Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 1. Tumor-associated soluble uPAR (s-uPAR) enhances HUVEC invasion, migration and angiogenesis. (a) Conditioned medium (CM) was collected from tumor cells (parental and stably expressing empty vector (EV), uPAR-cDNA (UR) and uPAR siRNA (UR-Si)). Immunoblot analyses were performed for s-uPAR and DDK using specific antibodies. (b) s-uPAR levels in CM were quantified using uPAR Quantikine Immunoassay kit. Columns: mean; bars: s.d.; n ¼ 3; *po0.01 vs parental control. (c) Cells were labeled (tumor cells: Qtracker-525-Green and HUVECs: Qtracker- 655-Red) and seeded into separate chambers of culture inserts. After 16 h, the culture inserts were removed and cells were allowed to migrate for a further 24 h. Images were captured at 0 and 24 h of incubation and cell migration was quantified using ImageJ software (NIH). The levels of HUVEC migration were normalized to HUVEC migration in parental cells and are represented as arbitrary units. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental control. (d) HUVEC invasion experiments were performed using ThinCertTM inserts as described in Materials and methods. The levels of HUVEC invasion was quantified and normalized to HUVEC invasion in parental-CM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM. (e, f) In vitro angiogenesis assay was performed as described in Materials and methods. The degree of angiogenic induction by CM was quantified by ImageJ software (NIH) for the numerical value of the product of the relative capillary length per microscopic field. Serum-free medium (SFM) and recombinant human uPAR (rh-uPAR) in SFM were used as controls (insets). Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **po0.01 vs UR-CM. uPAR antibody, uPAR-Ab; isotype control, NSp.IgG. (g) Migration assay was performed using CM. In this case, both chambers of culture inserts were seeded with HUVECs. After 16 h, the culture inserts were removed, CM was added and cells were allowed to migrate for 24 h. Invasion assay was performed as described above. uPAR-Ab. or Nsp.IgG were added to UR-CM before adding onto cells. rh-uPAR was added to SFM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Migration, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Labeling, Incubation, Software, In Vitro, Angiogenesis Assay, Recombinant, Invasion Assay

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 2. s-uPAR recruits onto HUVEC membrane. Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. (a) HUVECs were cultured on CM for 24 h, labeled with anti-uPAR antibody, followed by Alexa Fluor-488-conjugated secondary antibody and were analyzed by fluorescence-activated cell sorting (FACS) for uPAR expression. Serum-free medium (SFM) and rh-uPAR were used as controls. Isotype control (Neg.). (b) HUVECs were cultured in chamber slides on CM for 24 h and fixed in 4% paraformaldehyde and 0.2% glutaraldedyde in phosphate-buffered saline for 1 h. Immunocytochemical analysis was performed as described in Materials and methods. Isotype control (Neg.; inset). Slides were mounted and photographed. (c) Equal amounts of proteins were used for the extraction of HUVEC membrane fractions and were subjected to immunoblot analysis for uPAR expression using specific antibodies. The blot was re-probed for DDK-tag expression.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Membrane, Cell Culture, Labeling, FACS, Expressing, Control, Saline, Extraction, Western Blot

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 3. s-uPAR colocalizes in lipid rafts on HUVECs. Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. (a) HUVECs were cultured in chamber slides on CM for 24 h and incubated with anti-uPAR antibody followed by Alexa Fluor-488- conjugated secondary antibody at 4 1C. Cells were again labeled with Alexa Fluor-595-CTxB subunit. Slides were mounted and analyzed by confocal microscopy. Negative controls, using an isotype antibody, showed no staining (inset). Serum-free medium (SFM) and DDK-tag containing rh-uPAR were used as controls. To disrupt lipid rafts, HUVECs were pretreated with MBCD, as described in Materials and methods. (b) HUVECs lipid rafts were isolated as described in Materials and methods. Lipid raft-enriched fractions were analyzed for uPAR and DDK-tag levels using immunoblot analysis. Flotillin-1 and caveolin-1 served as controls. Protein band intensities were quantified by densitometric analysis using ImageJ software (NIH). The levels of uPAR protein were normalized to protein levels in HUVECs cultured on parental-CM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM. (c) Invasion and migration assays were performed as described in Figure 1d In vitro angiogenesis assay was performed as described in Figure 1. To deplete cholesterol, HUVECs were pretreated with MBCD as described in Materials and methods (c and d). Columns: mean; bars: s.d.; n ¼ 3; *po0.01 vs parental-CM; **po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Cell Culture, Incubation, Labeling, Confocal Microscopy, Staining, Isolation, Western Blot, Software, Migration, In Vitro, Angiogenesis Assay

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 4. s-uPAR induces ERK/Rac1-mediated migration and tube formation in HUVECs. Conditioned medium (CM) was collected from tumor cells, as described in Materials and methods. (a) HUVECs lysates were used to perform GST-Rac1 pull-down assay. The protein complexes were subjected to immunoblot analysis to detect active Rac1. Rac1 from total cell lysates was used as a control. (b) Total cell lysates were subjected to immunoblot analysis for phospho-ERK1/2 (pERK1/2) and total ERK1/2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. HUVECs grown on rh-uPAR were used as a control. (c) HUVECs were cultured on CM alone and/or supplemented with functional blocking anti-uPAR antibody (uPAR-Ab) or isotype control (Nsp.IgG.) or MEK inhibitor (U0126) for 24 h. Cell lysates or GST-Rac1 pull- down protein complexes were subjected to immunoblot analysis to detect active Rac1, Rac1 pERK1/2 and ERK1/2. GAPDH served as a loading control. (d) HUVECs were transfected with dominant-negative mutant Rac1 (Dn-Rac1) for 24 h and cultured on UR-CM. Micrographs were captured for green fluorescent protein (GFP) expression (green) and phase contrast (gray) immediately after the addition of UR-CM (magnification 60). (e) HUVECs were transfected with Dn-Rac1 for 24 h, cultured on CM for another 24 h, collected and lysed. GST-Rac1 pull- down protein complexes were subjected to immunoblot analysis to detect active Rac1. GFP and Rac1 from total cell lysates were used as controls. (f) HUVECs were transfected with Dn-Rac1 for 24 h and cultured on CM alone and/or supplemented with uPAR-Ab., or Nsp.IgG or U0126 for another 24 h. Invasion and migration assays were performed as described in Figure 1. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **Po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Migration, Pull Down Assay, Western Blot, Control, Cell Culture, Functional Assay, Blocking Assay, Transfection, Dominant Negative Mutation, Expressing

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 5. Diverse forms of tumor-associated s-uPAR in vitro and in vivo. (a) Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. CM was subjected to deglycosylation using a deglycosylation kit and analyzed by immunoblot for uPAR using specific antibodies. (b) Equal amount of proteins containing HUVEC lysates were used for extraction of cell membrane fractions and were subjected to deglycosylation, and analyzed by immunoblot for uPAR using specific antibodies. (c) In vivo angiogenic assay was performed by using the dorsal air sac model. 4910EV (EV), 4910UR (UR), 4910UR-Si (UR-Si) cells or a recombinant human uPAR (rh-uPAR) containing chamber was implanted in the dorsal cavity of mice. The micrographs for the presence of tumor-induced neovasculature (microvessels with curved thin structures and many tiny bleeding spots) and pre-existing vasculature (straight) were captured. Representative micrographs are shown. (d, e) Blood was collected from mice orthotopically xenografted with stably expressing EV, UR and UR-Si cells. Total uPAR levels were estimated using a commercial human uPAR Quantikine Immunoassay kit according to the manufacturer’s instructions. The data quantification for a set I (n ¼ 4; d) and set II (n ¼ 6; e), on day 15 and 40, respectively, after cell implantation are shown. Columns: mean; bars: s.d.; *Po0.01 vs parental control. (f) Blood serum (from mice 1–6; on day 40) was subjected to deglycosylation and analyzed by immunoblot for uPAR using specific antibodies. D2-D3, D2-D3 domain containing truncated s-uPAR; D3, D3 domain containing truncated s-uPAR; FL, full-length s-uPAR; .

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: In Vitro, In Vivo, Western Blot, Extraction, Membrane, Recombinant, Stable Transfection, Expressing, Control

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 6. uPAR overexpression enhances tumor growth, vascularity and s-uPAR recruits onto endothelial cells in vivo. (a) Stably expressing EV, UR and UR-Si cells were injected intracerebrally into mice. Mice were euthanized and brains were collected and fixed as described in Materials and methods. Brain sections were stained with hematoxylin and eosin (H&E) solution, and representative micrographs are shown (upper panel). H&E-stained micrographs showing the tumor invasive front ( 20; lower panel). (b) Brain tumor areas were calculated using Image Pro Discovery Program software (Media Cybernetics, Inc., Rockville, MD, USA). Columns: mean; bars: s.d.; n ¼ 6; *Po0.01 vs parental controls. (c) Immunohistochemical analysis of brain sections using anti-uPAR and anti-vascular endothelial growth factor (VEGF). Blood vessels in tumor sections were visualized with biotin-labeled tomato lectin. Inset: isotype control. (d, e) Fluorescence microscopy for colocalization of an endothelial cell marker (von Willebrand factor (vWF)/anti-CD31) and DDK-tag in tumor sections from mice that were implanted with 4910 EV (EV) and 4910UR (UR) cells. Inset, isotype control.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Over Expression, In Vivo, Stable Transfection, Expressing, Injection, Staining, Software, Immunohistochemical staining, Labeling, Control, Fluorescence, Microscopy, Marker

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 1. The IJM region is necessary for collagen-induced DDR2 activation. (a) Overall topology and alignment of the transmembrane (TM) domain and the intracellular juxtamembrane region (IJM) of DDR1a and DDR2. The IJM was divided into three regions: JM1, JM2 and JM3. (b) Schematic diagram of various DDR2 constructs used in our study. (c) HEK293T cells transiently transfected with plasmids encoding the full-length DDR2 and F-DJM1-JM2 mutant were stimulated by Type I collagen for 60 min. Tyrosine phosphorylation of the F-DJM1-JM2 mutant was inhibited compared to that of the full-length DDR2. (d) HEK293T cells were transfected with plasmids encoding full-length DDR2, F- DJM1 and F-DJM2 and were stimulated by collagen. The F-DJM2 mutant showed a significant decrease in tyrosine phosphorylation. **p < 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Activation Assay, Construct, Transfection, Mutagenesis, Phospho-proteomics

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 2. DDR2 dimerizes via the JM2 of the IJM region. (a and b) HEK293T cells were transiently cotransfected with plasmids encoding TM- JM1-JM2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blot analysis showed that the cytoplasmic domains of DDR2 bind to each other via the intact TM-JM1-JM2 domain and form homodimers. Asterisks indicate the expected size of TM-JM1-JM2. (c) HEK293T cells were transfected with plasmids encoding F-DJM1 and F-DJM2 mutants. A crosslinking assay showed that dimers of F-DJM1 were not changed compared to full-length DDR2, whereas dimers were significantly decreased for F-DJM2.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Immunoprecipitation, Western Blot, Transfection

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 3. JM2 has a dominant-negative effect on DDR2 activation. (a and b) HEK293T cells were cotransfected with plasmids encoding full- length DDR2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blotting showed that the full-length DDR2 and TM-JM1-JM2 bind to each other to form heterodimers. The immunoprecipitates obtained with anti-IgG antibodies were used as a negative control. (c) H1299 cells transfected with plasmids encoding full-length DDR2 and TM-JM1-JM2 and HeLa cells were lysed, and the whole cell lysates (W) were separated into the plasma membrane (P) and cytosol (C) fractions. EGFR and a-tubulin were used as positive controls for the plasma mem- brane and cytosol fractions, respectively. Endogenous full-length DDR2 (HeLa cells), forced-expressed full-length DDR2 and TM-JM1-JM2 pro- teins were appropriately localized in the plasma membrane. (d) HEK293T cells were transfected with plasmids encoding a C-terminally myc- tagged full-length DDR2 and TM-JM1-JM2. Only under the permeabilized condition, full-length DDR2-myc and TM-JM1-JM2-myc were visual- ized, indicating that the C-termini of these proteins were located in the cytosol and not extracellular space. Full-length DDR2 was used as a positive control. Bar, 50 mm. (e) HEK293T cells were cotransfected with plasmids encoding full-length DDR2 (500 ng) and an increasing amount of TM-JM1-JM2 (100, 300 and 500 ng) as indicated and then stimulated with collagen. Tyrosine phosphorylation gradually decreased with an increasing amount of TM-JM1-JM2. (f) HeLa cells were transiently transfected with a plasmid encoding TM-JM1-JM2 and were then stimulated. Tyrosine phosphorylation was significantly decreased in endogenous DDR2. **p < 0.01, Student’s t-test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Dominant Negative Mutation, Activation Assay, Immunoprecipitation, Western Blot, Negative Control, Transfection, Clinical Proteomics, Membrane, Positive Control, Phospho-proteomics, Plasmid Preparation

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 4. JM2 regulates the collagen-binding affinity of DDR2. (a and b) HEK293T cells transiently transfected with various DDR2 constructs were harvested, and protein expression was verified by Western blot- ting (a). Collagen-binding affinities were reduced in F-DJM2, F-DJM1- JM2 and extra mutants but not the F-DJM1 mutant in a dose- dependent manner (b). Nontransfected (NC) and TM-JM1-JM2 samples were used as negative controls. **p< 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Binding Assay, Transfection, Construct, Expressing, Western Blot, Mutagenesis

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 5. Colony formation and proliferation of tumor cells are suppressed by overexpression of JM2. (a and b) Formalin-fixed tissue micro- array slides were used in immunohistochemistry experiments. DDR2 was overexpressed in bladder, testis, lung, kidney, prostate and stom- ach cancers. Bar, 50 mm. (c) Stable TM-JM1-JM2–expressing H1299 cells were stimulated by collagen and then harvested. Immunoprecipitation and Western blot analysis showed that tyrosine phosphorylation of DDR2 was decreased by TM-JM1-JM2 overexpres- sion (labeled JM1/2), but phosphorylation of DDR1 was unaffected. (d) A colony-forming assay of H1299 cells showed that the number and projected area of colonies were decreased by TM-JM1-JM2 overexpression. Bar, 100 mm. (e) Proliferation of H1299 cells was assessed by cell counting (left) and an MTT assay (right). Cell proliferation was inhibited by TM-JM1-JM2 overexpression. **p < 0.01, Student’s t-test; control, nontransfected cells; Mock, empty vector stably transfected cells; JM1/2, TM-JM1-JM2 stably transfected cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Over Expression, Microarray, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics, Labeling, Cell Counting, MTT Assay, Control, Plasmid Preparation, Stable Transfection, Transfection

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Internalization of fluorescein isothiocyanate-conjugated monoclonal anti-human CCR7 induced by recombinant CCL19 in intact HEK 293a cells that express recombinant CCR7. nt, non-transfected cells.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Recombinant, Transfection

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Endocytosis of the anti-myc monoclonal antibody (clone 4A6, conjugated to AlexaFluor-488, final concentration in the culture medium 3.3 nM) as determined by co-treatment with the CCL19-myc construction in intact HEK 293a cells that optionally and transiently expressed CCR7. A control conditioned medium (CM) or authentic CCL19 were used as control stimuli. The undiluted CM were transferred for a 30-min incubation period before rinsing and observation. Original magnification ×1000.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Concentration Assay, Control, Incubation

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Cytofluorometry of HEK 293a cells that optionally expressed CCR7, sequentially detached and further stimulated with the undiluted CM of other cells producing CCL19-myc, authentic CCL19, or CM of untransfected cells, as indicated (30 min incubation at 37°C). Left: distributions based on the counting of 10,000 cells. A threshold of autofluorescence was defined using control cells with no fluorophore. It was surpassed only under one set of experimental conditions (arrow). Right: mean fluorescence of cells in replicated experiment. ANOVA indicated that the values were heterogeneous ( p < 10 -4 ). * p < 0.01 vs. top-most value (control CM, untransfected recipient cells) by Dunnett’s test.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Incubation, Control, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Epifluorescence microscopy studies in cells co-expressing β-arrestin 1 -Cherry and CCR7 and stimulated as indicated (stimulant and duration). Stimulation always included the fluorescent anti-myc monoclonal antibody 4A6 (green signal). Either CCL19-myc CM or authentic CCL19 rarely condensed β-arrestin 1 at the level of plasma membranes of endosomes (arrowhead indicates possible co-localization with the CCL19-myc-antibody cargo). Cells that expressed well the red-emitting transgene were assumed to co-express co-transfected CCR7 and were evaluated for morphology ( n = 16–49 eligible cells per group). The average number of red condensed structures was 0.4 ± 0.4 in cells treated with control CM and did not vary significantly in all other groups (Kruskal–Wallis test). The average number of green condensed structures in cells treated with control CM was 0 per cell and varied significantly according to treatments ( p < 10 -4 , Kruskal–Wallis test; p values for Dunn’s multiple comparison test for each value vs. that of control CM reported thereafter). There were 8.3 ± 1.7 green specs per cell ( p < 0.05), 25.8 ± 3.2 ( p < 0.001), 33.1 ± 4.4 ( p < 0.001), and 0 (N.S.) in cells treated with CCL19-myc CM for 5, 10, or 30 min, or with recombinant CCL19, respectively.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Epifluorescence Microscopy, Expressing, Clinical Proteomics, Transfection, Control, Comparison, Recombinant

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Epifluorescence microscopy studies in cells co-expressing CCR7 and β-arrestin 2 -GFP and stimulated as indicated (stimulant and duration). Stimulation did not include the fluorescent anti-myc antibody. Either CCL19-myc CM or authentic CCL19 frequently condensed β-arrestin 2 at the level of plasma membrane or endosomes. Right: number of green intracellular condensed structures (“specs”) per cell ± SEM. Numbers close to bars indicate the number of evaluated HEK 293a cells. The Kruskal–Wallis test indicated that the values were heterogenous ( p < 10 -4 ). The effect of each treatment vs. the effect of control CM was evaluated using Dunn’s multiple comparison test. * p < 0.01; ** p < 0.001.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Epifluorescence Microscopy, Expressing, Clinical Proteomics, Membrane, Control, Comparison

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Epifluorescence microscopy studies in cells co-expressing one of three forms of Rab5-Cherry fusion proteins and CCR7 and stimulated for 30 min with either CCL19-myc or control CM. Co-localization between Rab5-positive corpuscles and the CCL19-myc-antibody cargo was observed (arrowheads). The dominant positive (GTP-locked) mutant of the fusion protein produced typical giant vacuoles that included the green light-emitting cargo. The dominant negative Rab5-GDP-locked-Cherry inhibited the endocytosis of the green cargo (see text for numerical analysis).

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Epifluorescence Microscopy, Expressing, Control, Mutagenesis, Produced, Dominant Negative Mutation

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Epifluorescence microscopy studies in cells co-expressing Rab7-Cherry and CCR7 and stimulated as indicated (stimulant and duration). Co-localization between Rab7-positive corpuscles and the CCL19-myc-antibody cargo was not highly frequent (arrowheads), but increased in frequency as a function of incubation duration (see text for numerical analysis).

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Epifluorescence Microscopy, Expressing, Incubation

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Presence of CCR7 in A375 melanoma cells. (A) RT-PCR for CCR7 in the human melanoma A375 cell line. (B) c-Fos induction in A375 cells stimulated as indicated for 1 h (typical immunoblot and histograms representing the densitometry of replicated experiments).

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Detection of endogenous CCR7 in A375 cells using detection of the endocytosed CCL19-myc-4A6 antibody complex using theTyramide Signal Amplification (TSA) system that enzymatically generates AlexaFluor-488 labeling. Epifluorescence and transmission, original magnification: ×1000. Right: proportion of cells with a mean fluorescence intensity above a set threshold (Photoshop level ≥ 50/255) in large photographic records of A375 cells stained as in the left of figure (the number of evaluated cells is indicated next to each histogram). The proportions were compared with that of control cells without antibody (top-most histogram, χ 2 test; * p < 10 -4 ).

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Amplification, Labeling, Transmission Assay, Fluorescence, Staining, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Immunohistochemistry staining with antibody against phosphorylated Smad2 (pSmad2) and TGFβ receptor II (TβRII) showed increased nuclear signal for pSmad2 in the invasive ECdnT organotypic cultures. Scale bar 50 micron. (b) Analysis of immunohistochemistry staining for TβRII and pSmad2 in 83 ESCC cases in a tissue microarray shows no significant correlation. Fisher’s exact test, two tailed p= 0.3182. (c) Five paired normal adjacent and ESCC tissues (GSE17531) were analyzed for INHBA mRNA expression, which identified upregulation of INHBA in four ESCC samples. (d) Waterfall plot of a publically available dataset (GSE23400) represented upregulation of INHBA in the ESCC (grey bars) samples vs. normal (black bars).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Staining, Microarray, Two Tailed Test, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures with either fetal esophageal fibroblasts (FEF) or cancer-associated fibroblasts (CAF) embedded in the underlying matrix. Immunofluorescence staining with antibody against αSMA (green) and podoplanin (red) showed similar expression pattern in the cultures. Scale bar is 50 micron. (b) Activin A concentration in conditioned media from organotypic cultures is higher in invasive cultures as measured using indirect ELISA. * p=0.003, ** p= 0.005, *** p=0.03 (c) Stimulation of epithelial cells with Act A in monolayer plastic culture demonstrated phosphorylation of Smad. Neutralizing antibody against Activin (nAb) prevented the induction of pSmad2 by Act A. Following stimulation with Act A or with conditioned media from organotypic culture increased expression of vimentin was detected after 48 hours by Western Blot. The increase was reversed in the presence of neutralizing antibody (nAb). (d) Inhibition with the Act A antagonist, Follistatin, or a pan-TGFβ inhibitor A83-01 suppressed MMP-9 secretion in E, EC and ECdnT cells as measured by gelatin zymography. Upper bands reflect pro-MMP, lower bands activated, cleaved MMP (arrow).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Immunofluorescence, Staining, Concentration Assay, Indirect ELISA, Phospho-proteomics, Western Blot, Inhibition, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures in the presence of recombinant Activin A (Act A), its antagonist Follistatin or a neutralizing antibody against Activin A (nAb); H&E staining. Stimulation with Act A inhibited invasion of E and EC cells, but failed to suppress ECdnT cell invasion. Follistatin increased cell invasion in all cell types, while the neutralizing antibody prevented invasion of E and EC cells, without an effect on ECdnT cells. (b) Immunohistochemistry staining with ki67-antibody showed no differences in cell proliferation. Scale bars are 50 micron. (c) Indirect ELISA with antibody against Act A measured increased levels after addition of recombinant Act A in fibroblasts (FEF) and ECdnT. Untreated ECdnT cells (Control) secreted higher baseline levels of Act A than FEF, which were reduced by Follistatin. (d) TGFβ1 concentration was increased in response to stimulation with Act A and overall baseline secretion was higher in control ECdnT cells than fibroblasts as measured by indirect ELISA. Follistatin inhibited TGFβ1 secretion.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Recombinant, Staining, Immunohistochemistry, Indirect ELISA, Control, Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Separating the cellular matrix and epithelium of the organotypic cultures growing ECdnT cells through a collagen I layer, dashed lines, prevented cell invasion in the absence (control) and presence of Act A (+Act A). When the cellular matrix of the organotypic culture was treated with puromycin to kill the embedded fibroblasts before the ECdnT cells were seeded, epithelial formation occurred but invasion was inhibited with and without Act A stimulation. (b) Treatment of ECdnT organotypic cultures with a pan-MMP inhibitor, GM6001, suppressed cell invasion, which was not restored in the presence of Act A. Untreated (no tx) control ECdnT cells in organotypic culture invaded into the underlying matrix. TGFβ1 treatment inhibited epithelial cell invasion. Scale bars are 50 micron. (c) Immunohistochemistry showed nuclear localization of phosphorylated Smad (pSmad2, red) in control and Act A stimulated conditions. Collagen IV, red, was disrupted in invasive cultures after Act A treatment. Loss of the fibroblasts (FEF), labeled green with antibody against vimentin (no staining in the lower panels), had no effect on the nuclear localization of pSmad2. The collagen IV layer was not disrupted in non-invasive cultures in the absence of FEFs.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Control, Immunohistochemistry, Labeling, Staining

Journal: Journal of Biological Chemistry

Article Title: Regulation of Cyclin D1 and Wnt10b Gene Expression by cAMP-responsive Element-binding Protein during Early Adipogenesis Involves Differential Promoter Methylation

doi: 10.1074/jbc.m806423200

Figure Lengend Snippet: FIGURE4.Bt2cAMPblocksWnt10bexpressionin3T3-L1cells.3T3-L1prea- dipocytes were treated with 0.3 mM Bt2cAMP (A and B) or MDI (B) for 6 (A) or 24 h (A and B). A, total RNA was recovered from the cells and subjected to RT-PCR for Wnt10b or -actin. The analysis was repeated three times, and a representative figure shows that Bt2cAMP treatment lowers Wnt10b RNA lev- els. B, prior to MDI or Bt2cAMP treatment, the cells were transfected with a plasmid containing a 900-bp portion of the Wnt10b gene promoter linked to firefly luciferase. The cells were cotransfected with a plasmid from which Renilla luciferase was expressed from a constitutive thymidine kinase pro- moter as a transfection control. Firefly luciferase levels were measured (and corrected for Renilla luciferase levels) as an index of Wnt10b promoter activ- ity. The figure shows data averaged from three experiments, indicating that MDI and Bt2cAMP inhibit transcription from the Wnt10b gene promoter.

Article Snippet: A mouse monoclonal neutralizing antibody to Wnt10b was purchased from R & D Systems (Minneapolis, MN).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Luciferase, Control

Journal: Journal of Biological Chemistry

Article Title: Regulation of Cyclin D1 and Wnt10b Gene Expression by cAMP-responsive Element-binding Protein during Early Adipogenesis Involves Differential Promoter Methylation

doi: 10.1074/jbc.m806423200

Figure Lengend Snippet: FIGURE 5. CREB stimulates Wnt10b gene transcription. A–C, 3T3-L1 preadipocytes were transfected with plasmids containing the Wnt10b gene promoter linked to firefly luciferase and the enhancerless thymidine kinase promoter linked to Renilla luciferase (transfection control). A, cells were cotransfected with plasmids for the expression of wild type (WT), dominant negative (KCREB), or constitutively active (VP16-CREB or CREB DIEDML)formsofCREB.B,plasmid-transfectedcellsweresubsequentlytransfectedwithno(Cntrl)oligonucleo- tide or single-stranded scrambled or CREB-specific phosphorothioate antisense oligonucleotides as described under “Experimental Procedures.” C, plasmid-transfected cells were transfected with luciferase or CREB-spe- cific siRNA and then treated with MDI or 0.3 mM Bt2cAMP or left untreated (Cntrl). A–C, 48 h post-transfection/ treatment,fireflyluciferasewasmeasuredincelllysatesandcorrectedforRenillaluciferaselevels.A,datashows that dominant negative KCREB suppresses while constitutively active CREB isoforms stimulate Wnt10b gene promoter activity. Additional results indicate that depletion of CREB with antisense (B) or siRNA (C) inhibits Wnt10b gene promoter activity. D, preadipocytes were transfected with luciferase (Cntrl) or CREB-specific siRNA.48hlaterthecellsweretreatedwith0.3mMBt2cAMPfor0or6h.TotalRNAwasrecoveredandsubjected to RT-PCR for Wnt10b or -actin. The data indicate that CREB depletion reduces Wnt10b RNA levels. The figure shows data averaged from three experiments.

Article Snippet: A mouse monoclonal neutralizing antibody to Wnt10b was purchased from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Control, Expressing, Dominant Negative Mutation, Plasmid Preparation, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Regulation of Cyclin D1 and Wnt10b Gene Expression by cAMP-responsive Element-binding Protein during Early Adipogenesis Involves Differential Promoter Methylation

doi: 10.1074/jbc.m806423200

Figure Lengend Snippet: FIGURE 6. Putative CRE sites within the Wnt10b gene proximal promoter. The sequences of seven oligonucleotides used for subsequent DNA binding studies are shown with the putative CRE sites in alignment with the consensus CREor12-O-tetradecanoylphorbol-13-acetateresponseelementsequences.The percentage of homology of each putative CRE to the consensus CRE or 12-O- tetradecanoylphorbol-13-acetate response element (TRE) is indicated.

Article Snippet: A mouse monoclonal neutralizing antibody to Wnt10b was purchased from R & D Systems (Minneapolis, MN).

Techniques: Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Regulation of Cyclin D1 and Wnt10b Gene Expression by cAMP-responsive Element-binding Protein during Early Adipogenesis Involves Differential Promoter Methylation

doi: 10.1074/jbc.m806423200

Figure Lengend Snippet: FIGURE7.CREBstimulatestranscriptionfromtheWnt10bgenepromoterthroughtwoCREsites.A,CREBDNA binding assays were performed with biotinylated forms of each of the oligonucleotides shown in Fig. 6. Control assays were performed with a biotinylated oligonucleotide containing the consensus CRE sequence, an oligonu- cleotidewithascrambledsequence,ornooligonucleotide.Theabsorbanceofeachreactionismeasuredat450nm asanindexofCREBbinding.ThedataaveragesfromthreeseparateassaysindicatethatCREBbindstotheCREs5and 6 (Fig. 6) in the Wnt10b gene promoter. B, competition binding assays were performed by binding a biotinylated consensusCREoligonucleotidetotheassaywells.Thebindingreactionscontaineda10-foldexcessofeachWnt10b CRE oligonucleotide or consensus CRE-containing or scrambled oligonucleotides (as controls). Absorbance at 450 nmrevealedsignificantcompetitionofCREBbindingtothebiotinylatedCREsitebyWnt10bCREs5and6(averaged from three studies). C, CREs 5 and 6 were mutated, either individually or in combination, in the Wnt10b gene promoter. Wild type (WT) or mutated promoters were ligated into a plasmid upstream of the firefly luciferase gene. These plasmids were cotransfected in to 3T3-L1 preadipocytes with a plasmid containing the enhancerless thymi- dinekinasegenepromoterlinkedtoRenillaluciferase(transfectioncontrol).Thecellswerethentreatedwith0.3mM Bt2cAMP or left untreated (NA) for 24 h. Luciferase levels were measured in cell lysates as an index of promoter activity. The results averaged from three experiments indicate that mutation of CREs 5 and 6, individually or in combination, attenuates transcription from the Wnt10b gene promoter.

Article Snippet: A mouse monoclonal neutralizing antibody to Wnt10b was purchased from R & D Systems (Minneapolis, MN).

Techniques: Binding Assay, Control, Sequencing, Plasmid Preparation, Luciferase, Activity Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Regulation of Cyclin D1 and Wnt10b Gene Expression by cAMP-responsive Element-binding Protein during Early Adipogenesis Involves Differential Promoter Methylation

doi: 10.1074/jbc.m806423200

Figure Lengend Snippet: FIGURE 8. Bt2cAMP regulates binding of CREB to the Wnt10b and cyclin D1 gene promoters. Chromatin immunoprecipitation was performed on 3T3-L1 preadipocytes treated with Bt2cAMP for 0, 6, and 24 h. Formaldehyde- cross-linked chromatin was fragmented and immunoprecipitated with either isotype matched IgG (negative control) or CREB-specific antibodies. Precipi- tated DNA was PCR-amplified with primers to the Wnt10b and cyclin D1 gene promoters. Representative gel photographs show that Bt2cAMP inhibits bindingofCREBtotheWnt10bgenepromoterwhileincreasingCREBbinding to the cyclin D1 gene promoter. Similar results were obtained in three sepa- rate assays.

Article Snippet: A mouse monoclonal neutralizing antibody to Wnt10b was purchased from R & D Systems (Minneapolis, MN).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Amplification

Journal: Journal of Biological Chemistry

Article Title: Regulation of Cyclin D1 and Wnt10b Gene Expression by cAMP-responsive Element-binding Protein during Early Adipogenesis Involves Differential Promoter Methylation

doi: 10.1074/jbc.m806423200

Figure Lengend Snippet: FIGURE 9. Bt2cAMP regulates methylation of the Wnt10b gene promoter. 3T3-L1 preadipocytes were treated with Bt2cAMP for the times indicated. Methylation of the Wnt10b and cyclin D1 gene promoters was assessed using commercial reagents as described under “Experimental Procedures.” The fig- ure shows representative agarose gels on which Wnt10b and cyclin D1 pro- moter fragments were resolved. Bt2cAMP increases Wnt10b gene promoter methylation. No methylation of the cyclin D gene promoter was noted under any condition/time point.

Article Snippet: A mouse monoclonal neutralizing antibody to Wnt10b was purchased from R & D Systems (Minneapolis, MN).

Techniques: Methylation

Journal: Journal of Biological Chemistry

Article Title: Regulation of Cyclin D1 and Wnt10b Gene Expression by cAMP-responsive Element-binding Protein during Early Adipogenesis Involves Differential Promoter Methylation

doi: 10.1074/jbc.m806423200

Figure Lengend Snippet: FIGURE 10. Inhibition of DNA methyltransferases activity prevents Bt2cAMP-induced loss of CREB binding to the Wnt10b gene promoter and repression of Wnt10b expression but has no effect on CREB binding to the cyclin D1 gene promoter. A and C, preadipocytes were treated with Bt2cAMP for 24 h in the presence or absence of 5-azacytidine. The cells were treated briefly with formaldehyde to cross-link proteins to DNA, and the DNA was recovered and fragmented. The DNA fragments were immunoprecipi- tated with isotype-matched IgG or CREB-specific antibodies. The precipitated DNA was PCR-amplified with primers to the Wnt10b (A) or cyclin D1 (C) gene promoters. The figure demonstrates that the down-regulation of CREB bind- ing to the Wnt10b gene promoter by Bt2cAMP is inhibited by blockade of DNA methyltransferase activity. However, azacytidine had no affect on Bt2cAMP-induced CREB binding to the cyclin D1 gene promoter. B, preadipo- cytes were treated as in A, and total RNA was recovered and subjected to RT-PCR using primers to the Wnt10b coding region. The data show that whereas Bt2cAMP decreases Wnt10b gene expression, this affect is repressed by inhibition of DNA methyltransferases activity. Results are representative of three separate experiments.

Article Snippet: A mouse monoclonal neutralizing antibody to Wnt10b was purchased from R & D Systems (Minneapolis, MN).

Techniques: Inhibition, Activity Assay, Binding Assay, Expressing, Amplification, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Regulation of Cyclin D1 and Wnt10b Gene Expression by cAMP-responsive Element-binding Protein during Early Adipogenesis Involves Differential Promoter Methylation

doi: 10.1074/jbc.m806423200

Figure Lengend Snippet: FIGURE 11. Model for the regulation of cyclin D1 and Wnt10b gene tran- scription by cAMP during early adipogenesis. Treatment of confluent 3T3-L1 preadipocytes with cAMP mimetics stimulates the phosphorylation and binding of CREB to the cyclin D1 gene promoter, resulting in transient stimulation of cyclin D1 expression. Alternatively, cAMP induces the methyl- ation of the Wnt10b gene promoter, which attenuates CREB binding to this promoter and reduces Wnt10b expression.

Article Snippet: A mouse monoclonal neutralizing antibody to Wnt10b was purchased from R & D Systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Binding Assay, Expressing