Journal: PLoS ONE

Article Title: Construction and analyses of the microRNA-target gene differential regulatory network in thyroid carcinoma

doi: 10.1371/journal.pone.0178331

Figure Lengend Snippet: A. Putative mir-152-3p-binding site at the 3'-UTR of ERBB3(TargetScan). B. The luciferase assay of HEK293T cells. The relative luciferase activity was determined after the described reporter constructs (pMIR-ERBB3-WT or pMIR-ERBB3-Mut) were co-transfected with the NC or mir-152-3p mimic into HEK293T cells. The luciferase activity was normalized to Renilla luciferase activity. Columns, mean; bars, standard deviation. ** P<0.01. C. The mir-152-3p negatively regulates protein expression of ERBB3 in TPC-1 cells. β-Actin was used as an internal control for ERBB3.

Article Snippet: After blocking, the blots were probed with primary antibodies to actin, ERBB3 (1: 200 dilution, Santa Cruz, Biotechnology, Santa Cruz, CA, USA), and caspase-3 (1: 500 dilution, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Transfection, Standard Deviation, Expressing, Control

Journal: PLoS ONE

Article Title: Construction and analyses of the microRNA-target gene differential regulatory network in thyroid carcinoma

doi: 10.1371/journal.pone.0178331

Figure Lengend Snippet: A. The mRNA expression of ERBB3 in TPC-1 cells transfected with ERBB3-siRNA or NC. β-Actin served as an internal control. *P<0.05. B. The protein expression of ERBB3 in TPC-1 cells transfected with ERBB3-siRNA or NC. β-Actin served as an internal control. C. The MTT assay TPC-1 cells transfected with ERBB3-siRNA or NC. *P<0.05. D . The colony formation assay of TPC-1 cells transfected with ERBB3-siRNA or NC. *P<0.05, E. The acridine range/ethidium bromide ratio was used to determine the apoptosis of TPC-1 cells. F. The expression of caspase-3 in TPC-1 cells after treatment with NC or ERBB3-siRNA. * P<0.05.

Article Snippet: After blocking, the blots were probed with primary antibodies to actin, ERBB3 (1: 200 dilution, Santa Cruz, Biotechnology, Santa Cruz, CA, USA), and caspase-3 (1: 500 dilution, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Transfection, Control, MTT Assay, Colony Assay

Journal: The Journal of Clinical Investigation

Article Title: ErbB3 downregulation enhances luminal breast tumor response to antiestrogens

doi: 10.1172/JCI66764

Figure Lengend Snippet: (A) cDNA microarray analysis of clinical breast cancers (UNC337 data set). Heat map shows expression of EGFR, ERBB2, and ERBB3. Each colored square represents relative transcript abundance (in log2 space) for each sample: lowest expression is green, highest is red, and average expression is black. The colored array tree denotes the intrinsic subtype of each sample. Dark blue, luminal A/B; green, normal-like; red, basal-like; pink, HER2-enriched; yellow, claudin-low). (B and C) ERBB3 relative transcript levels across intrinsic molecular subtypes in the UNC337 data set (B) and an independently derived microarray data subset (C) (20, 21). BL, basal-like; CL, claudin-low; H2, HER2-enriched; LA, luminal A; LB, luminal B; NL, normal-like; Str, stromal. *P < 0.01 (Student’s t test). Midline shows the average ± SD. P values compare expression means across all groups. Midline shows the average. Whiskers show SD. Expression values in each tumor are shown by plus signs. P values compare expression means across all groups. (D and E) Luminal breast cancer cells were transduced with retrovirus expressing ErbB3-IRES-RFP. Puromycin-resistant cells overexpressing ErbB3 (1 × 103/well) were suspended in Matrigel (100 μl), cultured 10–14 days, photodocumented (D), and quantitated (E) using ImageJ software (NIH). Original magnification, ×100. Values shown represent the average number of colonies/well ± SD. n = 3. *P > 0.05; **P > 0.01; ***P > 0.001. (F and G) ERBB3 gene copy number and mRNA expression levels were determined in TCGA-curated luminal A and luminal B breast cancers (23) and used to organize samples into those exhibiting ERBB3 copy number gain or high ErbB3 expression (shown in red). Overall survival analysis was performed using cBio Portal (22).

Article Snippet: Cells were collected by gentle trypsinization, fixed and permeabilized (BD Cytofix/Cytoperm), and stained with phycoerythrin-conjugated mouse anti-human ErbB3 (1:50; R&D Systems).

Techniques: Microarray, Expressing, Derivative Assay, Transduction, Cell Culture, Software

Journal: The Journal of Clinical Investigation

Article Title: ErbB3 downregulation enhances luminal breast tumor response to antiestrogens

doi: 10.1172/JCI66764

Figure Lengend Snippet: ERBB3 copy number in breast cancer subtypes

Article Snippet: Cells were collected by gentle trypsinization, fixed and permeabilized (BD Cytofix/Cytoperm), and stained with phycoerythrin-conjugated mouse anti-human ErbB3 (1:50; R&D Systems).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: ErbB3 downregulation enhances luminal breast tumor response to antiestrogens

doi: 10.1172/JCI66764

Figure Lengend Snippet: (A) Cells were cultured in medium with 10% serum plus U3-1287 (5 μg/ml) or isotype-matched human IgG for 24 hours, then used for Western blot analysis of whole-cell lysates using antibodies against ErbB3 and E-cadherin. (B and C) Cells were cultured in Matrigel for 14 days with the anti-ErbB3 antibody U3-1287 or isotype-matched human IgG. Colonies were imaged by photomicroscopy (B) and quantitated (C) using ImageJ software (NIH). Original magnification, ×100 (B). Representative images are shown in B. Values shown (C) are average ± SD, n = 3 per treatment group. **P < 0.01; ***P < 0.001. (D–F) MCF7 tumor-bearing mice were randomized to receive normal human IgG or U3-1287 (5 mg/kg twice weekly). (D) Histological analysis of tumors on treatment day 28. Scale bars: 50 μm; Original magnification, ×400. (E) Average tumor volume ± SD for each treatment group is shown, n = 10. P value calculated using Student’s t test to compare tumor volumes at day 28. (F) Whole-tumor lysates were assessed by Western analysis. Antibodies are shown at right. (G) Immunohistochemical detection of ErbB3-, Ki67-, and TUNEL-positive cells (arrows) is shown in tumor samples collected after 8 days of treatment. Representative images are shown. Scale bars: 50 μm. Values shown are the average ± SD of [(number of Ki67+ or TUNEL+) ÷ (number of epithelial tumor cells]. n = 5 samples, 4 fields per sample, Student’s unpaired t test.

Article Snippet: Cells were collected by gentle trypsinization, fixed and permeabilized (BD Cytofix/Cytoperm), and stained with phycoerythrin-conjugated mouse anti-human ErbB3 (1:50; R&D Systems).

Techniques: Cell Culture, Western Blot, Software, Immunohistochemical staining, TUNEL Assay

Journal: The Journal of Clinical Investigation

Article Title: ErbB3 downregulation enhances luminal breast tumor response to antiestrogens

doi: 10.1172/JCI66764

Figure Lengend Snippet: (A and B) Immunofluorescence (A) and flow cytometry (B) of ErbB3 in cells cultured for 24 hours with or without fulvestrant. Original magnification, ×200. (B) Left panel shows the ErbB3hi percentage of cells (n = 50,000). Right panel shows average fluorescent intensity per cell. (C–E) Western blot analysis of cell lysates harvested after 0–72 hours of 1 μM fulvestrant. (F) Cells transfected with ErbB3-siRNA or control-siRNA were treated 24 hours with fulvestrant or DMSO prior to Western blot analysis for ErbB3 and ERα. (G) Real-time qPCR analysis of ERBB3 in cells treated 0–24 hours with fulvestrant (1 μM). Average relative ErbB3 levels shown ± SD. *P < 0.05, 1-way ANOVA. (H) Increased mRNA encoding ErbB receptors and ligands ± SD in fulvestrant-treated MCF7 cells measured in publicly available expression data (GSE14986). (I) MCF7 cells transfected with a human ERBB3 gene promoter (–1000/+1) luciferase reporter plasmid were treated with increasing fulvestrant doses for 24 hours. Average (n = 3) RLUs per μg protein ± SD are shown. One-way ANOVA. (J) Real-time qPCR analysis of ERBB3 mRNA in MCF7 cells treated 24 hours ± fulvestrant and actinomycin D (ActD). Average fold change is shown ± SD. ***P < 0.001. (K) Western blot analysis of fulvestrant-treated MCF7 lysates. Cycloheximide (CHX) was added for the final 0–4 hours.

Article Snippet: Cells were collected by gentle trypsinization, fixed and permeabilized (BD Cytofix/Cytoperm), and stained with phycoerythrin-conjugated mouse anti-human ErbB3 (1:50; R&D Systems).

Techniques: Immunofluorescence, Flow Cytometry, Cell Culture, Western Blot, Transfection, Control, Expressing, Luciferase, Plasmid Preparation

Journal: The Journal of Clinical Investigation

Article Title: ErbB3 downregulation enhances luminal breast tumor response to antiestrogens

doi: 10.1172/JCI66764

Figure Lengend Snippet: Cells expressing lentiviral ErbB3-RFP or RFP alone were cultured 14 days in Matrigel ± fulvestrant. Original magnification, ×100. Average number of colonies ± SD was evaluated. n = 5. One-way ANOVA.

Article Snippet: Cells were collected by gentle trypsinization, fixed and permeabilized (BD Cytofix/Cytoperm), and stained with phycoerythrin-conjugated mouse anti-human ErbB3 (1:50; R&D Systems).

Techniques: Expressing, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: ErbB3 downregulation enhances luminal breast tumor response to antiestrogens

doi: 10.1172/JCI66764

Figure Lengend Snippet: (A) MCF7 xenografts treated with U3-1287 or IgG (each at 20 mg/kg, twice weekly) in the presence or absence of fulvestrant (once weekly) were collected at day 8 of treatment (24 hours after the second fulvestrant treatment). Immunohistochemical analysis was used to detect ErbB3, P-S6, and ERα. Scale bars: 50 μm. (B–D) Western blot analysis of tumor xenografts from mice treated with fulvestrant with and without U3-1287 for 8 days. Tumors were harvested 24 hours after the final treatment with U3-1287 and fulvestrant. Antibodies used are indicated.

Article Snippet: Cells were collected by gentle trypsinization, fixed and permeabilized (BD Cytofix/Cytoperm), and stained with phycoerythrin-conjugated mouse anti-human ErbB3 (1:50; R&D Systems).

Techniques: Immunohistochemical staining, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: ErbB3 downregulation enhances luminal breast tumor response to antiestrogens

doi: 10.1172/JCI66764

Figure Lengend Snippet: (A) Matched pairs of clinical breast cancer specimens collected 24 hours prior to treatment (day 0) and 21 days after a single treatment with fulvestrant on day 1 followed by daily treatment with anastrozole (day 21) were assessed by IHC. ErbB3 and P-S6 expression was assessed by IHC. Intensity of total ErbB3 immunostaining (cytoplasmic and membrane) and P-S6 immunostaining were quantified. Values shown represent the average ± SD (n = 4). P values trended toward significance (P = 0.06, Student’s paired t test). A representative matched pair is shown. Scale bars: 50 μm. (B) TCGA-curated luminal A breast cancers were separated into those tumors exhibiting ERBB3 copy number gain on those with diploid ERBB3, then assessed for differences in phospho-proteins as detected by RPPA analysis. P-S6 and P-S6 kinase were significantly increased in tumors exhibiting ERBB3 copy number gain. (Student’s t test). (C) Model of how luminal breast cancers use ErbB3-PI3K signaling to limit their intrinsic sensitivity to fulvestrant. ERα targeting with fulvestrant drives ErbB3 upregulation at the cell surface through transcriptional, posttranscriptional, and posttranslational mechanisms. Increased ErbB3 enhances PI3K signaling through its 6 PI3K interaction motifs. PI3K activates PDK1, which phosphorylates Akt. Once activated, Akt promotes mTORC1 activation, driving increased S6-kinase and S6 phosphorylation.

Article Snippet: Cells were collected by gentle trypsinization, fixed and permeabilized (BD Cytofix/Cytoperm), and stained with phycoerythrin-conjugated mouse anti-human ErbB3 (1:50; R&D Systems).

Techniques: Expressing, Immunostaining, Membrane, Activation Assay, Phospho-proteomics

Journal: BMC Genomics

Article Title: A large-scale proteomic analysis of human embryonic stem cells

doi: 10.1186/1471-2164-8-478

Figure Lengend Snippet: Tight junction proteins and ERBB2/3 expression in hESCs . BG01 hESCs were disaggregated to single cells using accutase [52] and cultured in defined conditions. (A) ZO1 expression four and (B) seven days after plating, indicating progressive tight junction formation. (C) Occludin expression 5 days after plating. (D) General cell surface expression of ERBB2, in the same field of view as (A). (E) Localized expression of ERBB3, in the same field of view as (B). (F) Higher magnification of ERBB3 localization in ZO1 expressing BG01 cells, 5 days after plating. Nuclei were stained with DAPI.

Article Snippet: The following antibodies were used: ABP-280 (1:250, BD Biosciences 610798), CtBP1 (1:1000, BD Biosciences 612042), CtBP2 (1:1000, BD Biosciences 612044), GS-28 (1:2000, BD Biosciences 611184), HDJ-2 (1:100, BD Biosciences 611872), L-Caldesmon (1:2000, BD Biosciences 610660), Rabaptin-5 (1:500, BD Biosciences 611080), phospho-p130 Cas (Tyr165) (1:50, Cell Signaling Technology 4015), phospho-Ras-GAP (pY460) (1:250, BD Biosciences 612736), Ras-GAP (1:250, BD Biosciences 610043), Shc-C (1:1000, BD Biosciences 610642), Oct-4 (Santa Cruz biotechnology, 1:200 SC-8628), TNIK (1:100, BD Biosciences, 612250), p130 Cas (1:100, BD Biosciences, 610272), ERBB2 (1:100, Lab Vision, 9G6.10), ERBB3 (1:100, R&D Systems, MAB348), ZO1 (1:100, Invitrogen, 61–7300), or Occludin (1:100, Invitrogen, 71–1500).

Techniques: Expressing, Cell Culture, Staining