Journal: Biotechnology progress

Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm

doi: 10.1002/btpr.3102

Figure Lengend Snippet: (a) Amino acid sequences of the variable domains of heavy and light chains obtained from Herceptin36 and synthetic linker. (b) Schematic diagram of the pET28a vector containing anti-HER2 scFv antibodies in different orientations.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

Techniques: Plasmid Preparation

Journal: Biotechnology progress

Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm

doi: 10.1002/btpr.3102

Figure Lengend Snippet: (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, Tind=30°C, expressing anti-HER2 scFv-VLVH. Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-VLVH construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

Techniques: Western Blot, Expressing, Binding Assay, Activity Assay, Purification, Construct, Standard Deviation

Journal: Biotechnology progress

Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm

doi: 10.1002/btpr.3102

Figure Lengend Snippet: (a) Soluble/insoluble ratio of anti-HER2 scFv-VLVH expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-VLVH. Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-VLVH were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P-value<0.01 and P-value<0.001, respectively.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

Techniques: Western Blot, Expressing, Recombinant, Produced, Bradford Assay, Software, Standard Deviation

Journal: Biotechnology progress

Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm

doi: 10.1002/btpr.3102

Figure Lengend Snippet: SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-VLVH (lane 2), and pET28a-anti-HER2-scFv-VHVL (lane 4). (b) Purified anti-HER2 scFv-VLVH (lane 5) and anti-HER2 scFv-VHVL (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

Techniques: SDS Page, Construct, Purification, Molecular Weight, Marker

Journal: Biotechnology progress

Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm

doi: 10.1002/btpr.3102

Figure Lengend Snippet: SEC analysis anti-HER2 scFv constructs in different orientations using a Superdex 75 10/300 GL gel filtration column.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

Techniques: Construct, Filtration

Journal: Biotechnology progress

Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm

doi: 10.1002/btpr.3102

Figure Lengend Snippet: Antigen binding of purified anti-HER2 scFv constructs against commercial HER2. Histidine-tagged scFv13-R4 was used as negative control. All data are the average of triplicates and error bars are the standard deviation of the mean.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

Techniques: Binding Assay, Purification, Construct, Negative Control, Standard Deviation

Journal: Biotechnology progress

Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm

doi: 10.1002/btpr.3102

Figure Lengend Snippet: Binding affinity analysis of purified (a) anti-HER2 scFv-VLVH and (b) anti-HER2 scFv-VHVL measured by SPR. Commercial HER2 was immobilized on CM5 sensor chip and the response of different concentrations of anti-HER2 scFv constructs (ranging from 0.625–80 nM) was compared with an empty flow cell. Equilibrium binding responses at each anti-HER2 scFv concentration were plotted with binding curve obtained from the Hill slope non-linear regression analysis. The calculated Kd values are given in the table.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

Techniques: Binding Assay, Affinity Purification, Construct, Concentration Assay

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) Significant mRNA overexpression of t-DARPP and ERBB2 in adenocarcinomas of the esophagus and stomach (141 tumors and 51 normal tissue samples) (p<0.001). B) Spearman’s correlation coefficient and correlation test where the cutoff gene expression is ≥log(5,2)=2.32, show that t-DARPP and ERBB2 overexpression levels are significantly correlated in tumors (r=0.58, p=0.003). C) The multivariate regression model analysis indicates that tumor stage has a significant effect on t-DARPP mRNA gene expression levels (p=0.02). D) Left panel, cell viability of OE19 and OE33 cells in response to trastuzumab treatment was evaluated by Trypan blue staining. OE19 cells were two-fold more sensitive to trastuzumab than OE33 cells (p<0.001). Right panel, Western blot analysis demonstrates higher protein expression of ERBB2 in OE19 cells than OE33 cells. In contrast, t-DARPP expression was undetectable in OE19 cells but highly expressed in OE33 cells.

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Over Expression, Expressing, Staining, Western Blot

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) ERBB2 protein stability in OE19 cells stably expressing t-DARPP or pcDNA3 empty vector was evaluated by Western blot analysis after treatment with 80 μg/ml CHX to block new protein synthesis for the indicated times. The protein degradation data indicate that t-DARPP expression extended the protein half-life of ERBB2 from 30.8 h to 42.2 h relative to control (lower panel). B) ERBB2 protein stability in parental and trastuzumab resistant OE19 cells was assessed by Western blot analysis after treatment with CHX (80 μg/ml) for the indicated times. The protein degradation data show that endogenous t-DARPP expression in resistant cells was associated with increased ERBB2 protein half-life (60.8 h) relative to parental cells (31.3 h) (lower panel). C) Western blot analysis of p-ERBB2 (Y1248), ERBB2, p-AKT (S473), AKT, and t-DARPP proteins in OE19 cells infected with control (10 MOI) or t-DARPP (10 MOI) adenoviruses after treatment with vehicle or trastuzumab (20 μg/ml) for 24 h. The data indicate that transient expression of t-DARPP increased p-ERBB2(Y1248) and p-AKT(S473) basal protein levels, and blocked trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins. D) Western blot analysis of p-ERBB2 (Y1248), ERBB2, p-AKT (S473), AKT, and t-DARPP proteins in OE19 cells stably expressing t-DARPP or pcDNA3 vector after treatment with vehicle or trastuzumab (20 μg/ml) for 24 h. The results show that stable expression of t-DARPP increased basal levels of p-ERBB2(Y1248) and p-AKT(S473), and inhibited trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins. E) Western blot analysis of p-ERBB2(Y1248), ERBB2, p-AKT(S473), AKT, and t-DARPP proteins in parental or trastuzumab resistant OE19 cells following treatment with vehicle or trastuzumab (20 μg/ml) for 24 h. The results indicate that endogenous t-DARPP expression was associated with increased basal levels of p-ERBB2(Y1248) and p-AKT(S473), and suppression of trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins.

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Blocking Assay, Infection, De-Phosphorylation Assay

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) Western blot analysis of p-ERBB2(Y1248), ERBB2, p-AKT(S473), AKT, and t-DARPP proteins in OE33 cells transfected with control siRNA or t-DARPP siRNA and treated with vehicle or trastuzumab (20 μg/ml) for 48 h. The data indicate that knockdown of endogenous t-DARPP increased trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins. B) Cell viability of OE33 cells transfected with control siRNA or t-DARPP siRNA in response to treatment with vehicle or trastuzumab (20 μg/ml) for 48 h, was evaluated by CellTiter-Glo Luminescent CellViability Assay. The results revealed that knockdown of endogenous t-DARPP with treatment induced a significant decrease in cell survival (p<0.01).

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Western Blot, Transfection, De-Phosphorylation Assay

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) Western blot analysis of co-immunoprecipitated exogenous t-DARPP and endogenous ERBB2 proteins with M2-flag or trastuzumab antibodies in OE19 cells infected with t-DARPP-flag adenovirus (10 MOI). The data demonstrate protein association of ERBB2 with t-DARPP. B) Western blot analysis of immunoprecipitated endogenous ERBB2 protein with trastuzumab antibody in OE19 cells infected with control (10 MOI) or t-DARPP (10 MOI) adenoviruses. Pulled-down ERBB2 band intensity was depicted as a ratio relative to input ERBB2 protein. The results show that exogenous t-DARPP expression blocked binding of trastuzumab to ERBB2 receptor relative to control. C) Western blot analysis of immunoprecipitated endogenous ERBB2 protein with trastuzumab antibody in parental or trastuzumab resistant OE19 cells. The band intensity of immunoprecipitated ERBB2 protein was shown as a ratio relative to input ERBB2. The data indicate that endogenous t-DARPP expression in trastuzumab-resistant cells was associated with a significant decrease in trastuzumab/ERBB2 protein interaction relative to control.

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Western Blot, Immunoprecipitation, Infection, Expressing, Binding Assay

Journal: PLoS ONE

Article Title: Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

doi: 10.1371/journal.pone.0088983

Figure Lengend Snippet: (A) 8MOP interacts with three peptide regions within the ErbB2 catalytic kinase domain. Qualitative peptide identifications within the ErbB2 catalytic kinase domain following LC-MS/MS analysis of a streptavidin pull-down of biotinylated-8MOP bait (see ). The transmembrane domain is indicated (red diamond) and the five C-terminus tyrosine autophosphorylation sites are indicated (p). (B) Non-reducing Western blot analysis of the interaction of 8MOP with ErbB2. BT474 cells were treated with 800dye-8MOP (Promega) or with vehicle (0.01% DMSO) alone served as control for 48 hr and then exposed to UV irradiation (2J) prior to Western blot analysis. The image on the left shows the Western blot for ErbB2 (red). The image on the right shows the same membrane directly scanned for the presence of 800dye-8MOP (green), which overlays the ErbB2 signal. The results are representative of three independent experiments.

Article Snippet: ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, Irradiation, Membrane

Journal: PLoS ONE

Article Title: Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

doi: 10.1371/journal.pone.0088983

Figure Lengend Snippet: The growth and viability of BT474 and SKBR3 cells (top bar graphs) after being subjected to the indicated treatment conditions. The combination of PUVA plus neratinib: P <0.0005 (BT474 and SKBR3 cells). Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments. (B) Western blot analysis showing steady-state ErbB2, ErbB3, and phospho-Akt (S473) protein levels in BT474 and SKBR3 cells treated according to the indicated treatment conditions. Vehicle alone (0.01% DMSO) served as a control. Steady-state actin protein levels served as a control for equal loading of protein. The results are representative of three independent experiments.

Article Snippet: ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Western Blot, Control

Journal: PLoS ONE

Article Title: Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

doi: 10.1371/journal.pone.0088983

Figure Lengend Snippet: Top bar graph shows the results of the growth assays performed in T47D and stably transfected T47D cell line. T47D cells expressing p85 ErbB2 were pretreated with 5 µM lapatinib or 5 µM 8MOP for 4 hr followed by irradiation in a UV Stratalinker 1800 (Statagene). Cells transfected with empty vector (T47D/Vector), and those treated with vehicle alone (0.01% DMSO) served as controls. The effects of the treatments on cell growth and viability are shown in the bar graph. P<0.0071 (8MOP + UVA irradiation). Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments. Steady-state phospho-p85 ErbB2 protein levels (dotted arrow) and phospho-p185 ErbB2 (solid arrow) are shown by Western blot. Actin steady-state protein levels served as a control to ensure for equal loading of protein. Results are representative of three independent experiments.

Article Snippet: ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Stable Transfection, Transfection, Expressing, Irradiation, Plasmid Preparation, Western Blot, Control

Journal: Cancers

Article Title: Transcriptomic Changes Associated with ERBB2 Overexpression in Colorectal Cancer Implicate a Potential Role of the Wnt Signaling Pathway in Tumorigenesis

doi: 10.3390/cancers15010130

Figure Lengend Snippet: Validation of successful ectopic overexpression of ERBB2 in the CRC (HCT116 and HT29) and normal colon (CCD33 and CCD841) cell lines. ( A ) Relative ERBB2 expression in the normal colon cell lines CCD33 and CCD841 and the CRC cell lines HT29 and HCT116 as determined by qRT-PCR. Data were normalized to the expression of the internal control 18S rRNA gene and fold expressions were plotted relative to expression in the CCD33 cells. Data represent the mean ± SD of three independent experiments. ( B ) Relative ERBB2 expression in non-transfected and either empty pcDNA3 vector or pcDNA3- ERBB2 transfected HCT116, HT29, CCD33, and CCD841 cells as determined by qRT-PCR. Data were normalized to the expression of the internal control 18S rRNA gene and fold expressions were plotted relative to expression in the non-transfected cells. Data represent the mean ± SD of three independent experiments. *** p < 0.001; ns: not significant. ( C ) Same as B, but relative HER2 protein expression was determined in the different experimental conditions. Blots were re-probed with anti-β-Actin antibody to confirm equal loading across the lanes. The representative blots from three independent experiments are shown.

Article Snippet: The wild-type ERBB2 expression construct was a gift from Mien-Chie Hung (Addgene plasmid #16257; https://www.addgene.org/16257 , accessed on 15 December 2020) [ ].

Techniques: Biomarker Discovery, Over Expression, Expressing, Quantitative RT-PCR, Control, Transfection, Plasmid Preparation

Journal: Cancers

Article Title: Transcriptomic Changes Associated with ERBB2 Overexpression in Colorectal Cancer Implicate a Potential Role of the Wnt Signaling Pathway in Tumorigenesis

doi: 10.3390/cancers15010130

Figure Lengend Snippet: Heatmap of the differentially expressed genes in CRC (HCT116 and HT29) and normal colon (CCD33 and CCD841) cell lines transfected with empty vector or ERBB2, either clustered based on expression ( A ) or grouped based on transfection and phenotype ( B ).

Article Snippet: The wild-type ERBB2 expression construct was a gift from Mien-Chie Hung (Addgene plasmid #16257; https://www.addgene.org/16257 , accessed on 15 December 2020) [ ].

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: Cancers

Article Title: Transcriptomic Changes Associated with ERBB2 Overexpression in Colorectal Cancer Implicate a Potential Role of the Wnt Signaling Pathway in Tumorigenesis

doi: 10.3390/cancers15010130

Figure Lengend Snippet: Genome-wide gene expression changes between ERBB2 + and ERBB2 − CRC patients. ( A ) Principal component analysis (PCA) was performed to determine batch effects among the 14 patients’ samples. Comparison of PC1 and PC2 variation sequestered the samples based on ERBB2 expression. ( B ) Volcano plot of differentially expressed genes between ERBB2 - and ERBB2 + patients’ samples from input RNA-seq. Genes that are expressed significantly higher and lower based on log2 fold change in HER2+ samples are highlighted by green and blue dots, respectively. Unchanged transcripts are demarcated as grey circles ( p > 0.05). ( C ) Heatmap of the top 100 differentially expressed genes.

Article Snippet: The wild-type ERBB2 expression construct was a gift from Mien-Chie Hung (Addgene plasmid #16257; https://www.addgene.org/16257 , accessed on 15 December 2020) [ ].

Techniques: Genome Wide, Gene Expression, Comparison, Expressing, RNA Sequencing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Differential regulation of human monocytes and NK cells by antibody-opsonized tumors

doi: 10.1007/s00262-018-2179-z

Figure Lengend Snippet: Monocyte phagocytosis is increased with HER2 gene-amplified targets. Antibody-dependent phagocytosis, degranulation, and tumor cell death were evaluated on HER2-amplified and -non-amplified tumor cell lines. a Titration curves of antibody-dependent phagocytosis, degranulation, and tumor cell death from SKBR3 (HER2 amplified) and T47D (HER2 non-amplified) tumor cell lines. All samples were performed in triplicate; means ± SEM and regression line are plotted on the graph. Two-way ANOVA with Sidak’s multiple comparisons test was used to determine significance. Asterisk represents significance between SKBR3 and T47D. b Comparison between antibody-dependent phagocytosis, degranulation, and tumor cell death of breast cancer cell lines that are amplified and non-amplified for HER2. Each point on the graph represents a different cell line and is the mean of a minimum of two experiments at the trastuzumab concentration of 316 ng/ml. Two tailed t tests were used to obtain P values for phagocytosis (P < 0.03), NK cell degranulation (P < 0.01), and tumor cell death (P > 0.8)

Article Snippet: The MCF-7 cell line was modified with pcDNA3.1 (V79020), purchased from Thermo Fisher Scientific (Waltham, MA), or pcDNA3 HER2 WT, which was a gift from Mien-Chie Hung (Addgene plasmid # 16257) [ 20 ].

Techniques: Amplification, Titration, Comparison, Concentration Assay, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Differential regulation of human monocytes and NK cells by antibody-opsonized tumors

doi: 10.1007/s00262-018-2179-z

Figure Lengend Snippet: Antigen density is important for phagocytosis. The MCF-7 breast cancer cell line was transfected with pcDNA HER2, and individual clones were selected by serial dilution. a Characterization of the protein expression by flow cytometry. Filled histogram is pcDNA HER2, bold line is empty vector, and normal line is unstained control. b Phagocytosis was performed as previously described in Fig. 1. Phagocytosis was performed in triplicate; means ± SEM and regression line are plotted on the graph. Two-way ANOVA with Sidak’s multiple comparisons test was used to determine significance. Asterisk represents significance between MCF7-HER2 and MCF7-Vector

Article Snippet: The MCF-7 cell line was modified with pcDNA3.1 (V79020), purchased from Thermo Fisher Scientific (Waltham, MA), or pcDNA3 HER2 WT, which was a gift from Mien-Chie Hung (Addgene plasmid # 16257) [ 20 ].

Techniques: Transfection, Clone Assay, Serial Dilution, Expressing, Flow Cytometry, Plasmid Preparation, Control

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A A gene-centric heatmap representing copy number abundance (a minimum of 10% frequency in at least one single type of cancer). Color legend represents % frequency. B HEK293 cells were co-transfected with Super Nanoluciferase reporter vectors containing non-ARE control or RPS30-nLuc-ARE reporter together with of control Firefly luciferase vector, for 18 h. Cells were re-seeded into 96-well microplates and were transfected with empty vector control or ERBB2 -HA vector for 18 h. Cells were lysed, and luciferase activity was quantitated as the ratio of Nanoluciferase/Firefly luciferase intensity. The screen is at least from two independent experiments with Mean ± SEM of triplicate readings. ANOVA with Dunnett’s multiple comparisons was used to compare the effects of each of the indicated vectors and the empty vector on ARE-reporter readings. ** p < 0.01, *** p < 0.001, **** p < 0.0001. C Cancer-centric heatmap representing clustering of cancer type according to copy number variations.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Transfection, Luciferase, Plasmid Preparation, Activity Assay

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A The ERBB2-amplification associated expressed genes (≥1.7 fold, Q value <0.00005) were intersected with the AU-rich element database (ARED-Plus). B Functional enrichment of the ERBB2-associated ARE-coding genes. The most significant results with false discovery rate (FDR) P < 0.05 are shown. C Left panel: the mean of mRNA abundance for 160 ARE-coding genes (Fig. data) was compared between TCGA ERBB2-negative and ERBB2-positive patients’ tissue samples. Data are Mean ± SEM. *** p < 0.001 using Student’s t-test with Welch’s correction. Right panel: the interaction between the ARE-mRNA cluster or all mRNAs with ERBB2-positive gene expression data is represented by an odd ratio using Fisher’s exact test (Oncomine software). D Total RNA from the ERBB2-positive SKBR3 cell line and MCF10A normal-like ERBB2-negative cell lines were extracted and subjected to RT-QPCR using specific primers to the indicated genes. Inset shows an immunoblot of two for the ERBB2 protein among different types of breast cancer cell lines. E The SKBR3 cells were transfected with 200 nM control siRNA or ERBB2 siRNA for 24 h, followed by RNA extraction and RT-QPCR as indicated. Inset is a representative immunoblot of three, for the ERBB2 protein. The data in D and E are normalized to the housekeeping gene, RPL0. Data are Mean ± SEM (triplicate) from one representative experiment of at least two. ANOVA test demonstrates overall statistical significance. The student’s t-test was used to compare expression data for each gene.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Amplification, Functional Assay, Expressing, Software, Quantitative RT-PCR, Western Blot, Transfection, RNA Extraction

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A SKBR3 cells were treated with increasing doses of lapatinib for 24 h, and lysed for immunoblotting with anti-phospho ERBB2 protein and β-actin as a loading control. WB is one representative from three blots. B RT-QPCR expression analysis for the ERBB2-regulated ARE-mRNAs in SKBR3 cells (left panel) or MCF-10A cells ( right panel ) treated with DMSO or Lapatinib (300 nM) for overnight. Data are normalized to the housekeeping gene, RPL0. The normalized values were used to calculate % remaining relative to DMSO control. Statistical analysis was performed using two-tailed Student’s t -test for each gene reduced in expression in comparison to DMSO data. Data are Mean ± SEM (triplicate of one experiment of three). ** P < 0.001, *** P < 0.0001. C RT-QPCR for Nanoluciferase mRNA abundance or Nanoluciferase protein activity ( D ) in SKBR3 cells transfected with nLuc-ARE or control nLuc-non-ARE expression plasmid for overnight, and then treated with DMSO (control) or lapatinib for 6 h or 24 h, respectively. Data are presented as the mean ± SEM ( n = 2 experiments). ANOVA is used to compare overall significance while post hoc sidak’s test compares the DMSO and lapatinib data. *** p < 0.0001. ** p < 0.005, Student’s test. E The mRNA half-life determination in response to lapatinib. The SKBR3 cells were treated with DMSO or lapatinib (300 nM) overnight, then actinomycin D (AcD) (5 μg/ml) was added for the indicated time points. Total RNA was extracted, and cDNA samples were subjected to RT-QPCR using specific primers to the indicated genes. One-phase exponential decay curves for relative mRNA half-life measurements in the two treated groups are shown. Data are normalized to RPL0 transcript abundance, and the normalized values were used to calculate the mRNA half-life as a percentage to time 0. Data are presented as the mean ± SEM ( n = 2 experiments). F , G Immunoblot analysis for CDC6 and NEK2 protein abundance in SKBR3 cells treated as in ( A ). Representative blot each is from at least two experiments is shown.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Two Tailed Test, Activity Assay, Transfection, Plasmid Preparation

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A immunoblot analysis for ZFP36/TTP phosphorylated protein abundance in MCF10A and SKBR3 using specific primers to ZFP36/TTP and β-Actin protein as a loading control. B SKBR3 cells were transfected with an empty vector or ZFP36/TTP expression vector. Left panel: proteins were extracted, and immunoblotting was performed using a ZFP36/TTP antibody. Right panel: protein lysates were first treated with the phosphatase, CIP, or CIP phosStop C before immunoblotting with anti-ZFP36/TTP. The WB shows both phosphorylated and unphosphorylated bands. WB was performed with low exposure to avoid high background and overexposed signal of the transfected TTP (lower panel). D) SKBR3 cells were transfected with 200 nM ERBB2 siRNA or control siRNA for overnight, and ERBB2 and ZFP36/TTP protein abundance were assessed by immunoblotting. A representative blot from at least two experiments is shown. Immunoblot analysis of ERBB2 and ZFP36/TTP protein phosphorylation abundance in MCF10A ( E ) or MCF-7 ( F ) transfected with ERBB2 expression vector or empty vector. Representative blots are from at least three experiments.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A Dose-dependent activity of lapatinib on the abundance of phosphorylated ZFP36/TTP in the ERBB2-positive SKBR3 cells. The relative amounts of the proteins from three immunoblots were quantified using Image J software ( left panel ). Fold changes (Mean ± SEM, n = 3) of phosphorylated ZFP36/TTP abundance normalized to β-Actin using Image J program (right panel). B Effect of ERBB2 inhibition on ZFP36/TTP protein stability. SKBR3 cells were treated with lapatinib (300 nM) for 16 h, followed by 10 µg/ml of cycloheximide (CHX) treatment for the indicated durations (left panel). Protein lysates were subjected to immunoblotting with anti-TTP. Right panel: quantification of ZFP36/TTP abundance, normalized to GAPDH, was determined using ImageJ software and the protein half-life was assessed using the Graph Prism. Data are mean +/− SEM of two experiments. C Effect of lapatinib on the abundance of ZFP36/TTP protein in the ERBB2-positive cells, BT-474. WB is a representative of two blots from independent experiments.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Activity Assay, Western Blot, Software, Inhibition

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A Immunoblot analysis of ERBB2, AKT, MEK1/2, and ERK1/2 signaling inhibition after treatment with increasing doses of lapatinib for 24 h. Status of ERBB2 (phospho-tyrosine 1221/1222 and total), AKT (phospho-serine 473 and total pan AKT), ERK1/2 (Phospho-threonine 202/phospho-tyrosine 204 and total), MEK1/2 (Ser217/221), and ZFP36/TTP are shown. A blot from at least two experiments is shown. B Immunoblot analysis for the inhibition of MK2 activation after treatment with lapatinib. SKBR3 cells were treated with increasing doses of lapatinib, as indicated for 24 h. Phospho-threonine MK2 (T222) and total MK2 protein abundance are shown. A representative blot from two experiments is shown. C Immunoblot analysis for ZFP36/TTP phosphorylation in MAPKAPK-2 silenced SKBR3 cells. SKBR3 cells transfected with 100 nM of si Ctrl or si MAPKAPK-2 for 24 h were tested for ZFP36/TTP protein abundance. Total MK2, phosphorylated ZFP36/TTP, and β-actin protein abundances are shown. A representative blot from three experiments is shown. Effect of the MK2 inhibitor PF-3644022 (1 µM, 4 h) on the abundance of phosphorylated ZFP36/TTP in ERBB2-expressing SKBR3 ( D ) or SKBR3 transfected with ZFP36 expression plasmid ( E ). Western blots are from two independent experiments. F Immunoblot analysis of total and phosphorylated ZFP36/TTP, and MK2 in MK2 -knockout MEF cells in the absence or presence of overnight transfected ERBB2.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Western Blot, Inhibition, Activation Assay, Transfection, Expressing, Plasmid Preparation, Knock-Out

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A HEK293 cells were co-transfected with ERBB2 -HA along with ZFP36/TTP -HA or vector control (0.5 µg each) for 24 h. Proteins were extracted, and immunoblotting was performed using antibodies to ERBB2, ZFP36/TTP, and β-actin. A representative blot from three experiments is shown. B Total RNA samples were extracted and subjected to RT-QPCR using specific primers to the ERBB2-regulated ARE-mRNA s (as in Fig. D, E). Data are Mean ± SEM of average 18SRNA normalized expression of ARE-mRNA abundance remaining from vector control. C , D RT-QPCR using specific primers to the indicated genes RT-QPCR using specific primers to the indicated genes. Data in B to D are Mean ± SEM from an experiment with triplicate reactions of two independent experiments. Two-way ANOVA with Tukey’s multiple tests between the control and treatment test as indicated were used. *** p < 0.001. E Immunoblot analysis for ZFP36/TTP protein in HEK293 cells over-expressing cancer-amplified genes. HEK293 cells were co-transfected with cancer amplified genes and ZFP36/TTP -HA or vector control (0.5 µg each) for 24 h. The results of the two experiments are shown.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Expressing, Amplification

Journal:

Article Title: Improved Hepatic Gene Transfer by Using an Adeno-Associated Virus Serotype 5 Vector

doi: 10.1128/JVI.76.20.10497-10502.2002

Figure Lengend Snippet: Estimation of vector gene copy number in AAV-transduced livers by quantitative PCR (3 months after vector administration). A 350-bp fragment of the hF.IX cDNA as present in the AAV-EF1α-hF.IX vector was coamplified with a 1.1-kb fragment from the endogenous murine HPRT gene using biotinylated primers (20 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, separated on a 2% agarose gel, transferred to a nylon membrane, and visualized with the Southern light detection system from Applied Biosystems. Template for PCR was as follows: 100 ng of genomic DNA extracted from several random pieces of liver and subsequently combined for each individual animal. Each sample column represents an individual animal. standards, linearized plasmid pAAV-EF1α-hF.IX (0.01, 0.1, or 1 pg) mixed with 100 ng of genomic mouse DNA (extracted from untransduced animal); NC, negative control (template, genomic DNA from untransduced animal). Bands were analyzed by densitometric scanning, and intensities were quantitated with NIH Image 6.16 software. Shown is one representative blot. Ratios of band intensities (hF.IX band/mAAT band) are for the blot shown. Gene copy number estimates are average for two experiments.

Article Snippet: A 350-bp fragment of the hF.IX cDNA as present in the AAV-EF1α-hF.IX vector was coamplified with a 1.1-kb fragment from the endogenous murine HPRT gene using biotinylated primers (20 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, separated on a 2% agarose gel, transferred to a nylon membrane, and visualized with the Southern light detection system from Applied Biosystems.

Techniques: Plasmid Preparation, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control, Software

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 regulates the expression of HER receptors and the activities of their downstream signaling pathways in gastric cancer cells. ( a ) qRT-PCR assay was used to evaluate mRNA expression of EHF and HER receptors in primary gastric cancers ( n =30). Linear regression analysis was performed to assess the correlations between them. 18S rRNA was used as a normalized control. ( b ) qRT-PCR assay was performed to investigate the effect of EHF knockdown on the expression of HER receptors. Expression levels of these genes were normalized with 18S rRNA levels. Data were presented as mean±S.E. ( c ) The effect of EHF depletion on the expression of HER2-4 was determined in the indicated cells by western blot analysis. GAPDH was used as loading control. ( d ) Cells transfected with si-EHF-979 or si-NC were lysed and lysates were subjected to western blot analysis. The antibodies against phospho-Erk (p-Erk), total Erk (t-Erk), phospho-Akt (p-Akt) and total Akt (t-Akt) were used to determine the effect of EHF knockdown on the activities of the MAPK/Erk and PI3K/Akt cascades. GAPDH was used as a loading control

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: Expressing, Quantitative RT-PCR, Western Blot, Transfection

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: 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: HER2 depletion attenuates proliferation-promoting effect of EHF in gastric cancer cells. Inhibitory effect of HER2 depletion on cell proliferation in GES-1 and MGC803 cells overexpressing EHF were evaluated by MTT assay. The data were presented as mean±S.E. 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: MTT Assay