Journal: Clinical Cancer Research

Article Title: Expression and Nuclear Localization of ErbB3 in Prostate Cancer

doi: 10.1158/1078-0432.ccr-05-2242

Figure Lengend Snippet: Fig. 3. Activation and subcellular localization of ErbB3 in prostate cancer cell lines. The phosphorylation of ErbB3 was observed byWestern blotting of whole-cell extracts of prostate cancer cell lines (LNCaP, PC-3, DU145, and 22Rv1) and normal prostate cells RWPE-1treated with NRG1 (A). Due to loading problems (*), the 22Rv1neuregulin1-h1 (NRG1)^ treated sample is presented in duplicate (note difference in h-actin levels). Cytoplasmic extracts (B) and nuclear extracts (C) of prostate cancer cells and normal prostate cells (RWPE-1) treated with NRG1show that ErbB3 is phosphorylated in cytoplasmic-enriched fractions and particularly in androgen-sensitive cell lines LNCaP and 22Rv1 (B). P, neuregulin1-h1-stimulated MCF-7 whole-cell extract used as positive control for phospho-ErbB3. h-Actin serves as the loading control in all experiments.

Article Snippet: The medium was replaced, and 20 ng/mL recombinant human neuregulin1-h1 (R&D Systems, Minneapolis, MN) was added for 60 minutes; 5 ng/mL of bovine serum albumin was added to control plates.

Techniques: Activation Assay, Phospho-proteomics, Positive Control, Control

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: TGF-β-induced, ShcA-dependent gene expression profiling in ErbB2-expressing breast cancer cells identifies Chrdl1. (A) Immunoblot analysis of NMuMG-ErbB2/rtTA cells following doxycycline treatment for the indicated times using antibodies against ShcA, ErbB2, and α-tubulin. (B) Schematic of experimental design. (C) Gene ontology analysis of differentially expressed genes. (D) Gene expression heat maps of the most differentially expressed genes in ErbB2-expressing breast cancer cells following TGF-β treatment (2.5 ng/ml) (3 or 24 h) in the context of high (−Dox) or low (+Dox) ShcA levels. A yellow to blue gradient represents low to high gene expression levels. The red box highlights Chrdl1 Agilent gene expression data. (E and F) TGF-β-induced, ShcA-dependent gene expression of BMP family members or BMP antagonists in ErbB2-expressing breast cancer cells. Gene expression heat maps of BMP genes (E) or BMP antagonist genes (F) in breast cancer cells following TGF-β treatment (3 or 24 h) in the context of high (−Dox) or low (+Dox) ShcA levels are shown. A yellow to blue gradient represents low to high gene expression levels. The red box highlights Chrdl1 Agilent gene expression data. (G) RT-qPCR analysis of Chrdl1 mRNA expression levels following TGF-β stimulation in the presence or absence of ShcA. *, P < 0.029. (H) Chrdl1 secreted protein expression levels were monitored by ELISA using conditioned medium harvested under the conditions described for panel G. Secreted Chrdl1 concentrations were normalized to protein concentrations measured in whole-cell protein lysates. *, P < 0.023.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Gene Expression, Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Chrdl1 expression is elevated following TGF-β stimulation of HER2+ and basal breast cancer cells in the context of reduced ShcA levels. (A to C) Immunoblot analysis of ShcA expression was performed in NIC (A), MDA-MB-231 (B), and BT549 (C) breast cancer cells transfected with scrambled or ShcA-specific siRNAs. Cells were stimulated with TGF-β (+) or not stimulated (−) for 24 (NIC and MDA-MB-231) or 48 h (BT549). In all cases, α-tubulin was used as a loading control. (D to F) ELISAs were performed to detect secreted Chrdl1 levels in the conditioned media of NIC (D), MDA-MB-231 (E), and BT549 (F) breast cancer cells. Secreted Chrdl1 concentrations were normalized to protein concentrations from whole-cell protein lysates. *, P = 0.044; **, P = 0.015; ***, P = 0.023.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Expressing, Western Blot, Transfection, Control

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Recombinant Chrdl1 suppresses BMP7-induced Smad1/8 phosphorylation in mouse breast cancer cells and Smad1/9 phosphorylation in human breast cancer cells but fails to block TGF-β-induced signaling. (A to C) Immunoblot analyses using antibodies against phospho-Smad and total Smad1 in MDA-MB-231 (phospho-Smad1/9) (A), HCC1954 (phospho-Smad1/9) (B), and NMuMG-ErbB2 (phospho-Smad1/8) (C) breast cancer cells treated with BMP7 (60 min, 50 ng/ml) and increasing concentrations of rChrdl1 (60 min, 100 to 400 ng/ml). (D to F) The ratio of phosphorylated Smads to total Smad1 protein in MDA-MB-231 (D), HCC1954 (E), and NMuMG-ErbB2 (F) breast cancer cells was measured using the Odyssey infrared imaging system, and the averaged fold increases relative to untreated controls (−BMP7 and −rChrdl1) from three independent experiments are shown. (G to I) BT549 (G), HCC1954 (H), and NMuMG-ErbB2 (I) breast cancer cells were serum starved overnight and stimulated with TGF-β (2 ng/ml) (+) or not stimulated (−) in the presence (+) or absence (−) of recombinant Chrdl1 (100 ng/ml) for 30 min. Immunoblot analyses were performed with antibodies against phospho-Smad2 and total Smad2/3. In all cases, α-tubulin was used as a loading control.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Recombinant, Phospho-proteomics, Blocking Assay, Western Blot, Imaging, Control

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Stable reduction of ShcA levels increases Chrdl1 expression within ErbB2-expressing mammary tumors in vivo. Representative images of ErbB2/ShcAhigh (A) and ErbB2/ShcAlow (B) mouse mammary tumors following immunohistochemical (IHC) staining with antibodies against ShcA or Chrdl1 are shown. Scale bar in ×4 panel, 100 μm (applies to all panels). Scale bar in ×20 inset, 20 μm (applies to all panels).

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Expressing, In Vivo, Immunohistochemical staining, Immunohistochemistry

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Conditioned medium containing Chrdl1 suppresses BMP4-induced Smad phosphorylation in breast cancer cells. (A) Immunoblot analysis with antibodies specific for phospho-Smad and total Smad1 in cells with high or low Chrdl1 levels following BMP4 treatment (30 min, 2.5 ng/ml). (B) ELISAs were performed on conditioned media from the indicated cell populations. Secreted Chrdl1 concentrations were normalized to protein concentrations from whole-cell protein lysates. (C and D) Immunoblot analysis with antibodies against ShcA was performed on NIC (C) and MDA-MB-231 (D) breast cancer cells that were transfected with scrambled control or ShcA-specific siRNAs and stimulated with TGF-β (2 ng/ml) or not stimulated for 24 h. Conditioned medium was collected and added to serum-starved NIC and MDA-MB-231 cells, which were left untreated or immediately stimulated with BMP4 (2.5 ng/ml) for 30 min. (E and F) NIC (E) or MDA-MB-231 (F) breast cancer cells were lysed and subjected to immunoblot analysis with antibodies specific for phospho-Smad1/5/8 (NIC), phospho-Smad1/5/9 (MDA-MB-231), or total Smad1. In all cases, α-tubulin was used as a loading control.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Phospho-proteomics, Western Blot, Transfection, Control

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Recombinant Chrdl1 diminishes BMP4-induced Smad phosphorylation in breast cancer cells. (A) Immunoblot analyses using antibodies against phospho-Smad1/8 and total Smad1 in NMuMG-ErbB2 breast cancer cells treated with BMP4 (30 min, 2.5 ng/ml) and increasing concentration of rChrdl1 (30 min, 25 to 250 ng/ml). (B) The ratio of phosphorylated Smad1/8 to total Smad1 protein in NMuMG-ErbB2 cells was measured using the Odyssey infrared imaging system, and the averaged fold increases relative to untreated controls (−BMP4 and −rChrdl1) from three independent experiments are shown. (C to E) Immunoblot analyses using antibodies against phospho-Smad and total Smad1 in NMuMG-ErbB2 (phospho-Smad1/8) (C), BT549 (phospho-Smad1/9) (D), and HCC1954 (phospho-Smad1/9) (E) breast cancer cells treated with BMP4 (30 min, 2.5 ng/ml) and rChrdl1 (30 min, 100 ng/ml). The ratio of phosphorylated Smads to total Smad1 protein in NMuMG-ErbB2 (C), BT549 (D), and HCC1954 (E) cells was measured using the Odyssey infrared imaging system and the fold increases relative to untreated controls (−BMP4 and −rChrdl1) are indicated at the bottom of each panel.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Recombinant, Phospho-proteomics, Western Blot, Concentration Assay, Imaging

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Chrdl1 blocks BMP4-induced but not TGF-β-induced cell migration of NMuMG-ErbB2 cells and BT549 breast cancer cells. Analysis of cellular velocity as measured by manual tracking of NMuMG-ErbB2 and BT549 cells is shown. Cells were stimulated with BMP4 (2.5 ng/ml) (A and C) or TGF-β (2 ng/ml) (B) in the absence (left panels) or presence (right panels) of rChrdl1 (100 ng/ml). The graphs represent the mean cellular velocity from one representative experiment (four experiments for NMuMG-ErbB2 cells and three experiments for BT549 cells). The arrow in each panel indicates the time that ligand (BMP4 or TGF-β), in the presence or absence of rChrdl1, was added to the cultures. *, P < 0.001.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Migration

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Clinical correlations between Chrdl1 expression and overall survival of lung, ovarian, and gastric cancer patients. Kaplan-Meier survival curves comparing Chrdl1 expression with overall survival in lung cancer (n = 1,926) (A), ovarian cancer (n = 1,581) (B) and gastric cancer (n = 1,305) (C) patients are shown. P values were calculated using the log rank test between patients with high versus low Chrdl1 expression by using a median split.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Expressing

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Chrdl1 impairs BMP4-induced breast cancer invasion and gelatin degradation. (A) Boyden chamber invasion assays were performed with HCC1954 and BT549 cells. The data represent the average from three (HCC1954) or four (BT549) independent experiments performed in triplicate. Representative images from each breast cancer cell line, under each condition, are shown. *, P < 0.001. (B) NMuMG-ErbB2 and BT549 cells treated with or without BMP4, in the presence or absence of Chrdl1, for 24 h were cultured on glass coverslips coated with green fluorescent protein (GFP) fluorescent-gelatin matrix. Cells were fixed and images captured using confocal microscopy. Quantification of matrix degradation is illustrated in the graph, and representative images from each experimental condition are shown. The data represent two independent experiments (20 images were quantified for each condition in each independent experiment). Arrows indicate areas of gelatin degradation. NMuMG-ErbB2: *, P = 0.0164; **, P = 0.044. BT549: *, P = 0.0373; ** P = 0.027.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Cell Culture, Confocal Microscopy

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Chrdl1 impairs BMP4-induced MMP2 and MMP9 enzymatic activity. (A) NMuMG-ErbB2 and BT549 breast cancer cells were stimulated with BMP4 (2.5 ng/ml) or not stimulated for 24 and 48 h. MMP2, MMP9, and MMP14 mRNA levels were measured by RT-qPCR. The data represent the means from three independent experiments performed in triplicate. No statistically significant differences in MMP mRNA expression were observed following BMP4 stimulation. (B and C) Conditioned media (CM) from NMuMG-ErbB2 (B) and BT549 (C) breast cancer cells stimulated with BMP4 (2.5 ng/ml) or not stimulated, in the presence or absence of rChrdl1 (100 ng/ml), for 48 h were harvested and analyzed for MMP activity by gelatin zymography. The gels were photographed, and band intensities were digitally quantified. The data represent the mean band intensity for each condition from three (B) or four (C) independent experiments. (B, left) *, P < 0.0022; **, P < 0.024; (B, middle) *, P < 0.022; **, P < 0.029; (C, left) *, P < 0.045; (C, middle) *, P < 0.016; **, P < 0.045.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Zymography

Journal: Molecular and Cellular Biology

Article Title: Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

doi: 10.1128/MCB.00600-15

Figure Lengend Snippet: Correlations between Chrdl1 expression and clinical outcomes in breast cancer patients. (A) Chrdl1 mRNA expression is elevated in the basal and luminal A subtypes. The average Chrdl1 mRNA levels in all breast cancer subtypes was compared to the average Chrdl1 mRNA levels in each individual subtype (Lum A, luminal A; Lum B, luminal B). (B to D) Kaplan-Meier survival curves comparing Chrdl1 expression with overall survival (OS) (B), disease-free survival (DFS) (C), or distant metastasis-free survival (DMFS) (D) in breast cancer (n = 1,115 patients). P values were calculated using the log rank test between patients with high versus low Chrdl1 expression by using a median split.

Article Snippet: To directly examine the effects of Chrdl1, breast cancer cells were treated with human recombinant Chrdl1 (rChrdl1) (catalog number 1808-NR/CF; R&D Systems).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells

doi: 10.3390/ijms262311340

Figure Lengend Snippet: Expression of tag-less Norrin K86P in BL21(DE3) cell inclusion body protein fractions. DISC-PAGE protein gel of inclusion body (IB) preparations from four separate batches are shown. IB material was dissolved and reduced by heating (100 °C × 10 min) in SDS sample buffer with 2-mercaptoethanol. Some dimeric and tetrameric bands can be formed during this treatment. Two different loading volumes (1 µL and 5 µL) were used for each of the four batches shown. Gels were stained with colloidal Coomassie blue. IB protein content was >98% recombinant Norrin K86P .

Article Snippet: The cells were weaned to media containing no Hydrocortisone Hemisuccinate prior to stimulation with Norrin K86P or rh Norrin (R&D Systems, Minneapolis, MN, USA; 3014-NR).

Techniques: Expressing, Staining, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells

doi: 10.3390/ijms262311340

Figure Lengend Snippet: Fundus and fluorescein angiography images before and after injection of Norrin K86P . Long Evans rats were imaged pre injection and 3 weeks post injection of 250 ng Norrin K86P . An example of the same Norin K86P -treated eye (OS) and vehicle-treated contralateral eye (OD) are shown. Brightfield fundus images are shown for each eye with their corresponding fluoresceine angiography image below. Angiography shows the perfused microvasculature of the neural retina (green). No impact on the retinal vasculature was noted for any eyes, whether Norin K86P - or vehicle-injected, across all rats tested. (N = 4). The same retinas were also analyzed for retinal thickness using SD-OCT.

Article Snippet: The cells were weaned to media containing no Hydrocortisone Hemisuccinate prior to stimulation with Norrin K86P or rh Norrin (R&D Systems, Minneapolis, MN, USA; 3014-NR).

Techniques: Injection

Journal: International Journal of Molecular Sciences

Article Title: Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells

doi: 10.3390/ijms262311340

Figure Lengend Snippet: Retinal thickness before and after intraocular injection of Norrin K86P . Long Evans rats received intraocular injections of vehicle (Veh) OD and 250 ng of Norrin K86P (Nor) OS. Average retinal thickness was calculated from 24 measurements around the optic disk for each retina, before injection and then again three weeks post injection. Thickness was measured between the inner limiting membrane (ILM) and outer limiting membrane (OLM) of the neural retina. The retinal thickness values (mm) averaged from four different rats are shown; bars show standard deviation.

Article Snippet: The cells were weaned to media containing no Hydrocortisone Hemisuccinate prior to stimulation with Norrin K86P or rh Norrin (R&D Systems, Minneapolis, MN, USA; 3014-NR).

Techniques: Injection, Membrane, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells

doi: 10.3390/ijms262311340

Figure Lengend Snippet: Mixed Rod–Cone ERG response of Long Evans rats. Long Evans rats received intraocular injections of vehicle (Veh) OD and 250 ng of Norrin K86P (Nor) OS. ( A ) Examples of Rod and mixed Rod–Cone ERG recordings from vehicle and contralateral Norrin K86P -injected eyes, 3 weeks post-injection. The left vertical marker indicates the light pulse flash at 0 milliseconds (ms). ( B ) Mixed Rod–Cone ERGs were measured, comparing vehicle-injected (OD) and Norrin-injected eyes, after dark adaptation. Average magnitudes of A-wave and B-wave (uV) are shown, using full-field white-light stimulation of 3.0 cd-sec/m 2 . Bars show standard deviation (N = 4 rats). No inhibition of Cone–Rod ERG response was detected in Norrin-treated versus vehicle-treated eyes.

Article Snippet: The cells were weaned to media containing no Hydrocortisone Hemisuccinate prior to stimulation with Norrin K86P or rh Norrin (R&D Systems, Minneapolis, MN, USA; 3014-NR).

Techniques: Injection, Marker, Standard Deviation, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells

doi: 10.3390/ijms262311340

Figure Lengend Snippet: Norrin-based protein sequences inserted into the pD454-MBP vector. Inserted sequences were added in frame following the MBP sequence. The resulting proteins were MBP N-terminal fusion proteins. ( A ) Nor-2 contained the mature human Norrin K86P , sequence underlined. The HRV3C protease cleavage site is shown in bold font. ( B ) Nor-w-Linker included extra linker sequences to improve the steric exposure of the HRV3C cleavage site. Three rigid helical linkers (blue font) and three flexible linkers (red font) are indicated. Two polypeptides identified by mass spectroscopy after electrophoresis of a Norrin-sized band (15 kDa) obtained after HRV3C protease treatment are highlighted with yellow.

Article Snippet: The cells were weaned to media containing no Hydrocortisone Hemisuccinate prior to stimulation with Norrin K86P or rh Norrin (R&D Systems, Minneapolis, MN, USA; 3014-NR).

Techniques: Plasmid Preparation, Sequencing, Mass Spectrometry, Electrophoresis