Journal: Nature communications

Article Title: HSF1 phosphorylation establishes an active chromatin state via the TRRAP-TIP60 complex and promotes tumorigenesis.

doi: 10.1038/s41467-022-32034-4

Figure Lengend Snippet: Fig. 1 | Identification of the co-activator TRRAP that interacts with HSF1. a Relative enrichment of proteins identified in HSF1 ChIP preparations from heat-shocked cells. HeLa cells were untreated or treated with heat shock at 42 °C for 60 min, and HSF1-interacting proteins were identified by ChIP-MS. Thirty- one proteins highly enriched upon HS (difference of peptide numbers >3) are shown. Proteins related to histone modifications are indicated in red. b HSF1 interacts with TRRAP in the nucleus during heat shock. Cytoplasmic (Cyto) and nuclear (Nucl) extracts were prepared and complexes co-immunoprecipitated using anti-IgG or anti-HSF1 and subjected to immunoblotting. c Expression of HSP72 mRNA in TRRAP-KD cells during heat shock. Levels of HSP72 mRNA were quantified, and the levels relative to that in control SCR-treated cells are shown. Extracts of cells were subjected to immunoblotting. d Venn diagram of HSF1 and TRRAP ChIP-seq binding peaks in HeLa cells untreated (Cont.) or treated with

Article Snippet: The following antibodies were used: rabbit antibodies for mHSF1 (antimHSF1n, Nakai lab, 1/1000), hTRRAP (anti-hTRRAP-2, Nakai lab, 1/ 1000), hTRIM24 (anti-hTRIM24-3, Nakai lab, 1/1000), hTRIM33 (antihTRIM33-2, Nakai lab, 1/1000), hPLK1 (anti-hPLK1-1, Nakai lab, 1/1000), GST (anti-GST, Nakai lab, 1/1000),mHSP110 (anti-mHSP110a, Nakai lab, 1/1000), hHSP90 (anti-hHSF90d, Nakai lab, 1/1000), hHSP40 (antihHSP40a,Nakai lab, 1/1000)15, p300 (SantaCruz, sc-585, 1/1000), p400 (Novus Biologicals, NB200-210, 1/1000), H2B (Abcam, ab1790, 1/ 1000), ubiquityl-histone H2B (Lys120) (Cell Signaling, 5546, 1/1000), RNF40 (GeneTex, GTX115295, 1/1000), histone H3 (Abcam, ab1791, 1/ 1000), H3 (acetyl K18) (Abcam, ab1191, 1/1000), Lamin B1 (Abcam, Ab16048, 1/1000), HSF1 (phosphor S326) (Abcam, ab115702, 1/1000) and HSF1 (phosphoS419) (anti-HSF1 phospho-S419b, Nakai lab, 1/ 1000), mouse antibodies for PLK1 (Santa Cruz, sc-17783, 1/1000), HSP72 (Santa Cruz, sc-24, 1/1000), ubiquitin (Santa Cruz, sc-8017, 1/ 1000), CSNK1A1 (Santa Cruz, sc-74582, 1/1000), NEK7 (Santa Cruz, sc393539, 1/1000), RNF20 (Santa Cruz, sc-517358, 1/1000), β-actin (Milipore Sigma, A5441, 1/1000), and GFP (Nacalai Tesque, GF200, 1/ 1000), rat antibody for HA (Roche, ROAHAHA, 1/1000), and goat Nature Communications | (2022) 13:4355 12 antibody for TIP60 (Santa Cruz, N-17, 1/200).

Techniques: Immunoprecipitation, Western Blot, Expressing, Control, ChIP-sequencing, Binding Assay

Journal: Acta pharmacologica Sinica

Article Title: Huperzine A derivative M3 protects PC12 cells against sodium nitroprusside-induced apoptosis.

doi: 10.1038/aps.2011.147

Figure Lengend Snippet: Figure 6. Effect of M3 on the expression of Hsp70 at different time. (A) Analysis of Hsp70 protein expression in PC12 cells by western blotting at different time after exposure to M3 or HupA and the quantification of protein levels compare to the 0 h treating group by density scanning.

Article Snippet: The anti-Hsp70 antibody and antimouse secondary antibody were bought from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot

Journal: Acta pharmacologica Sinica

Article Title: Huperzine A derivative M3 protects PC12 cells against sodium nitroprusside-induced apoptosis.

doi: 10.1038/aps.2011.147

Figure Lengend Snippet: Figure 7. Quercetin inhibited the protective effect of M3 against SNP- induced apoptosis in PC12 cells. PC12 cells were pre-treated with 10 μmol/L HupA, M3, or 10 μmol/L quercetin for 1 h then exposed to 200 μmol/L SNP for the next 24 h. (A) Effect of quercetin on the cell viability in PC12 cells by MTT method. The percentage of cell viability was presented as mean±standard deviation for six replicates. (B) Effect of quercetin on the ratio of Caspase-3 and Hsp70 in PC12 cells. The percentage of density was presented as mean±standard deviation for three individual experiments. cP<0.01 vs control group. eP<0.05 vs SNP group. hP<0.05 vs M3 pretreated group.

Article Snippet: The anti-Hsp70 antibody and antimouse secondary antibody were bought from Santa Cruz Biotechnology.

Techniques: Standard Deviation, Control

Journal: Journal of Korean Neurosurgical Society

Article Title: S100ß, Matrix Metalloproteinase-9, D-dimer, and Heat Shock Protein 70 Are Serologic Biomarkers of Acute Cerebral Infarction in a Mouse Model of Transient MCA Occlusion

doi: 10.3340/jkns.2017.0200

Figure Lengend Snippet: Plasma HSP70 concentration in all groups. HSP70 concentration increases significantly in both infarction groups (p=0.001) when compared to that of the sham group (A); however, there is no correlation between HSP70 concentration and infarct area (r=0.174, p=0.406; b) or neurological score (r=0.138, p=0.510; c). * p <0.05. HSP70 : heat shock protein 70, McAO : middle cerebral artery occlusion.

Article Snippet: The plasma concentrations of S100β, MMP-9, D-dimer, and HSP70 were calculated using mouse protein S100β (CSB-EL-0206643MO; CUSABIO, Wuhan, China), mouse total MMP-9 (MMPT90; R&D Systems, Minneapolis, MN, USA), mouse D-dimer (E03D0224; BlueGene, Shanghai, China), and mouse HSP70 (CSB-E08311m; CUSABIO) ELISA kits.

Techniques: Clinical Proteomics, Concentration Assay

Journal: Journal of Korean Neurosurgical Society

Article Title: S100ß, Matrix Metalloproteinase-9, D-dimer, and Heat Shock Protein 70 Are Serologic Biomarkers of Acute Cerebral Infarction in a Mouse Model of Transient MCA Occlusion

doi: 10.3340/jkns.2017.0200

Figure Lengend Snippet: Sensitivity and specificity of the biomarkers. MMP-9 and HSP70 both have an AUc of 1. S100β has an AUc of 0.98. The AUc for D-dimer is 0.58. MMP-9 : matrix metalloproteinase-9, HSP70 : heat shock protein 70, AUc : the area under the receiver operating characteristic curve.

Article Snippet: The plasma concentrations of S100β, MMP-9, D-dimer, and HSP70 were calculated using mouse protein S100β (CSB-EL-0206643MO; CUSABIO, Wuhan, China), mouse total MMP-9 (MMPT90; R&D Systems, Minneapolis, MN, USA), mouse D-dimer (E03D0224; BlueGene, Shanghai, China), and mouse HSP70 (CSB-E08311m; CUSABIO) ELISA kits.

Techniques:

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: HSP70 and eIF4G expression are significantly higher in hepatocellular carcinoma (HCC) tumor specimens. ( A ) Representative pictures of H&E and IF staining. Magnification, 20×; Scale bar, 50μm. ( B ) The protein levels of HSP70 and eIF4G in HCC tumor specimens were significantly higher than those of adjacent non-tumor specimens. ( C ) Heatmap showing the relative protein expression of eIF4A, eIF4E, eIF4G, 4EBP1, and HSP70 in all HCC patients tissue samples based on IF staining results. ( D ) Heatmap showing the average relative protein expression of eIF4A, eIF4E, eIF4G, 4EBP1, and HSP70 in all patients in each TNM stage. ( E , F ) Protein expression of HSP70 and eIF4G in HCC patients displayed according to the TNM stage. ( G , H ) The scatter plot of correlation between the AFP level and the protein expression of HSP70 and eIF4G. * p < 0.05, *** p < 0.001 compared with the adjacent non-tumor groups.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Expressing, Staining

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: Clinicopathological Characteristics of HCC Patients.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Expressing

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: Correlation between HSP70 Expression and Clinicopathological Characteristics in HCC Patients.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Expressing

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: The expression of HSP70 and eIF4G positively correlated in tumor specimens of patients with HCC. High and low expression of HSP70 or eIF4G are defined in Materials and Methods. ( A ) Correlation plot showing the relationships between HSP70 and eIF4A, eIF4E, eIF4G, and 4EBP1, respectively. Light blue indicates the highest correlation while light red indicates the lowest correlation. ( B ) The relative protein expression of HSP70 and eIF4G in HCC tissues was positively correlated. ( C , D ) The OS and PFS in HCC patients with high and low HSP70 expression were evaluated. ( E , F ) The OS and PFS of HCC patients were assessed based on high and low eIF4G expression. ( G , H ) HCC patients were divided into four groups of low HSP70 expression/low eIF4G expression, high HSP70 expression/low eIF4G expression, low HSP70 expression/high eIF4G expression, and high HSP70 expression/high eIF4G expression. The OS and PFS of each group were analyzed. ( I – K ) The PFS of HCC patients was compared according to clinical features of AFP level ( I ), tumor size ( J ), and TNM stage ( K ). The survival curves were plotted by the Kaplan-Meier method, and p values were calculated by the log-rank test.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Expressing

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: HSP70 interacts with eIF4G in vitro. ( A , B ) Cell lysates were analyzed with a Western blot to detect the protein level by using primary antibodies targeting eIF4A, eIF4E, eIF4G, 4EBP1, and HSP70. β-actin was used as an internal control. ( C ) Flag-HSP70 immunoprecipitated eIF4G from the cell lysates of HepG2 and Huh7 cells transfected with or without Flag-HSP70 plasmid. Immunoprecipitates were analyzed with Flag or eIF4G antibodies via Western blot. ( D ) The HSP70-eIF4G interaction was detected by in situ PLA. Flag-HSP70 transfected cells and control cells were incubated with mouse anti-HSP70 mAb and rabbit anti-eIF4G mAb. Red signal spots are shown to indicate the protein interaction between HSP70 and eIF4G. Magnification 20×; Scale bar 50 μm. Quantitative data from three experiments with similar results were analyzed. Data are presented as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with corresponding control groups.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: In Vitro, Western Blot, Control, Immunoprecipitation, Transfection, Plasmid Preparation, In Situ, Incubation, Standard Deviation

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: The HSP70 and eIF4G interaction increases under hypoxia. ( A ) HepG2 and Huh7 cells were treated as described in the methods section. IC50 of CoCl2 in HepG2 and Huh7 cells were calculated based on cell viability assessed by MTT assay, and the results presented as a ratio with the control group. ( B , C ) Cells were treated with 10 µM CoCl 2 for 12 h and the protein levels of the eIF4F complex and HSP70 detected by Western blot. β-actin was used as an internal control. ( D ) Cells were incubated with 10 µM CoCl2 for 12 h and the protein of eIF4G immunoprecipitated from the cell lysates of HepG2 and Huh7 cells with HSP70 antibody. The immunoprecipitates were analyzed via Western blot with HSP70 and eIF4G antibodies. ( E ) Cells were stimulated with 10 µM CoCl 2 for 12 h and the HSP70–eIF4G interaction in situ was detected by PLA assay. Representative images are shown. Magnification 20×; Scale bar 50 μm. Quantitative data from three experiments with similar results were analyzed. Data are presented as mean ± SD. ** p < 0.01 compared with corresponding control groups.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: MTT Assay, Control, Western Blot, Incubation, Immunoprecipitation, In Situ

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: HSP70–eIF4G interaction promotes cellular protein synthesis. ( A , B ) Cells were incubated with 10 µM CoCl 2 for 12 h; each group was then examined via protein synthesis assay. Representative pictures of protein synthesis are shown. Magnification 20×; Scale bar 50 μm. ( C , D ) Cells were cultivated with 10 µM CoCl 2 for 12 h and the eIF4G-eIF4E interaction examined in situ for each group. Representative images of the PLA assay are presented. Magnification 20×; Scale bar 50 μm. Quantitative data from three experiments with similar results were analyzed. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with corresponding control groups.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Incubation, In Situ, Control

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: HSP70–eIF4G interaction promotes cell proliferation. ( A , B ) Cells were incubated with 10 μM CoCl 2 for 5 days; cell viability was measured daily. ( C , D ) Cells treated with 10 μM CoCl 2 ; colony units were counted after 14 days of incubation. Quantitative data from three experiments with similar results were summarized. Data are presented as mean ± SD. ns: not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001 compared with corresponding control groups.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Incubation, Control

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: HSP70–eIF4G interaction inhibits cell apoptosis. ( A , B ) Cells were incubated with 10 μM CoCl 2 for 48 h. Annexin-V and PI were used to stain the treated cells and flow cytometry analysis was performed to assess the rate of cell apoptosis. ( C , D ) Cells treated with 10 μM CoCl 2 for 48 h. The percentage of the Sub-G1 portion of the cell cycle distribution was quantified by flow cytometry analyses after PI staining. Quantitative data from three experiments with similar results were summarized. Data presented as mean ± SD. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to corresponding control groups.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Incubation, Staining, Flow Cytometry, Control

Journal: PloS one

Article Title: Biological responses of three-dimensional cultured fibroblasts by sustained compressive loading include apoptosis and survival activity.

doi: 10.1371/journal.pone.0104676

Figure Lengend Snippet: Figure 5. The secretions of HSP90a, HA, and PGE2 into cell culture medium were increased by compressive loading. Fibroblasts were seeded to collagen sponge and incubated for 24 h. And then they were subjected to 0, 50, 100, or 200 mmHg compression for 6 h. Culture supernatants were collected after 6 h loading experiments. The level of each substance (A: HSP90a, B: HA, C: PGE2) was measured by ELISA. A value of concentration was normalized by WST-1 value. The results are represented as the mean 6 SEM (error bars) of five experiments. Statistical analysis was performed using the Dunnett’s multiple test between non-loaded group and each of loaded group, and statistical significance was taken as p,0.05. A value of p was expressed as: *; p,0.05, **; p,0.01, and ***; p,0.001. doi:10.1371/journal.pone.0104676.g005

Article Snippet: HSP90a concentration was measured using the Rat Heat Shock Protein 90a ELISA kit (CUSABIO BIOTECH, Wuhan, China).

Techniques: Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay