Journal: International journal of molecular medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple‑negative breast cancer metastasis via the inhibition of CXCR4 expression.

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: Figure 1. The HER3 pathway is associated with the DMFS of patients with TNBC. (A) c‑Met, EGFR, IGF1R, IR and HER3 protein phosphorylation levels were measured using Luminex® 200. The experiments were performed in duplicate and repeated three times. Data are presented as the mean ± SD. *P<0.05, compared with 4T1 cells. (B) The expression levels of p‑HER3, HER3, p‑Akt, Akt, p‑mTOR, mTOR, p‑ERK and ERK were detected using western blot analysis. The expression levels of β‑actin were used as internal controls. Quantification of signals is presented as fold of changes relative to phosphory lated proteins vs. total proteins. The experiments were repeated three times. Data are presented as the mean ± SD. *P<0.05, compared with 4T1 cells. (C) Kaplan‑Meier Plotter analysis indicated that DMFS was associated with HER3 expression (low expression group, n=212; high expression group, n=212) among 424 TNBC cases (log‑rank, P=0.0035). DMFS, distant metastasis‑free survival; TNBC, triple‑negative breast cancer; HER3, human epidermal growth factor receptor 3; EGFR, epidermal growth factor receptor; IGF1R, insulin‑like growth factor 1 receptor; IR, insulin receptor; mTOR, mammalian target of rapamycin; p‑phosphorylated.

Article Snippet: The membranes were blocked with 5% skim milk for 30 min at room temperature and incubated with antibodies against phosphorylated (p‐)HER3 (cat. no. 2235; dilution 1:1,000), HER3 (cat. no. 4754; dilution 1:1,000), p‐Akt (cat. no. 9271; dilution 1:3,000), Akt (cat. no. 9272; dilution 1:3,000), p‐mTOR (cat. no. 2971; dilution 1:3,000), mTOR (cat. no. 2972; dilu‐ tion 1:3,000), p‐extracellular signal‐regulated kinase (p‐ERK) (cat. no. 4370; dilution 1:3,000), ERK (cat. no. 9102; dilution 1:3,000), chemokine receptor (CCR)2 (cat. no. 12199; dilution 1:3,000) (all from Cell Signaling Technology, Inc.), CCR7 (cat. no. sc‐9701; dilution 1:3,000), C‐X‐C chemokine receptor type 4 (CXCR4) (cat. no. sc‐9046; dilution 1:3,000) (all from Santa Cruz Biotechnology, Inc.) and β‐actin (cat. no. A2228; dilu‐ tion 1:3,000; MilliporeSigma) overnight at 4 ̊C.

Techniques: Phospho-proteomics, Luminex, Expressing, Western Blot

Journal: International journal of molecular medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple‑negative breast cancer metastasis via the inhibition of CXCR4 expression.

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: Figure 2. Knockdown of HER3 decreases the migration, invasion and metastasis of metastatic TNBC cells by inhibiting Akt and mTOR. (A‑C) The 4T1‑L8 cells were transfected with HER siRNA (50 nM) or control siRNA. (A) The expression levels of HER3, p‑Akt, Akt, p‑mTOR and mTOR were detected using western blot analysis. The expression levels of β‑actin were used as internal controls. Quantification of signals is presented as fold of changes relative to phosphorylated protein vs. total proteins or total proteins vs. β‑actin. The experiments were repeated four times. Data are presented as the mean ± SD. *P<0.05, compared with control siRNA. (B) The number of cells stained with trypan blue counted on days 1, 2 and 3. The experiments were performed in triplicate and repeated three times. Data are presented as the mean ± SD. *P<0.05, compared with control siRNA. (C) Cell migration and invasion analysis was performed using the cell culture insert. The cells passing through the cell culture insert were counted. The experiments were repeated three times. Data are presented as the mean ± SD. *P<0.05, compared with control siRNA. Representative images of cells transfected with control siRNA and HER3 siRNA are shown. Scale bars, 100 µm. (D) The 4T1‑L8 cells transfected with HER siRNA (50 nM) or control siRNA were injected into BALB/c mice via the tail vain. Metastasis in the lungs was monitored using IVIS. After 8 days, the mice were sacrificed and the number of metastatic nodules in the lungs were counted. The results are expressed as the mean ± SD. *P<0.05, compared with control siRNA. TNBC, triple‑negative breast cancer; HER3, human epidermal growth factor receptor 3; mTOR, mammalian target of rapamycin; p‑phosphorylated.

Article Snippet: The membranes were blocked with 5% skim milk for 30 min at room temperature and incubated with antibodies against phosphorylated (p‐)HER3 (cat. no. 2235; dilution 1:1,000), HER3 (cat. no. 4754; dilution 1:1,000), p‐Akt (cat. no. 9271; dilution 1:3,000), Akt (cat. no. 9272; dilution 1:3,000), p‐mTOR (cat. no. 2971; dilution 1:3,000), mTOR (cat. no. 2972; dilu‐ tion 1:3,000), p‐extracellular signal‐regulated kinase (p‐ERK) (cat. no. 4370; dilution 1:3,000), ERK (cat. no. 9102; dilution 1:3,000), chemokine receptor (CCR)2 (cat. no. 12199; dilution 1:3,000) (all from Cell Signaling Technology, Inc.), CCR7 (cat. no. sc‐9701; dilution 1:3,000), C‐X‐C chemokine receptor type 4 (CXCR4) (cat. no. sc‐9046; dilution 1:3,000) (all from Santa Cruz Biotechnology, Inc.) and β‐actin (cat. no. A2228; dilu‐ tion 1:3,000; MilliporeSigma) overnight at 4 ̊C.

Techniques: Knockdown, Migration, Transfection, Control, Expressing, Western Blot, Staining, Cell Culture, Injection

Journal: International journal of molecular medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple‑negative breast cancer metastasis via the inhibition of CXCR4 expression.

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: Figure 3. The HER3/Akt/mTOR pathway promotes the migration and invasion of metastatic triple‑negative breast cancer cells by increasing CXCR4 expres sion. (A) The expression levels of CCR2, CCR7 and CXCR4 were detected using western blot analysis. The expression levels of β‑actin were used as internal controls. Quantification of signals is presented as fold of changes relative to total proteins vs. β‑actin. The experiments were repeated three times. Data are presented as the mean ± SD. *P<0.05, compared with 4T1 cells. (B) Cell migration and invasion analysis was performed using the cell culture insert. The cells passing through the cell culture insert were counted. The experiments were repeated three times. Data are presented as the mean ± SD. *P<0.05, compared with SDF‑1(‑). Representative images of SDF‑1‑untreated 4T1 or 4T1‑L8 and SDF‑1‑treated 4T1 or 4T1‑L8 cells are shown. Scale bars, 100 µm. (C and D) 4T1‑L8 cells were transfected with HER siRNA (50 nM) or control siRNA (Stealth™ RNAi Negative Control). (C) The expression levels of CXCR4 were detected using western blotting. The expression levels of β‑actin were used as internal controls. Quantification of signals is presented as fold change or relative levels of CXCR4 vs. β‑actin. The experiments were repeated three times. Data are presented as the mean ± SD. *P<0.05, compared with control siRNA. (D) Cell migration and invasion analysis was performed using the cell culture insert. The cells passing through the cell culture insert were counted. The experiments were repeated three times. Data are presented as the mean ± SD. *P<0.05, compared with control siRNA. Representative images of control siRNA and HER3 siRNA are shown. Scale bars, 100 µm. HER3, human epidermal growth factor receptor 3; mTOR, mammalian target of rapamycin; CCR, chemokine receptor; CXCR4, C‑X‑C chemokine receptor type 4; SDF‑1, stromal derived factor‑1.

Article Snippet: The membranes were blocked with 5% skim milk for 30 min at room temperature and incubated with antibodies against phosphorylated (p‐)HER3 (cat. no. 2235; dilution 1:1,000), HER3 (cat. no. 4754; dilution 1:1,000), p‐Akt (cat. no. 9271; dilution 1:3,000), Akt (cat. no. 9272; dilution 1:3,000), p‐mTOR (cat. no. 2971; dilution 1:3,000), mTOR (cat. no. 2972; dilu‐ tion 1:3,000), p‐extracellular signal‐regulated kinase (p‐ERK) (cat. no. 4370; dilution 1:3,000), ERK (cat. no. 9102; dilution 1:3,000), chemokine receptor (CCR)2 (cat. no. 12199; dilution 1:3,000) (all from Cell Signaling Technology, Inc.), CCR7 (cat. no. sc‐9701; dilution 1:3,000), C‐X‐C chemokine receptor type 4 (CXCR4) (cat. no. sc‐9046; dilution 1:3,000) (all from Santa Cruz Biotechnology, Inc.) and β‐actin (cat. no. A2228; dilu‐ tion 1:3,000; MilliporeSigma) overnight at 4 ̊C.

Techniques: Migration, Expressing, Western Blot, Cell Culture, Transfection, Control, Negative Control, Derivative Assay

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: After centrifugation at 16,000 × g for 10min, aliquots of the supernatant containing 5mg of proteins were incubated with 3 μl of rat anti-HSF1 monoclonal antibody (Novus Biologicals, 10H4) at 4 °C for 3 h, and then mixed with 50 μl protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 2 h. Beads were washed five times with NP-40 lysis buffer and 50 μl of 2 × Laemmli sample buffer was then added.

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

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. 5 | PLK1 phosphorylates HSF1-S419 and triggers histone modifications. HeLa cells were infected with adenovirus expressing shRNA for PLK1, CSNK1A1, NEK7 or SCR (a), or treated with BI6727 (PLK1 inhibitor), FR180204 (ERK1/2 inhibitor), AZD6244 (MEK1/2 inhibitor), or rapamycin (mTOR inhibitor) (b). These cells were then treated with heat shock. Cell extracts were subjected to HSF1 immunoprecipitation and immunoblotting using antibody for HSF1 phospho- S419 or HSF1. Blue arrows indicate the positions of HSF1-S419 phosphorylated bands. The intensity of the upper band was markedly enhanced during heat shock. c PLK1 interacts with HSF1 in the nucleus during heat shock. Cell extracts were prepared as described in Fig. 1b and subjected to immunoblotting. Cells, in

Article Snippet: After centrifugation at 16,000 × g for 10min, aliquots of the supernatant containing 5mg of proteins were incubated with 3 μl of rat anti-HSF1 monoclonal antibody (Novus Biologicals, 10H4) at 4 °C for 3 h, and then mixed with 50 μl protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 2 h. Beads were washed five times with NP-40 lysis buffer and 50 μl of 2 × Laemmli sample buffer was then added.

Techniques: Infection, Expressing, shRNA, Immunoprecipitation, Western Blot

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. 6 | HSF1-S419 phosphorylation maintains proteostasis capacity. a TRRAP promotes cell survival. Cells were infected with adenovirus expressing shRNA for HSF1, TRRAP or SCR, and were heat-shocked at 45 °C for the indicated periods. Number of viable cells excluding trypan blue was counted. Extracts of cells were subjected to immunoblotting. b Phosphorylation of HSF1-S419 promotes cell survival. Cells, in which endogenous HSF1 was replaced with GFP, wild-type hHSF1-HA, or hHSF1-S419A mutants were heat-shocked at 45 °C for 4 h. Number of viable cells excluding trypan blue was counted, and MTT assay was performed. Extracts of cells were subjected to immunoblotting. c Phosphorylation of HSF1- S419 inhibits the accumulation of ubiquitylated proteins. Cells treated as described in b were heat-shocked at 45 °C for 4 h. The accumulation of insoluble ubiquitylated proteins was examined by immunoblotting using anti-Ub antibody and quantified. β-Actin levels in the soluble fraction were also shown. d TRRAP

Article Snippet: After centrifugation at 16,000 × g for 10min, aliquots of the supernatant containing 5mg of proteins were incubated with 3 μl of rat anti-HSF1 monoclonal antibody (Novus Biologicals, 10H4) at 4 °C for 3 h, and then mixed with 50 μl protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 2 h. Beads were washed five times with NP-40 lysis buffer and 50 μl of 2 × Laemmli sample buffer was then added.

Techniques: Phospho-proteomics, Infection, Expressing, shRNA, Western Blot, MTT Assay

Journal: Cells

Article Title: Transcriptional Regulation of Selenoprotein F by Heat Shock Factor 1 during Selenium Supplementation and Stress Response

doi: 10.3390/cells8050479

Figure Lengend Snippet: Heat Shock Factor 1 (HSF1) increased SELENOF transcriptional regulation. ( a ) Luciferase reporter activities of SELENOF core positive regulatory element, ( b ) transcriptional levels of SELENOF, protein levels of ( c ) SELENOF and ( d ) GPX4 in control and HSF1 over-expressed HEK293T cells. The empty vector pcDNA3.1 was used as the negative control to be compared with pcDNA3.1-HSF1. In Luciferase reporter assays, the empty vector pGL4.10 was used as the negative control, while the pGL4-818/-248 was used as core promoter of SELENOF. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Specific siRNA to knock-down HSF1 (sc-35611) and its control siRNA (sc-36869) were purchased from Santa Cruz.

Techniques: Luciferase, Control, Plasmid Preparation, Negative Control

Journal: Cells

Article Title: Transcriptional Regulation of Selenoprotein F by Heat Shock Factor 1 during Selenium Supplementation and Stress Response

doi: 10.3390/cells8050479

Figure Lengend Snippet: HSF1 interacted with the 5′-flanking regions of SELENOF. ( a ) PCR amplification results of chromatin immunoprecipitation (ChIP) primers after protein-chromatin cross-linkage and immunoprecipitation. Locations of JASPAR predicted HSF1 binding sites and the designed ChIP primers were presented in the schematic map. ( b ) Manually searched results of both typical and less conserved variant of HSF1 Sequence-binding Elements (HSEs) in the sequences of SELENOF 5′- flanking region from −1430 to −248. The data are representative of three independent experiments.

Article Snippet: Specific siRNA to knock-down HSF1 (sc-35611) and its control siRNA (sc-36869) were purchased from Santa Cruz.

Techniques: Amplification, Chromatin Immunoprecipitation, Immunoprecipitation, Binding Assay, Variant Assay, Sequencing

Journal: Cells

Article Title: Transcriptional Regulation of Selenoprotein F by Heat Shock Factor 1 during Selenium Supplementation and Stress Response

doi: 10.3390/cells8050479

Figure Lengend Snippet: Heat shock treatment increased SELENOF transcriptional regulation. ( a ) Luciferase reporter activities of SELENOF core positive regulatory element in transfected HEK293T cells with/without heat shock treatment. Transcriptional levels of SELENOF ( b ) and heat shock protein (HSP)70 ( c ) at several time intervals in HEK293T cells under heat shock treatment. ( d ) Protein levels of SELENOF in control and HSF1 over-expressed HEK293T cells with/without heat shock treatment. The data are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Specific siRNA to knock-down HSF1 (sc-35611) and its control siRNA (sc-36869) were purchased from Santa Cruz.

Techniques: Luciferase, Transfection, Control

Journal: Cells

Article Title: Transcriptional Regulation of Selenoprotein F by Heat Shock Factor 1 during Selenium Supplementation and Stress Response

doi: 10.3390/cells8050479

Figure Lengend Snippet: The effect of selenate and Tm treatment on the endogenous HSF1 activation and SELENOF protein levels. ( a ) Protein levels of SELENOF, p-HSF1 (S326), and HSF1 at several time intervals in HEK293T cells treated with selenate. ( b ) Protein levels of Binding immunoglobulin protein (BIP), SELENOF, p-HSF1 (S326), and HSF1 at several time intervals in HEK293T cells treated with Tm. BIP bands were used as protein markers for adaptive endoplasmic reticulum (ER) stress. The data are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Specific siRNA to knock-down HSF1 (sc-35611) and its control siRNA (sc-36869) were purchased from Santa Cruz.

Techniques: Activation Assay, Binding Assay

Journal: Cells

Article Title: Transcriptional Regulation of Selenoprotein F by Heat Shock Factor 1 during Selenium Supplementation and Stress Response

doi: 10.3390/cells8050479

Figure Lengend Snippet: HSF1 further increased SELENOF transcriptional regulation during selenate and Tm treatment. Luciferase reporter activities of SELENOF core positive regulatory element in control and HSF1 over-expressed HEK293T cells with/without ( a ) selenate and ( b ) Tm treatment. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Specific siRNA to knock-down HSF1 (sc-35611) and its control siRNA (sc-36869) were purchased from Santa Cruz.

Techniques: Luciferase, Control

Journal: Cells

Article Title: Transcriptional Regulation of Selenoprotein F by Heat Shock Factor 1 during Selenium Supplementation and Stress Response

doi: 10.3390/cells8050479

Figure Lengend Snippet: Involvement of HSF1 in the up-regulation of SELENOF during selenate and Tm treatment. Protein levels of SELENOF in ( a ) control and HSF1 over-expressed HEK293T cells with/without selenate treatment, ( b ) control and HSF1 over-expressed HEK293T cells with/without Tm treatment, ( c ) siRNA transfected HEK293T cells with/without selenate treatment, and ( d ) siRNA transfected HEK293T cells with/without Tm treatment. The data are representative of three independent experiments. The sham siRNAs were used as negative control to be compared with the siHSF1 siRNAs. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Specific siRNA to knock-down HSF1 (sc-35611) and its control siRNA (sc-36869) were purchased from Santa Cruz.

Techniques: Control, Transfection, Negative Control

Journal: bioRxiv

Article Title: Heat shock induces alternative polyadenylation through dynamic DNA methylation-regulated chromatin looping

doi: 10.1101/2023.08.25.554792

Figure Lengend Snippet: ( A ) Annotation of the CpG island (CGI) sequence in the APA control region. Predicted binding sites for CTCF (highlighted in orange), YY1 (underlined), ATF6 (blue letters), and HSF1 (highlighted in yellow) are marked. ( B ) Workflow of DNA pulldown experiment. ( C ) Images of DNA pulldown cell lysates resolved on acrylamide gels and stained with Coomassie (left panel) and probed with anti-CTCF antibody (right panel). WNE, whole nuclear extract; Beads only, elute from reactions without nuclear extracts; Methylated bait, elute from reactions containing methylated bait; Unmethylated bait, elute from reactions containing unmethylated bait. ( D ) ChIP-qPCR in untreated HCT116 cells and post-heat shock (HS) HCT116 cells at the indicated time point. Numbers indicate hours post heat shock. Asterisks identify heat shock (HS) samples that are statistically different from untreated (NT) HCT116 (p < 0.05). ( E ) Western blot for DNAJB6 protein isoforms, DNMT1, TET1, YY1, CTCF, and ACTB loading control in DKO, untreated HCT116 (NT), and post-heat shock HCT116 cells at the indicated time points.

Article Snippet: The antibodies and their respective dilutions are as follows: CTCF antibody (Cell Signaling Technology #2899) at 1:25 dilution, YY1 antibody (Cell Signaling Technology #46395) at 1:50 dilution, ATF6 antibody (Cell Signaling Technology #65880) at 1:50 dilution, TET1 antibody (ThermoFisher #61443) at 1:25 dilution, HSF1 antibody (Novus #NBP2- 42206V) at 1:50 dilution, and DNMT1 antibody (Sigma-Aldrich #D4692) at 1:50 dilution.

Techniques: Sequencing, Control, Binding Assay, Staining, Methylation, ChIP-qPCR, Western Blot