Journal: International Journal of Nanomedicine

Article Title: Investigation of antitumor activities of trastuzumab delivered by PLGA nanoparticles

doi: 10.2147/IJN.S152742

Figure Lengend Snippet: HER2 binding of released TZ from PLGA NPs. Notes: ( A ) TZ-FITC binding on SKBR3 cells after treatment with RIgG, CTZ and RTZ at two different concentrations (2 and 20 µg mL −1 ) for 24 and 48 h. Data are represented as the mean of fluorescence intensity of three independent replicates ± SD acquiring 10,000 events for each sample by flow cytometry. ( B – E ) Confocal microscopy images of TZ-FITC binding on SKBR3. ( B ) Basal binding of TZ-FITC; ( C , D and E ) binding of TZ-FITC after pre-treatment of RIgG, CTZ and RTZ, respectively, for 24 h at 37°C. Cells were labeled with DAPI (nuclei) and WGA-555 (membranes). Scale bar =50 µm. **** P <0.01 vs UNTR; ** P <0.05 vs UNTR. Abbreviations: HER2, human epidermal growth factor receptor 2; TZ, trastuzumab; PLGA NPs, poly(lactic- co -glycolic) acid nanoparticles; FITC, fluorescein isothiocyanate; RIgG, released IgG; CTZ, control TZ; RTZ, released TZ; SD, standard deviation; CNTR, cells without any labeling; UNTR, cells coated with TZ-FITC.

Article Snippet: The membranes were blocked with TBS – Tween 0.1% solution +5% milk and then immunodecorated alternatively with monoclonal anti-HER2/ErbB2 antibody (Cell Signaling Technology, Leiden, the Netherlands) or polyclonal anti-phosphorylated HER2 antibody (Tyr1248; Cell Signaling Technology) or monoclonal antibody anti-β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA) in TBS – Tween 0.1% solution +5% BSA overnight at 4°C.

Techniques: Binding Assay, Fluorescence, Flow Cytometry, Confocal Microscopy, Labeling, Standard Deviation

Journal: International Journal of Nanomedicine

Article Title: Investigation of antitumor activities of trastuzumab delivered by PLGA nanoparticles

doi: 10.2147/IJN.S152742

Figure Lengend Snippet: HER2 phosphorylation. Notes: ( A ) Analysis of pHER2 (Y1248) and HER2 expression on SKBR3 cells after treatment with control CTZ and RTZ at 2 µg mL −1 for 4 and 24 h; ( B ) Analysis of HER2 expression on SKBR3 cells after treatment with PLGA-TZ at 2 µg mL −1 for 4 and 24 h. Values were calculated as ratio between pHER2/HER2 and HER2/b-actin and normalized with untreated cells (UNTR). Abbreviations: HER2, human epidermal growth factor receptor 2; CTZ, control trastuzumab; RTZ, released trastuzumab; PLGA-TZ, trastuzumab-loaded poly(lactic- co -glycolic) acid nanoparticles; UNTR, cells without treatment.

Article Snippet: The membranes were blocked with TBS – Tween 0.1% solution +5% milk and then immunodecorated alternatively with monoclonal anti-HER2/ErbB2 antibody (Cell Signaling Technology, Leiden, the Netherlands) or polyclonal anti-phosphorylated HER2 antibody (Tyr1248; Cell Signaling Technology) or monoclonal antibody anti-β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA) in TBS – Tween 0.1% solution +5% BSA overnight at 4°C.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Identification of Nucleolin as New ErbB Receptors- Interacting Protein

doi: 10.1371/journal.pone.0002310

Figure Lengend Snippet: (A) COS7 cells were transiently co-transfected with expression vector of each of the ErbB receptors and Myc-Nucleolin. Cell lysates were separated into cytosolic (supernatant) and total membrane (pellet) fractions. The pellet fraction was subjected to immunoprecipitation with anti-Myc (nucleolin) or anti-HA (control) antibodies and immunoblotted with specific ErbBs antibodies. Total pellet and supernatant extracts are shown in the right lanes of each panel. (B) COS7 cells were transiently co-transfected with expression vector of ErbB1 and Myc-Nucleolin. Nuclear and non-nuclear cell extracts were immunoblotted with anti-ErbB1 antibodies. As control for the fractionation purity Blots were reacted with anti-PARP antibodies (as a nuclear marker) and anti-tubulin antibodies (as a cytosolic marker). (C) Cell lysates prepared from SKBR3 cells were subjected to immunoprecipitation with either anti-nucleolin or anti ErbB2 antibodies and immunoblotted with either anti- ErbB2 or anti-nucleolin antibodies as indicated, as a control total cell lysates are shown in the right lane of each panel.

Article Snippet: Polyclonal rabbit anti phosphorylated ErbB1 (Tyr1068) and phosphorylated ErbB2 (Tyr1248) were purchased from Cell Signaling technology.

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Fractionation, Marker

Journal: PLoS ONE

Article Title: Identification of Nucleolin as New ErbB Receptors- Interacting Protein

doi: 10.1371/journal.pone.0002310

Figure Lengend Snippet: (A) COS7 cells were transiently co-transfected with expression vector of either ErbB1 or ErbB2 receptors alone or with Myc-Nucleolin. Following 30 min serum deprivation, cells were either untreated or treated with EGF 100 ng/ml for 5 min. Cell lysates were immunoblotted with anti-phosphorylated EGFR or anti-phosphorylated ErbB2 antibodies respectively. As control, lysates were immunoblotted with anti-EGFR or anti-ErbB2 antibodies. Note that at time 0 phosphorylated receptors are detected in cells expressing nucleolin and EGFR or ErbB2. (B) COS7 cells were transiently co-transfected with expression vector of each of the ErbB receptors and Myc-Nucleolin. Following 30 min serum deprivation cells were untreated or treated with either EGF 100 ng/ml or NRG 100 ng/ml for 5 min as indicated. Cell lysates were subjected to immunoprecipitation with anti-phosphotyrosine antibodies (PY20) and immunoblotted with specific ErbB antibodies. Note that in untreated cells, phosphorylated receptor is detected in cells expressing nucleolin and ErbB receptor. The values represent fold induction compared to the receptor levels in untreated cells (1).

Article Snippet: Polyclonal rabbit anti phosphorylated ErbB1 (Tyr1068) and phosphorylated ErbB2 (Tyr1248) were purchased from Cell Signaling technology.

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation

Journal: Disease Markers

Article Title: Mig-6 Gene Knockout Induces Neointimal Hyperplasia in the Vascular Smooth Muscle Cell

doi: 10.1155/2014/549054

Figure Lengend Snippet: EGFR phosphorylation of vascular SMCs with regard to Mig-6 knockout. (a) Western blotting for phospho-EGFR in cultured vascular SMCs of Mig-6 d/d and Mig-6 f/f mice. pEGFR, phospho-EGFR; α SMA, α -smooth muscle actin. (b) Western blotting for EGFR and downstream signals according to transfection of siRNA into human vascular SMCs. siNS , transfection of nonspecific siRNA; siMIG-6 , transfection of siRNAs against MIG-6 ; pEGFR, phospho-EGFR; tEGFR, total EGFR; pERK, phospho-ERK; tERK, total ERK; pAAK, phospho-Akt; tAKT, total AKT.

Article Snippet: Primary antibodies against mouse Mig-6, phosphorylated EGFR, total EGFR, phosphorylated AKT, total AKT, phosphorylated ERK, and total ERK were purchased from Cell Signaling (Danvers, Massachusetts, USA).

Techniques: Knock-Out, Western Blot, Cell Culture, Transfection

Journal: BioMed Research International

Article Title: Pretreatment with Gemcitabine/5-Fluorouracil Enhances the Cytotoxicity of Trastuzumab to HER2-Negative Human Gallbladder Cancer Cells In Vitro and In Vivo

doi: 10.1155/2019/9205851

Figure Lengend Snippet: Microphotographs of dual-color FISH for HER2 detection in GBC cell lines (×1,000). SpectrumGreen probe for centromere 17 and SpectrumOrange probe for HER2 gene. The HER2:CEP17 ratio in NOZ and GB-D1 cell lines was 1.2 and 1.08, respectively.

Article Snippet: Primary antibodies against HER2, phosphorylated-HER2 (Tyr1248), and phosphorylated-AKT (Ser473) were purchased from Cell Signaling Technology, Inc. (Beverly, USA).

Techniques:

Journal: BioMed Research International

Article Title: Pretreatment with Gemcitabine/5-Fluorouracil Enhances the Cytotoxicity of Trastuzumab to HER2-Negative Human Gallbladder Cancer Cells In Vitro and In Vivo

doi: 10.1155/2019/9205851

Figure Lengend Snippet: Cell viability inhibition rates and apoptosis in GBC cell lines after treatment with various drugs . (a) Cell viability inhibition rates in NOZ cells and GB-D1 cells after treatment with trastuzumab (Herceptin, H) only, chemotherapy (GEM, G and 5-Fu, F) only, sequential therapy with chemotherapy followed by trastuzumab, concomitant therapy with chemotherapy and trastuzumab, and sequential therapy with trastuzumab followed by chemotherapy were evaluated. Sequential therapy with chemotherapy followed by trastuzumab demonstrated superiority over the others. ∗ P > 0.05. Others: P < 0.05. Cell viability inhibition rate was calculated as follows: (nontreated cells OD - treated cells OD) / (nontreated cells OD - blank OD). (b) Results relating to apoptosis in NOZ cells were similar to those of the cell viability assay. Sequential therapy with chemotherapy followed by trastuzumab demonstrated superiority over the others in terms of apoptosis. ∗ P > 0.05. Others: P < 0.05. (c) Effects of trastuzumab and/or GEM on cell cycle in NOZ cells: P > 0.05, G versus (H + G) in the G1 phase of the cell cycle; control versus H, and G versus (H + G) in the S phase of the cell cycle, all in the G2 phase of the cell cycle. Others: P < 0.05. Effects of trastuzumab and/or 5-Fu on cell cycle in NOZ cells: P > 0.05: H versus (H→F), and (H + F) versus (F→H) in the G1 phase of the cell cycle; control versus H, control, H or F versus (H→F) in the S phase of the cell cycle; control versus H, H versus F, (F→H) versus (H + F), control, H or F versus (H→F) in the G2 phase of the cell cycle. Others: P < 0.05. (d) Western blots were performed to detect the effects of trastuzumab and/or chemotherapeutic drugs on the expressions of key proteins HER2, pHER2, AKT, and pAKT in the HER2/AKT signaling pathway in NOZ cells. β -actin was used as a loading control. NOZ cells showed increases in pHER2, pAKT, HER2, and AKT expression following G/F alone or H→G/F treatment.

Article Snippet: Primary antibodies against HER2, phosphorylated-HER2 (Tyr1248), and phosphorylated-AKT (Ser473) were purchased from Cell Signaling Technology, Inc. (Beverly, USA).

Techniques: Inhibition, Viability Assay, Western Blot, Expressing

Journal: BioMed Research International

Article Title: Pretreatment with Gemcitabine/5-Fluorouracil Enhances the Cytotoxicity of Trastuzumab to HER2-Negative Human Gallbladder Cancer Cells In Vitro and In Vivo

doi: 10.1155/2019/9205851

Figure Lengend Snippet: Key protein expressions of HER2/AKT signaling pathway in tumor tissues of the xenograft mouse model by IHC assay (magnification of ×200) . GEM or 5-Fu alone increased the expression of HER2 protein. HER2 IHC scores of group G, H→G, F, and H→F were higher than those of other groups (P < 0.05). But there were no statistic differences between other groups.

Article Snippet: Primary antibodies against HER2, phosphorylated-HER2 (Tyr1248), and phosphorylated-AKT (Ser473) were purchased from Cell Signaling Technology, Inc. (Beverly, USA).

Techniques: Expressing

Journal: Orphanet Journal of Rare Diseases

Article Title: Biallelic ERBB3 loss-of-function variants are associated with a novel multisystem syndrome without congenital contracture

doi: 10.1186/s13023-019-1241-z

Figure Lengend Snippet: The c.1253 T > C (p.I418T) mutation has no effect on ERBB3 expression and interaction with ERBB2, whereas the c.3182dupA (p.N1061Kfs*16) mutation produces a novel truncated protein. a Western blotting results using anti-Myc antibody to detect ERBB3 in lysates of HEK293T cells transfected with 2 μg empty vector (EV) or WT, c.1253 T > C (M1), or c.3182dupA (M2) plasmids. b Results of co-immunoprecipitation to detect the interaction between ERBB2 and WT or I418T (M1) mutant ERBB3

Article Snippet: Antibodies against ERBB2 (#2242), phosphorylated (p-)ERBB2 (Tyr1248) (#2247), ERBB3 (C-terminus) (#12708), p-ERBB3 (Tyr1289) (#4791), ERK (#4695), p-ERK (#4370), AKT (#4685), p-AKT (#4060), GFP (#2555), Myc (#2272), and GAPDH (#5174) were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Expressing, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Orphanet Journal of Rare Diseases

Article Title: Biallelic ERBB3 loss-of-function variants are associated with a novel multisystem syndrome without congenital contracture

doi: 10.1186/s13023-019-1241-z

Figure Lengend Snippet: ERBB3 variants lack the capacity to activate PI3K/AKT and ERK signaling pathways. a Immunoblot analysis was performed using indicated antibodies to determine the effects of WT or mutant ERBB3 on PI3K/AKT and ERK pathway activation. To induce protein phosphorylation, HEK293T cells were treated with 10 ng/ml NRG-1β for 30 min after transfection with empty vector (EV), WT, M1, M2, or M3 (V104 L) plasmids. b–e Quantitative analysis of p-ERK, p-AKT, p-ERBB2, and p–ERBB3 expression. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT

Article Snippet: Antibodies against ERBB2 (#2242), phosphorylated (p-)ERBB2 (Tyr1248) (#2247), ERBB3 (C-terminus) (#12708), p-ERBB3 (Tyr1289) (#4791), ERK (#4695), p-ERK (#4370), AKT (#4685), p-AKT (#4060), GFP (#2555), Myc (#2272), and GAPDH (#5174) were purchased from Cell Signaling Technology.

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

Journal: Scientific Reports

Article Title: Genetically distinct glioma stem-like cell xenografts established from paired glioblastoma samples harvested before and after molecularly targeted therapy

doi: 10.1038/s41598-018-37437-2

Figure Lengend Snippet: Histopathological analysis reveals drastic changes in EGFR and MIB1 status in re-recurrent GBM after EGFR inhibitor therapy. Microscopic pictures of H&E staining (top row) and IHC for EGFR (2 nd row), phospho-EGFR (Tyr1068, 3 rd row) and MIB-1 (Ki-67, the bottom row) for, from left to right column, original tumour MGG70 (70), recurrent tumour MGG70R (70R), re-recurrent tumour MGG70RR (70RR), and autopsy material MGG70A (70A). Original magnification, x200. Note negative staining of EGFR and phospho-EGFR in tumour-associated blood vessels (arrows). pEGFR positivity (% area) and MIB-1 labeling indices of each tumour are plotted on the right.

Article Snippet: Primary antibodies used were MIB-1 (Dako, 1:150), EGFR, phosphorylated EGFR (phospho-EGFR), CD44, Mesenchymal-epithelial transition (Met) (all Cell Signaling, 1:500), and YKL40 (Quidel, 1:300).

Techniques: Staining, Negative Staining, Labeling

Journal: Scientific Reports

Article Title: Genetically distinct glioma stem-like cell xenografts established from paired glioblastoma samples harvested before and after molecularly targeted therapy

doi: 10.1038/s41598-018-37437-2

Figure Lengend Snippet: Paired GSC orthotopic xenografts recapitulate the molecular and phenotypic characteristics of patient tumours. ( A ) Cell counting assay showing different growth kinetics of MGG70R-GSCs (70R-GSC) and MGG70RR-GSCs (70RR-GSC) in culture. Shown right are representative microscopic pictures of a neurosphere generated from a single GSC. ( B ) H&E staining of coronal sections of the brains bearing GSC-derived orthotopic tumours. Scale bar, 5 mm. ( C ) H&E staining and IHC for EGFR, phospho-EGFR, and Ki-67 (MIB-1) in orthotopic GSC xenografts. Top row, MGG70R-GSC tumours. Bottom row, MGG70RR-GSC tumours. ( D ) MIB-1 labeling indices and pEGFR immunopositivity (% area) of MGG70R-GSC and MGG70RR-GSC tumours. ( E ) FISH images of the GSC xenografts, with EGFR probe in green and CEN7 control probe in red. Left, MGG70R (pre-dacomitinib) GSC tumour. Right, MGG70RR (post-dacomitinib) GSC tumour.

Article Snippet: Primary antibodies used were MIB-1 (Dako, 1:150), EGFR, phosphorylated EGFR (phospho-EGFR), CD44, Mesenchymal-epithelial transition (Met) (all Cell Signaling, 1:500), and YKL40 (Quidel, 1:300).

Techniques: Cell Counting, Generated, Staining, Derivative Assay, Labeling