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: CX3CL1 Overexpression Prevents the Formation of Lung Metastases in Trastuzumab-Treated MDA-MB-453-Based Humanized Tumor Mice (HTM)

doi: 10.3390/cancers13102459

Figure Lengend Snippet: CX3CL1 and HER2 shedding and expression in trastuzumab and TMI-1 treated MDA-MB-453 tumor cells. MDA-MB-453 and SK-BR-3 cells were stably transfected with human Cx3CL1 (Cx3CL1 + ) or vector control (empty). ( A ) SK-BR-3/MDA-MB-453 CX3CL1 and SK-BR-3/MDA-MB-453 empty were stained for ADAM 10 expression using western blot technology. MDA-MB-453 CX3CL1 and MDA-MB-453 empty were cultured with a total concentration of 5 µg/mL trastuzumab (Trast) and 10 µM TMI-1 for 48 h and supernatants were analyzed for CX3CL1 ( B ) or HER2 extracellular domain (HER2 ECD) ( C ) in comparison to DMSO treated control cells by ELISA. ( D ) HER2 expression intensity of the treated and untreated MDA-MB-453 cells was analyzed by flow cytometry and the stain index (MFI = mean fluorescence intensity; stain index = (MFI Her2 − MFI Isotype )/(2 (×) SD Isotype )) was calculated. ( E ) MFI and the stain index of the anti-HER2 staining after 60 min of trastuzumab pre-incubation (with increasing concentration: 0–10 µg/mL) on ice are displayed. All experiments were performed in triplicate. Data are shown as mean ± SEM and Tukey’s multiple comparisons test was applied and significances are indicated (** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: For the anti-HER2–trastuzumab competition assay, cells were stained for 60 min on ice with increasing trastuzumab concentrations (0–10 µg/mL), washed and afterward incubated for 20 min with the anti-HER2 FITC antibody (clone 24D2, BioLegend, San Diego, CA, USA).

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Control, Staining, Western Blot, Cell Culture, Concentration Assay, Comparison, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Incubation

Journal: Cancers

Article Title: CX3CL1 Overexpression Prevents the Formation of Lung Metastases in Trastuzumab-Treated MDA-MB-453-Based Humanized Tumor Mice (HTM)

doi: 10.3390/cancers13102459

Figure Lengend Snippet: Treatment efficiency of trastuzumab and TMI-1 in MDA-MB-453-transplanted HTM. MDA-MB-453 CX3CL1 and MDA-MB-453 empty were transplanted orthotopically in humanized NSG mice. Animals were randomized to receive trastuzumab (Trast), TMI-1, Trast+TMI-1 or DMSO control. ( A ) Immunhistological staining of tumor tissue (exemplarily shown for a one-time trastuzumab-treated HTM) for human HER2, cytokeratin 18, T helper (CD4), cytotoxic (CD8) T cells, macrophages (CD68) and B cells (CD20 in the periphery of the tumor and in the connective tissue). Red arrows indicate CD20 + -stained cells. Tumor growth over time in MDA-MB-453 CX3CL1 . Scale bar represents 50 μm. ( B ) and MDA-MB-453 empty ( C ) in the different treatment groups is shown and summarized in ( D ). Tumor volume ( E ) and tumor weight ( F ) of the treated HTM and the correlation ( G ) between both parameters were analyzed (Pearson’s correlation coefficient r = 0.9008). ( D – F ) Data are shown for HTM, which achieved the end of the experiments (control n = 6; Trast n = 6, TMI-1 n = 5; TMI-1 + Trast n = 5) as mean ± SEM. Dunnett’s multiple comparison test ( D – F ; * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001) and Sidak’s multiple comparisons test ( D , red bars; * p = 0.037) were applied and significances are indicated.

Article Snippet: For the anti-HER2–trastuzumab competition assay, cells were stained for 60 min on ice with increasing trastuzumab concentrations (0–10 µg/mL), washed and afterward incubated for 20 min with the anti-HER2 FITC antibody (clone 24D2, BioLegend, San Diego, CA, USA).

Techniques: Control, Staining, Comparison

Journal: Cancers

Article Title: CX3CL1 Overexpression Prevents the Formation of Lung Metastases in Trastuzumab-Treated MDA-MB-453-Based Humanized Tumor Mice (HTM)

doi: 10.3390/cancers13102459

Figure Lengend Snippet: Tumor cell phenotyping and HER2 shedding detected in the serum of MDA-MB-453 HTM. Humanized NSG mice were transplanted with 1 × 10 6 MDA-MB-453 CX3CL1 and MDA-MB-453 empty and tumor cells were analyzed by flow cytometry. ( A , B ) Histogram of tumor cells stained with HER2, CD47, cMET, CD24 and CD44 are displayed ( A : isotype control = grey; tumor isolated from HTM = black; B : cell culture = grey; tumor isolated from HTM = black). Dot plot shows the low percentage of CD44 + MDA-MB-453 tumor cells from the cell culture. ( C ) Percentage of CD44 expression on HER2+ tumor cells from cell culture compared with tumor cells isolated from the primary tumor of HTM are summarized. ( D ) Shed serum HER2 extracellular domain (sHER2 ECD) was detectable in the serum of HTM. Data are shown as mean ± SEM and significances were calculated using Tukey’s multiple comparisons test (* p < 0.05). ( E ) Graph outlines the correlation between sHER2 ECD and tumor volume (Pearson’s correlation coefficient r = 0.4130).

Article Snippet: For the anti-HER2–trastuzumab competition assay, cells were stained for 60 min on ice with increasing trastuzumab concentrations (0–10 µg/mL), washed and afterward incubated for 20 min with the anti-HER2 FITC antibody (clone 24D2, BioLegend, San Diego, CA, USA).

Techniques: Flow Cytometry, Staining, Control, Isolation, Cell Culture, Expressing

Journal: Cancers

Article Title: CX3CL1 Overexpression Prevents the Formation of Lung Metastases in Trastuzumab-Treated MDA-MB-453-Based Humanized Tumor Mice (HTM)

doi: 10.3390/cancers13102459

Figure Lengend Snippet: Metastases formation in MDA-MB-453-transplanted humanized tumor mice (HTM). (A) Immunohistochemical HER2 staining of a lung tissue exemplarily shown in a TMI-1-treated MDA-MB-453 CX3CL1 -transplanted HTM. Red arrows indicate tumor cells in the lung tissue. Metastases formation in the lung of HTM were analyzed by flow cytometry using an anti-HER2 antibody ( B ). ( Ci–iii ) Analysis of the bone marrow (bm) samples from the femurs revealed two different tumor cell populations (EPCAM + HER2 - and EPCAM - HER2 + ). CD44 expression on EPCAM - HER2 + ( D ) and EPCAM + HER2 - ( E ) were analyzed using a multicolor panel. Data are shown as mean ± SEM, and significances were analyzed using Tukey’s multiple comparisons (* p < 0.05; ** p < 0.01; **** p < 0.0001).

Article Snippet: For the anti-HER2–trastuzumab competition assay, cells were stained for 60 min on ice with increasing trastuzumab concentrations (0–10 µg/mL), washed and afterward incubated for 20 min with the anti-HER2 FITC antibody (clone 24D2, BioLegend, San Diego, CA, USA).

Techniques: Immunohistochemical staining, Staining, Flow Cytometry, Expressing

Journal: Cancers

Article Title: CX3CL1 Overexpression Prevents the Formation of Lung Metastases in Trastuzumab-Treated MDA-MB-453-Based Humanized Tumor Mice (HTM)

doi: 10.3390/cancers13102459

Figure Lengend Snippet: Detection of tumor cells in the lung of control versus treated HTM.

Article Snippet: For the anti-HER2–trastuzumab competition assay, cells were stained for 60 min on ice with increasing trastuzumab concentrations (0–10 µg/mL), washed and afterward incubated for 20 min with the anti-HER2 FITC antibody (clone 24D2, BioLegend, San Diego, CA, USA).

Techniques: Control

Journal: Cancers

Article Title: CX3CL1 Overexpression Prevents the Formation of Lung Metastases in Trastuzumab-Treated MDA-MB-453-Based Humanized Tumor Mice (HTM)

doi: 10.3390/cancers13102459

Figure Lengend Snippet: Detection of tumor cells in the bm of control versus treated HTM.

Article Snippet: For the anti-HER2–trastuzumab competition assay, cells were stained for 60 min on ice with increasing trastuzumab concentrations (0–10 µg/mL), washed and afterward incubated for 20 min with the anti-HER2 FITC antibody (clone 24D2, BioLegend, San Diego, CA, USA).

Techniques: Control

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: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A, B) Cells were transfected with HER2 or control (CON) siRNA for 72 hours and then analyzed by Western blot analysis for HER2, PARP1, and PARP2 protein levels. β-actin was used as a loading control. MDA-MB-231 cells that (C) transiently or (D) stably express the HER2 vector (231 HER2) as compared to cells expressing the control plasmid (231 NEO) were subjected to Western blot analysis for HER2, PARP1, PARP2, and β-actin. Data shown are representative immmunoblots from one of two independent experiments.

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

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

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: Expression of the Let-7 miRNA family is altered after HER2 overexpression in breast cancer cells lines.

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Expressing, Over Expression

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: Ten nanograms of isolated miRNA from 70% confluent (A) 231 NEO and 231 HER2 or (B) MCF7, BT-474, and SKBR3 human breast cancer cells were reverse transcribed to cDNA and then analyzed by qRT-PCR for let-7a, let-7i, and U6 expression. The figures are representative from (A, B) one of two independent experiments performed in triplicate. A t-test or one-way ANOVA test was performed. (C) Western blot analysis of HER2, PARP1, PARP2 and β-actin levels in untreated 231 NEO, 231 HER2, MCF7, BT-474, and SKBR3 cell lines. **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Isolation, Reverse Transcription, Quantitative RT-PCR, Expressing, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A–C) BT-474, SKBR3 and 231 HER2 cells were transiently transfected with a let-7a miRNA (hsa-let-7a-5p) or its neg control miRNA (negative control). Following transfection, cells were analyzed by Western blot analysis for PARP-1, PARP-2, and β-actin. (D) The 231 NEO cell line was transiently transfected with an anti-let-7a miRNA (let-7a antagomiR) or a neg control miRNA (negative control). Following transfection, cells were analyzed by Western blot analysis for PARP-1, PARP-2, and β-actin. Results shown are from one of two independent experiments performed duplicate.

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Transfection, Control, Negative Control, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A) The PARP1 3’UTR was aligned with the seed sequence of the let-7a miRNA. (B, C) BT-474 and 231 HER2 cells were co-transfected with a let-7a miRNA or its neg control miRNA and a plasmid containing a firefly luciferase gene and the 3’UTR of PARP1 or a control 3’UTR. (D) 231 NEO cell line was co-transfected with an anti-let-7a miRNA or its neg control miRNA and a plasmid containing a firefly luciferase gene and the 3’UTR of PARP1 or a control 3’UTR. Luciferase activity was normalized to cells co-transfected with the respective neg control miRNA and the plasmid containing the control 3’UTR. The fold change values were calculated back to the cells co-transfected with the respective neg control miRNA and the plasmid containing the 3’UTR of PARP1. Results shown are from one of two independent experiments performed in quadruplicate. *p<0.05, **p<0.01

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Sequencing, Transfection, Control, Plasmid Preparation, Luciferase, Activity Assay

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A) Let-7a levels were measured by qRT-PCR analysis in HER2+ and HER2− breast cancer specimens. U6 was used to normalize data. (B) Let-7a gene and PARP1 protein expression levels were correlated in 17 UAB HER2− and HER2+ breast cancer patients (rs=−0.6937, p=0.0020). ***p<0.001

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Quantitative RT-PCR, Expressing

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A) BT-474 and (B) 231 HER2 cells were transiently transfected with a let-7a miRNA or its negative control. Forty-eight hours after transfection, the cells were treated with DMSO or 10µM ABT-888 for 96 hours and then analyzed by a cellular proliferation assay. Results shown are from one of two independent experiments performed in quadruplicate. *p<0.05, **p<0.01, and ***p<0.001

Article Snippet: The following are the primary antibodies used in this study: anti-human HER2 (2165S; Cell Signaling, Danvers, MA, USA), PARP1 (9532S; Cell Signaling, Danvers, MA, USA), PARP2 (ab176330; Abcam, Cambridge, MA, USA), c-Myc (5605S; Cell Signaling, Danvers, MA, USA), and β-actin HRP (sc-47778 HRP; Santa Cruz, Dallas, TX, USA).

Techniques: Transfection, Negative Control, Proliferation 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 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: Advanced Science

Article Title: Semaphorin 3E–Plexin D1 Axis Drives Lung Fibrosis through ErbB2‐Mediated Fibroblast Activation

doi: 10.1002/advs.202415007

Figure Lengend Snippet: P61‐Sema3E produces a pro‐fibrotic effect through the activation of ErbB2. A) Western blot analysis of the levels of ErbB2, P‐ErbB2, AKT, P‐AKT, ERK1/2, and P‐ERK1/2 in primary human lung fibroblasts (PHLFs) after stimulation with different concentrations of P61‐Sema3E for 2 h. B) Western blot analysis of ErbB2, P‐ErbB2, AKT, P‐AKT, ERK1/2, and P‐ERK1/2 expression in PHLFs treated with PlexinD1 siRNA or Scrambled siRNA following P61‐Sema3E induction. C) Co‐immunoprecipitation analysis of Plexin D1 and ErbB2, FLAG‐tagged Plexin D1 plasmid were transfected into PHLFs. D) Co‐immunoprecipitation analysis of Plexin D1 and ErbB2, PHLFs were treated with P61‐Sema3E for 2 h. E) Western blot analysis of Fibronectin, Col1a1, and α‐SMA expression in PHLFs treated with the ErbB2 inhibitor Lapatinib or PBS following P61‐Sema3E stimulation for 48 h. Data are represented as the mean ± SEM of three independent experiments. Statistical analyses were performed using one‐way ANOVA test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The ErbB2 inhibitor Lapatinib, was obtained from MedChemExpress (CM00323).

Techniques: Activation Assay, Western Blot, Expressing, Immunoprecipitation, Plasmid Preparation, Transfection

Journal: Advanced Science

Article Title: Semaphorin 3E–Plexin D1 Axis Drives Lung Fibrosis through ErbB2‐Mediated Fibroblast Activation

doi: 10.1002/advs.202415007

Figure Lengend Snippet: Downregulation of Sema3E attenuates BLM‐induced pulmonary fibrosis. A,B) Histological analysis of lung fibrosis severity in mice following BLM induction. (A) Representative images of lung sections stained with H&E, Masson's trichrome, and Sirius red to assess fibrosis severity. (B) Bar graph showing the quantitative mean score of fibrosis severity. Samples include AAV9‐NC saline mice ( n = 5), AAV9‐ShSema3E saline mice ( n = 5), AAV9‐NC BLM mice ( n = 5), and AAV9‐ShSema3E BLM mice ( n = 5). C) Quantification of hydroxyproline contents in AAV9‐NC mice and AAV9‐ShSema3E mice after BLM challenge. D,E) Western blot and RT‐qPCR analysis of Fibronectin, Col1a1, and α‐SMA protein and mRNA expression in lung homogenates from the mentioned mouse groups. F) Western blot analysis of ErbB2, P‐ErbB2, AKT, P‐AKT, ERK1/2, and P‐ERK1/2 expression in lung homogenates from the mentioned mouse groups. Samples include AAV9‐NC saline mice ( n = 3), AAV9‐ShSema3E saline mice ( n = 3), AAV9‐NC BLM mice ( n = 3), and AAV9‐ShSema3E BLM mice ( n = 3). Data are represented as the mean ± SEM. Statistical analyses were performed using one‐way ANOVA tests. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The ErbB2 inhibitor Lapatinib, was obtained from MedChemExpress (CM00323).

Techniques: Staining, Saline, Western Blot, Quantitative RT-PCR, Expressing

Journal: Advanced Science

Article Title: Semaphorin 3E–Plexin D1 Axis Drives Lung Fibrosis through ErbB2‐Mediated Fibroblast Activation

doi: 10.1002/advs.202415007

Figure Lengend Snippet: Sema3E deficiency in fibroblasts protects mice from BLM‐induced lung injury and fibrosis. A,B) Representative lung sections from Sema3E‐C and Sema3E‐CKO mice treated with saline or BLM, stained with H&E, Masson's Trichrome, and Sirius Red (Panel A, left). Panel B (right) shows quantitative fibrosis scores. Each group consisted of five mice ( n = 5). C) Quantification of hydroxyproline contents in Sema3E‐CKO and Sema3E‐C mice after BLM challenge. D,E) Western blot and RT‐qPCR analyses of Fibronectin, Col1a1, and α‐SMA protein and mRNA levels in lung homogenates from each group ( n = 5). F) Western blot analysis of ErbB2, P‐ErbB2, AKT, P‐AKT, ERK1/2, and P‐ERK1/2 in lung homogenates from the same groups of three mice( n = 3). Data are expressed as the mean ± SEM. Statistical significance was determined by one‐way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The ErbB2 inhibitor Lapatinib, was obtained from MedChemExpress (CM00323).

Techniques: Saline, Staining, Western Blot, Quantitative RT-PCR

Journal: Stem cells (Dayton, Ohio)

Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

doi: 10.1002/stem.2109

Figure Lengend Snippet: Inhibition of growth and HER2 signaling by HER2Ab-NSCs cells. (A) Inhibition in growth of BT474 cells using supernatant of HER2Ab-NSCs. (B) Co-Culture assay using BT474 cells with either vector control or HER2Ab-NSCs cells. (C-D) Inhibition of PI3K-AKT signaling using purified anti-HER2Ab released by NSC. Right panel in C and D demonstrates significant decrease in relative densitometric units. Trastuzumab was used as positive control in A, C and D. The experiments were repeated three times. * indicates p<0.05 and ** indicates p<0.01.

Article Snippet: ELISA plates (Corning, Pittsburg, PA) were coated overnight with 2μg/mL of recombinant human HER2 Fc chimera (R&D Systems, Minneapolis, MN).

Techniques: Inhibition, Co-culture Assay, Plasmid Preparation, Control, Purification, Positive Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

doi: 10.1002/stem.2109

Figure Lengend Snippet: Binding of anti-HER2Ab to HER2 overexpressing breast cancer cells. (A) BT474Br were stained with trastuzumab as a positive control (B-F) MCF 7, MDA-MB-361, BT474Br, ZR-75-30 and SKBR3 cells stained with anti-HER2Ab secreted by NSC. The experiments were repeated two times.

Article Snippet: ELISA plates (Corning, Pittsburg, PA) were coated overnight with 2μg/mL of recombinant human HER2 Fc chimera (R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Staining, Positive Control

Journal: Cancer cell

Article Title: Neuregulin-1-mediated autocrine signaling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers.

doi: 10.1016/j.ccr.2011.07.011

Figure Lengend Snippet: Figure 2. HER3 Mediates Lapatinib Sensitivity in Non-HER2 Amplified Cancer Cell Lines (A) Immunoblots indicating levels of pHER2, HER2, pHER3, HER3, pEGFR, EGFR, pAKT, AKT, pERK, and ERK in the lapatinib-sensitive, moderately sensitive, and refractory cell lines. As a control for HER2 overexpression, the HER2-amplified SKBR3 breast cancer cell line was included. (B) Box and whisker plot comparing lapatinib sensitivity (cell proliferation, 72 hr) in the detectable pHER3 (n = 13) and nondetectable pHER3 (n = 9)-expressing cells. Statistical difference between the two groups was assessed using Student’s t test (two-tailed, p = 0.0308). Cell lines belonging to the detectable pHER3 group include: CHL-1, MDA-MB-175-VII, PCI-6A, SAT, FaDu, BHY, DoTC2-4510, CAL27, HSC-3, SNG-M, SN12C, HN, and NCI-H2347. Cell lines belonging to the pHER3 nondetectable group include: CS1R, LN18, NB69, HuCCT1, DLD-1, DU145, A2.1, NCI-H661, and HSC-2. (C) Immunoblots showing the effect of acute lapatinib (200 nM) and erlotinib (200 nM) treatment (2 hr) in a panel of lapatinib-sensitive, moderately sensitive, or refractory cell lines on HER3 phosphorylation and downstream AKT phosphorylation.

Article Snippet: The phospho-EGFR (#2236), phospho-HER2 (#2247), HER2 (#2242), phosphoHER3 (Y1289; #4791), AKT (#9272), phospho-ERK (T202/Y204; #9101), ERK (#9102), and HER4 (#4795) antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot, Control, Over Expression, Whisker Assay, Expressing, Two Tailed Test, Phospho-proteomics

Journal: Cancer cell

Article Title: Neuregulin-1-mediated autocrine signaling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers.

doi: 10.1016/j.ccr.2011.07.011

Figure Lengend Snippet: Figure 3. A HER3-Activating Ligand Is Secreted by Non-HER2 Amplified Lapatinib-Sensitive Cell Lines (A) Left view has immunoblots showing the expression of pHER2, HER2, pHER3, HER3, pAKT, AKT, pERK, and ERK in the HER2-amplified SKBR3, the NRG1- DOC4 fusion containing MDA-MB-175-VII, and the EFM-19 breast cancer cell lines. Right view is of immunoblots demonstrating HER3 activation (pHER3) in EFM-19 cells following the addition of media conditioned overnight from the MDA-MB-175-VII cell line. (B) Immunoblots showing HER3 activation in EFM-19 cells following the addition of conditioned media (CM) from a panel of lapatinib-sensitive (green) and lapatinib-refractory (red) cell lines (30 min). The effect of conditioned media from the HER2-amplified SKBR3 and the EGFR mutated PC9 cell lines on the activation of HER3 (pHER3) in EFM-19 cells is also shown. (C) Immunoblots showing the effects of cotreatment with lapatinib (500 nM) or erlotinib (500 nM) on the ability of CM from the panel of lapatinib-sensitive cell lines to activate HER3 in EFM-19 cells (30 min). (D) Immunoblots showing the effects of cotreatment with lapatinib (500 nM) or erlotinib (500 nM) on the ability of CM from KP4, HEC-1, and H2722 lapatinib- refractory cell lines to activate HER3 (pHER3) in EFM-19 cells (30 min). (E) Immunoblots showing the levels of pHER3, HER3, pAKT, and AKT in the lapatinib-refractory KP4, HEC-1, H2722, A2.1, NCI-H661, and HSC-2 cell lines. CHL-1 lysate is included as a positive control for pHER3 levels. WCE, whole-cell extract. See also Figure S3.

Article Snippet: The phospho-EGFR (#2236), phospho-HER2 (#2247), HER2 (#2242), phosphoHER3 (Y1289; #4791), AKT (#9272), phospho-ERK (T202/Y204; #9101), ERK (#9102), and HER4 (#4795) antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Activation Assay, Positive Control

Journal: Cancer cell

Article Title: Neuregulin-1-mediated autocrine signaling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers.

doi: 10.1016/j.ccr.2011.07.011

Figure Lengend Snippet: Figure 4. NRG1 Is Highly Expressed and Secreted by the Non-HER2 Amplified Lapatinib-Sensitive Cell Lines (A) Immunoblots showing the expression of NRG1, NRG2, HER3, and ERK in the panel of lapatinib-sensitive (green) cell lines and a panel of lapatinib-refractory (red) cell lines derived from multiple tissue types. Cell line histology is indicated below the immunoblots. Me, melanoma; K, kidney; C, cervical; H, head and neck; U, uterus; B, breast; P, pancreatic; G, gastric; E, endometrial; Li, liver; M, mesothelioma; I, intestine; Pr, prostate; L, lung. (B) Correlation between NRG1 protein and mRNA expression in the panel of lapatinib-sensitive (green squares) and lapatinib-refractory (red triangles) cell lines. Lapatinib-sensitive cell lines include: CHL-1, PCI-6A, FaDu, CAL27, HSC-3, SAT, SNG-M, SN-12C, and DoTC2-4510. Lapatinib-refractory cell lines include: EFM-19, MCF-7, MDA-MB-468, KP4, SUIT2, GTL16, HEC-1, ESS-1, H2722, and DV90. (C) Box and whisker plot comparing lapatinib response (cell proliferation, 72 hr) in the NRG1 and HER3-coexpressing cells (n = 10) compared to the NRG1 or HER3 negative-expressing cells (n = 21). Statistical difference between the two groups was assessed using Student’s t test (two-tailed, p < 0.0001). (D) Immunoblot demonstrating secreted NRG1 in the media from the panel of lapatinib-sensitive cell lines. Media from EFM-19, MCF-7, and SKBR3 cells are included as negative controls.

Article Snippet: The phospho-EGFR (#2236), phospho-HER2 (#2247), HER2 (#2242), phosphoHER3 (Y1289; #4791), AKT (#9272), phospho-ERK (T202/Y204; #9101), ERK (#9102), and HER4 (#4795) antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Derivative Assay, Whisker Assay, Two Tailed Test

Journal: Cancer cell

Article Title: Neuregulin-1-mediated autocrine signaling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers.

doi: 10.1016/j.ccr.2011.07.011

Figure Lengend Snippet: Figure 5. NRG1 Mediates Cell Survival in Non-HER2 Amplified Lapatinib-Sensitive Cell Lines (A) Immunoblots showing NRG1 protein expression in CHL-1 and SN-12C cell lines following infection with either a control vector (shGFP) or the NRG1-specific vectors (5 days). The numbers 613, 834, 868, and 923 correspond to four different RNAi sequences. (B) Cell viability assay demonstrating suppression of cell proliferation in a panel of lapatinib-sensitive cell lines following lentiviral infection of NRG1-specific shRNAs or a control (shGFP) vector (5 days). MCF-7 cells were used to control for the effects of RNAi. Error bars represent mean ± SEM. (C) Lentiviral rescue experiment in CHL-1 cells transduced with NRG1 shRNAs for 5 days. Following overnight infection, media was changed, and cells were treated daily with rhNRG1-b1 (100 ng/ml) as indicated. Error bars represent mean ± SEM. (D) Left view has immunoblots showing the expression of NRG1 in a panel of the lapatinib-sensitive cell lines following infection with either a control (shGFP) or NRG1 (834 and 868) shRNA vectors (5 days). WCE, whole-cell extract. Right view is of immunoblots showing the activation of HER3 (pHER3) in EFM-19 cells following the addition of conditioned media from the panel of lapatinib-sensitive cells on the left (30 min). Twenty-four hours prior, lentiviral-infected cells were washed, and the media was replaced with unsupplemented media. The volume of conditioned media added to EFM-19 cells was adjusted to control for the antiproliferative effect observed in the NRG1 shRNA-transduced cells. (E) Detection of secreted NRG1 in the media from SN-12C cells following lentiviral infection with a control vector (shGFP) or 4 different NRG1 shRNAs; 613, 834, 868, and 923 (5 days). Twenty-four hours prior to collection, the cells were washed and the media replaced with unsupplemented media. See also Figure S4.

Article Snippet: The phospho-EGFR (#2236), phospho-HER2 (#2247), HER2 (#2242), phosphoHER3 (Y1289; #4791), AKT (#9272), phospho-ERK (T202/Y204; #9101), ERK (#9102), and HER4 (#4795) antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Infection, Control, Plasmid Preparation, Viability Assay, Transduction, shRNA, Activation Assay

Journal: Cancer cell

Article Title: Neuregulin-1-mediated autocrine signaling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers.

doi: 10.1016/j.ccr.2011.07.011

Figure Lengend Snippet: Figure 7. A Lapatinib Sensitivity Biomarker Is Observed in Head and Neck Primary Tumors (A) Immunoblots showing the levels of pHER2, HER2, pHER3, HER3, NRG1, pAKT, and AKT in a panel of lapatinib-sensitive and -refractory head and neck tumor- derived cell lines. (B) Box and whisker plot comparing NRG1 mRNA expression between the lapatinib-sensitive and lapatinib-refractory head and neck cancer cell lines. Statistical difference between the two groups was assessed using Student’s t test (two-tailed, p = 0.026). (C) Quantitative PCR analysis showing the expression of NRG1 and HER3 in head and neck primary tumor samples. As a comparison for expression, lapatinib- sensitive and -refractory cell lines are included. The blue dashed line represents the median NRG1 expression calculated using the expression from the panel of lapatinib-sensitive cell lines. Error bars represent mean ± SEM. (D) Immunoblot showing the constitutive activation of HER3 (pHER3) in a subset of head and neck primary tumor samples. HER3 was immunoprecipitated as described in the Experimental Procedures and subsequently immunoblotted with antibodies directed against pHER3 and HER3, respectively. See also Figure S5.

Article Snippet: The phospho-EGFR (#2236), phospho-HER2 (#2247), HER2 (#2242), phosphoHER3 (Y1289; #4791), AKT (#9272), phospho-ERK (T202/Y204; #9101), ERK (#9102), and HER4 (#4795) antibodies were purchased from Cell Signaling Technology.

Techniques: Biomarker Discovery, Western Blot, Derivative Assay, Whisker Assay, Expressing, Two Tailed Test, Real-time Polymerase Chain Reaction, Comparison, Activation Assay, Immunoprecipitation

Journal: Cancer cell

Article Title: Neuregulin-1-mediated autocrine signaling underlies sensitivity to HER2 kinase inhibitors in a subset of human cancers.

doi: 10.1016/j.ccr.2011.07.011

Figure Lengend Snippet: Figure 8. Model Depicting the Proposed NRG1-Mediated Autocrine- Signaling Mechanism NRG1 secreted by cancer cells in an autocrine manner binds to HER3, re- sulting in its heterodimerization with HER2. HER2 subsequently phosphory- lates HER3 resulting in the activation of the PI3K/AKT pathway promoting cell survival and proliferation. Importantly, transphosphorylation of HER2 is not required for this signaling cascade. As proposed, this pathway can be inhibited at multiple steps: first, by preventing NRG1 from binding HER3 using HER3- targeted antibodies; second, by preventing HER2:HER3 heterodimerization using a heterodimer-blocking antibody, such as pertuzumab; and finally, by inhibiting HER2 kinase activity using a HER2 TKI such as lapatinib.

Article Snippet: The phospho-EGFR (#2236), phospho-HER2 (#2247), HER2 (#2242), phosphoHER3 (Y1289; #4791), AKT (#9272), phospho-ERK (T202/Y204; #9101), ERK (#9102), and HER4 (#4795) antibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Binding Assay, Blocking Assay, Activity Assay