Journal: Cell Death & Disease

Article Title: Increased expression of EHF via gene amplification contributes to the activation of HER family signaling and associates with poor survival in gastric cancer

doi: 10.1038/cddis.2016.346

Figure Lengend Snippet: EHF is identified as a new HER2 transcription factor and the modulator of HER3 and HER4 in gastric cancer. ( a ) BGC823 cells were transiently transfected with pGL3-Basic or luciferase reporter constructs containing various lengths of the promoter region of HER2 gene, as indicated (F1: −607/+11; F2: −175/+11; F3: −607/−175) (left panels). Cotransfection with empty vector was used as a control. The ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays (right panels). ( b ) The luciferase reporter gene assay was performed to evaluate the effect of EHF knockdown on promoter activity of HER2 in BGC823 cells. The ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays. ( c ) HEK293T cells were cotransfected pGL3-HER2-Luc-F1 and various amounts of pcDNA3.1(-)A-EHF or empty vector, respectively. Promoter activities of HER2 were measured by luciferase reporter gene assays. All the ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays. ( d ) Putative promoter regions of HER2 (−607/+11), HER3 (−997/+440) and HER4 (−697/+306) were inserted into the pGL3-Basic to construct the luciferase reporter plasmid pGL3-HER2-Luc, pGL3-HER3-Luc and pGL3-HER4-Luc (upper panels). P1-P7 represent the regions analyzed by ChIP assays for HER2 , HER3 and HER4 , respectively. BGC823 cells were transiently transfected with pcDNA3.1/myc-His(-)A-EHF or empty vector, and were subjected to ChIP-qRT-PCR assays using anti-Myc tag antibody. Flod enrichment was shown as means±S.E. of three independent assays (lower panels). ( e ) EMSA assay was performed to confirm the interaction between EHF and HER2 promoter. Shown are specific DNA-binding of in vitro translated EHF protein to an oligonucleotide sequence (SH2) containing ETS responsive element (GAGGAA) from the HER2 promoter. Unlabeled mutated probes contain specific mutations in the GGAA ETS core or flanking nucleotides of core sequence, as indicated by MT1 and MT2. Unlabeled wild-type (WT) and mutated (MT1 or MT2) competitor probes were added at 100-fold molar excess. Statistically significant differences were indicated: * P <0.05; ** P <0.01; *** P <0.001

Article Snippet: The membranes were blocked for 2 h in 5% bovine serum albumin (BSA) in 1 × TBS-T (0.5% Tween-20) and incubated with the indicated primary antibodies, including anti-EHF (Abcam, Inc), anti-total-Erk1/2 (Abcam, Inc), anti-phospho-Erk1/2 (Epitomics, Inc), anti-phospho-AktSer473 (Bioworld Technology, co, Ltd), anti-total-Akt (Bioworld Technology, co, Ltd), anti-HER2 (Sino Biological, Inc), anti-HER3 (Sino Biological, Inc), anti-HER4 (Sino Biological, Inc), anti-E-cadherin (Epitomics, Inc), anti-Vimentin (Epitomics, Inc) and anti-GAPDH (Abgent, Inc).

Techniques: Transfection, Luciferase, Construct, Cotransfection, Plasmid Preparation, Activity Assay, Reporter Gene Assay, Quantitative RT-PCR, Binding Assay, In Vitro, Sequencing

Journal: Journal of Neuroinflammation

Article Title: Targeted activation of ErbB4 receptor ameliorates neuronal deficits and neuroinflammation in a food-borne polystyrene microplastic exposed mouse model

doi: 10.1186/s12974-025-03406-6

Figure Lengend Snippet: ErbB4 small molecule agonist ameliorates cognitive behavioral impairments in mice exposed to PS-MPs. ( A ) Representative traces in open field experiment. ( B ) The statistical analysis related to open field test. ( C ) Representative heat map in Y maze exploration experiment, with arm A denoting the initial arm. ( D ) The rate of spontaneous alternation in Y maze experiment. ( E ) Representative traces from the new object recognition (NOR) experiment. ( F ) Statistical analysis related to the NOR experiment. ( G ) Representative swimming traces from the Morris water maze (MWM) exploration experiment. ( H ) The statistical analysis of MWM exploration test. Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons, and the data are presented as means ± standard error of the mean (SEM). Statistical significance is indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001, with n = 8 per group

Article Snippet: Specifically, fixed hippocampal neuronal cells were treated with mouse anti-β3-tubulin antibody (1:100, sc80016, Santa Cruz biotech, USA) in combination with the following antibodies: rabbit anti-p-ErbB4 (1:200, bs-3220R, Bioss, Beijing, China), and rabbit anti-PSD95 (1:200, GB11277-100, Servicebio, Wuhan, China).

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Targeted activation of ErbB4 receptor ameliorates neuronal deficits and neuroinflammation in a food-borne polystyrene microplastic exposed mouse model

doi: 10.1186/s12974-025-03406-6

Figure Lengend Snippet: ErbB4 small molecule agonist can improve the dendritic spine injury of hippocampal neurons in mice exposed to PS-MPs. ( A ) Representative images of neuronal dendritic segments. scale bar, 10 μm. ( B ) Total dendritic spine density of hippocampal neurons. ( C ) The density of mushroom spines in hippocampal neurons. Statistical analysis was conducted using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons.The results were presented as means ± SEM. Statistical significance is indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001, with n = 4 per group

Article Snippet: Specifically, fixed hippocampal neuronal cells were treated with mouse anti-β3-tubulin antibody (1:100, sc80016, Santa Cruz biotech, USA) in combination with the following antibodies: rabbit anti-p-ErbB4 (1:200, bs-3220R, Bioss, Beijing, China), and rabbit anti-PSD95 (1:200, GB11277-100, Servicebio, Wuhan, China).

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Targeted activation of ErbB4 receptor ameliorates neuronal deficits and neuroinflammation in a food-borne polystyrene microplastic exposed mouse model

doi: 10.1186/s12974-025-03406-6

Figure Lengend Snippet: ErbB4 small molecule agonist can improve synaptic dysfunction of hippocampal neurons in mice exposed to PS-MPs. ( A ) The ultrastructural analysis of hippocampal neuron synapses was performed using transmission electron microscopy (TEM). ( B ) Statistical analyses were conducted on the synaptic data of hippocampal neurons. ( C ) Immunofluorescence was employed to detect the levels of synaptophysin (SYP) and β3-tubulin. ( D ) Western blot analysis was utilized to assess the protein levels of synapse-associated proteins, including DOCK3, p-CREB, CREB, SYP, GAP43, PSD95, and SYT1 in hippocampal neurons. ( E ) Statistical analyses of these protein levels were performed using one-way ANOVA with Tukey’s multiple comparison test. The synaptic structure was examined at a magnification of 10,000×, with a scale bar of 1 μm. Results are expressed as means ± SEM. Immunofluorescence labeling was as follows: SYP in red and β3-tubulin in green, with a scale bar of 20 μm. Statistical significance was indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001, with a sample size of n = 4 per group

Article Snippet: Specifically, fixed hippocampal neuronal cells were treated with mouse anti-β3-tubulin antibody (1:100, sc80016, Santa Cruz biotech, USA) in combination with the following antibodies: rabbit anti-p-ErbB4 (1:200, bs-3220R, Bioss, Beijing, China), and rabbit anti-PSD95 (1:200, GB11277-100, Servicebio, Wuhan, China).

Techniques: Transmission Assay, Electron Microscopy, Immunofluorescence, Western Blot, Comparison, Labeling

Journal: Journal of Neuroinflammation

Article Title: Targeted activation of ErbB4 receptor ameliorates neuronal deficits and neuroinflammation in a food-borne polystyrene microplastic exposed mouse model

doi: 10.1186/s12974-025-03406-6

Figure Lengend Snippet: ErbB4 small molecule agonist can improve mitochondrial damage in hippocampal neurons of mice exposed to PS-MPs. ( A ) The ultrastructural integrity of hippocampal neuronal mitochondria was evaluated using transmission electron microscopy (TEM). ( B ) Statistical analyses were performed on data pertaining to the hippocampal neuronal mitochondria. ( C ) The expression levels of mitochondria-associated proteins, including phosphorylated mTOR (p-mTOR), mTOR, and SIRT3, in the hippocampus of mice were quantified via western blot analysis. ( D ) Statistical analyses of these protein expression levels were conducted using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. The original magnification for mitochondrial imaging was 2000×, with a scale bar of 5 μm. Data are presented as means ± SEM. Statistical significance was indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001, with a sample size of n = 4 per group

Article Snippet: Specifically, fixed hippocampal neuronal cells were treated with mouse anti-β3-tubulin antibody (1:100, sc80016, Santa Cruz biotech, USA) in combination with the following antibodies: rabbit anti-p-ErbB4 (1:200, bs-3220R, Bioss, Beijing, China), and rabbit anti-PSD95 (1:200, GB11277-100, Servicebio, Wuhan, China).

Techniques: Transmission Assay, Electron Microscopy, Expressing, Western Blot, Imaging

Journal: Journal of Neuroinflammation

Article Title: Targeted activation of ErbB4 receptor ameliorates neuronal deficits and neuroinflammation in a food-borne polystyrene microplastic exposed mouse model

doi: 10.1186/s12974-025-03406-6

Figure Lengend Snippet: ErbB4 small molecule agonist enhances the ErbB4 signaling pathway in hippocampus of mice exposed to PS-MPs. ( A ) Immunofluorescence was used to detect levels of phosphorylated ErbB4 (p-ErbB4) and β3-tubulin. ( B ) Protein levels associated with the ErbB4 signaling pathway in the hippocampus of mice were assessed through western blot analysis. ( C ) A statistical analysis was performed on the data derived from protein levels, employing one-way ANOVA with Tukey’s multiple comparison tests. The p-ErbB4 is represented in red, while β3-tubulin is depicted in green, with a scale bar of 20 μm. The results are expressed as means ± SEM, with significance levels indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001. Each experimental group comprised n = 4 samples

Article Snippet: Specifically, fixed hippocampal neuronal cells were treated with mouse anti-β3-tubulin antibody (1:100, sc80016, Santa Cruz biotech, USA) in combination with the following antibodies: rabbit anti-p-ErbB4 (1:200, bs-3220R, Bioss, Beijing, China), and rabbit anti-PSD95 (1:200, GB11277-100, Servicebio, Wuhan, China).

Techniques: Immunofluorescence, Western Blot, Derivative Assay, Comparison

Journal: Journal of Neuroinflammation

Article Title: Targeted activation of ErbB4 receptor ameliorates neuronal deficits and neuroinflammation in a food-borne polystyrene microplastic exposed mouse model

doi: 10.1186/s12974-025-03406-6

Figure Lengend Snippet: ErbB4 small molecule agonist can improve hippocampal neuroinflammation in mice exposed to PS-MPs via the TLR4/NLRP3 pathway. ( A ) Levels of IBA1 were detected through immunofluorescence staining. ( B ) Levels of GFAP were detected through immunofluorescence staining. ( C ) Western blot analysis was utilized to detect protein levels associated with the TLR4/NLRP3 signaling pathway in the hippocampus of mice. ( D ) Statistical chart was generated to display the protein levels of TLR4/NLRP3. GFAP and IBA1 are both indicated in red, with a scale bar of 20 μm. Statistical analysis was conducted using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons, and the data are reported as means ± SEM, with significance levels denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001. Each group comprised n = 4 samples

Article Snippet: Specifically, fixed hippocampal neuronal cells were treated with mouse anti-β3-tubulin antibody (1:100, sc80016, Santa Cruz biotech, USA) in combination with the following antibodies: rabbit anti-p-ErbB4 (1:200, bs-3220R, Bioss, Beijing, China), and rabbit anti-PSD95 (1:200, GB11277-100, Servicebio, Wuhan, China).

Techniques: Immunofluorescence, Staining, Western Blot, Generated

Journal: Journal of Neuroinflammation

Article Title: Targeted activation of ErbB4 receptor ameliorates neuronal deficits and neuroinflammation in a food-borne polystyrene microplastic exposed mouse model

doi: 10.1186/s12974-025-03406-6

Figure Lengend Snippet: The ErbB4 small molecule agonist ameliorates hippocampal neuron damage under pathological conditions induced by PS-MPs in vitro via the ErbB4 signaling pathway, and concurrently attenuates microglial activation through the TLR4/NLRP3 signaling pathway. ( A - D ) Following exposure to PS-MPs at a concentration of 100 µg/mL for 24 h, hippocampal neurons were subsequently treated with C11H7BrO3 at a concentration of 10 nM for an additional 24 h. ( A , C ) Protein expression levels associated with the ErbB4 signaling pathway, mitochondrial function, apoptosis, and synaptic integrity in hippocampal neurons were quantified using Western blot analysis. ( B , D ) Statistical representations of these protein expression levels were depicted in graphical form. ( E ) Oxidative stress markers in hippocampal neurons, including total superoxide dismutase (T-SOD), glutathione (GSH), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels, were evaluated. ( F ) Intracellular ATP levels in hippocampal neurons were also measured. ( G - H ) Microglial cells were co-cultured with supernatants derived from treated hippocampal neurons for 24 h. ( G ) Protein expression levels related to the TLR4/NLRP3 signaling pathway in microglial cells were assessed via Western blot analysis. ( H ) Statistical representations of these protein expression levels were also depicted in graphical form. Statistical analysis was conducted using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons, and data are presented as means ± SEM. Significance levels were denoted as * p < 0.05, ** p < 0.01, *** p < 0.001. A minimum sample size of n = 4–6 per group was utilized

Article Snippet: Specifically, fixed hippocampal neuronal cells were treated with mouse anti-β3-tubulin antibody (1:100, sc80016, Santa Cruz biotech, USA) in combination with the following antibodies: rabbit anti-p-ErbB4 (1:200, bs-3220R, Bioss, Beijing, China), and rabbit anti-PSD95 (1:200, GB11277-100, Servicebio, Wuhan, China).

Techniques: In Vitro, Activation Assay, Concentration Assay, Expressing, Western Blot, Cell Culture, Derivative Assay

Journal: Journal of Neuroinflammation

Article Title: Targeted activation of ErbB4 receptor ameliorates neuronal deficits and neuroinflammation in a food-borne polystyrene microplastic exposed mouse model

doi: 10.1186/s12974-025-03406-6

Figure Lengend Snippet: The schematic diagram illustrates the impact of targeted activation of ErbB4 receptor on hippocampal neuronal damage, neuroinflammation, and cognitive functions in mice exposed to PS-MPs. The small molecule ErbB4 receptor agonist (E4A) binds to the ErbB4 receptor, initiating its downstream signaling pathway. This activation leads to the upregulation of SIRT3 protein expression, which enhances mitochondrial redox homeostasis, increases ATP production, and ameliorates mitochondrial dysfunction. Additionally, there is an upregulation of synaptic-related proteins, which contributes to the repair of synaptic damage and enhancement of synaptic function. As hippocampal neuronal function improves, the activation of the TLR4 receptor by pro-inflammatory factors in microglia is reduced, resulting in diminished phosphorylation of NF-κB and decreased activation of the NLRP3 inflammasome towards Caspase-1. Consequently, there is a reduction in the conversion of the IL-1β precursor to its mature form, thereby alleviating hippocampal neuroinflammation and ultimately enhancing the learning and cognitive functions of PS-MPs-exposed mice. The diagram was created with MedPeer ( www.medpeer.cn )

Article Snippet: Specifically, fixed hippocampal neuronal cells were treated with mouse anti-β3-tubulin antibody (1:100, sc80016, Santa Cruz biotech, USA) in combination with the following antibodies: rabbit anti-p-ErbB4 (1:200, bs-3220R, Bioss, Beijing, China), and rabbit anti-PSD95 (1:200, GB11277-100, Servicebio, Wuhan, China).

Techniques: Activation Assay, Expressing

Journal: The Journal of biological chemistry

Article Title: Heregulin activation of extracellular acidification in mammary carcinoma cells is associated with expression of HER2 and HER3.

doi: 10.1074/jbc.270.38.22608

Figure Lengend Snippet: FIG. 3. RT-PCR detection of HER2, HER3, and HER4 mRNA expression in human mammary and ovarian carcinoma cells. Total RNA was isolated and RT-PCR was performed as described under “Materials and Methods.” A, the ethidium bromide-agarose gels of the PCR products using primers for HER2, HER3, and HER4 are shown here. The DNA size standard fX174/HaeIII is also included. B, PCR products from A were blotted to a nylon membrane, probed with the corresponding biotinylated probes, and detected as described under “Materials and Methods.”

Article Snippet: Western Blot Analysis—Cells were analyzed for the expression of HER2 (p185), HER3 (p160), and HER4 (p180) using Western blotting with mouse monoclonal anti-NEU (HER2) SC08, rabbit anti-erbB-3 peptide (HER3) SC285, and rabbit anti-erbB-4 peptide (HER4) SC283 antibodies (Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Membrane