation, Activity Assay

Journal: BMC Cancer

Article Title: Interaction between the BAG1S isoform and HSP70 mediates the stability of anti-apoptotic proteins and the survival of osteosarcoma cells expressing oncogenic MYC

doi: 10.1186/s12885-019-5454-2

Figure Lengend Snippet: Proposed model of MYC-mediated BAG1 regulation and distinct functions of BAG1 isoforms. MYC activates gene transcription of BAG1 which produces BAG1 mRNA with three translation start sites that encode distinct protein isoforms BAG1L, M, and S. Nuclear BAG1L and BAG1M cooperate with transcription factors to regulate gene expression. Cytoplasmic BAG1M and BAG1S interact with HSP70 to modulate chaperone activity. As reported here, HSP70/BAG1S complex upregulates an array of proteins and inhibits MYC-driven apoptosis

Article Snippet: The HSP70 inhibitor MKT-077 (Sigma-Aldrich) was used at a final concentration of 10uM for 48 h.

Techniques: Expressing, Activity Assay