Journal: Advanced Science

Article Title: Combinatorial Microgels for 3D ECM Screening and Heterogeneous Microenvironmental Culture of Primary Human Hepatic Stellate Cells

doi: 10.1002/advs.202303128

Figure Lengend Snippet: a) Representative maximum intensity projection of confocal images of HSCs cultured with 8C4 and 8FN scaffolds, the lowest and highest conditions for PDGFRβ expression. Light blue: DAPI. Purple: DiD membrane stain. Green: anti‐PDGFRβ. Scale bar: 100 µm. b) Box and whisker plots of the anti‐PDGFRβ fluorescence intensity per nucleus normalized to the average of all conditions per experimental replicate. n = 9 from three experimental replicates. c) Representative maximum intensity projection of confocal images of HSCs cultured with 8C1 and 8C4 scaffolds, the lowest and highest conditions for collagen I expression. Light blue: DAPI. Purple: DiD membrane stain. Yellow: anti‐collagen I. Scale bar: 100 µm. d) Box and whisker plots of the anticollagen I fluorescence intensity per nucleus normalized to the average of all conditions per experimental replicate. n = 9 from three experimental replicates. Two‐way interaction ANOVA. * p < 0.05 ** p < 0.01 *** p < 0.001 where * means between conditions and ^ means versus eight‐arm 8% counterpart.

Article Snippet: Microwells had their media removed and were fixed with 4% paraformaldehyde (RT15710, Electron Microscopy Sciences) in PBS for 20 min. Microwells were then washed twice with PBS and permeabilized with 0.5% Triton X‐100 (X100, MilliporeSigma) in PBS for 15 min. Microwells were washed once with PBS and blocked with 1% w/v bovine serum albumin (BSA, A2153, MiliporeSigma) in 0.1% Triton X‐100 in PBS for 1 h. Microwells were washed once with PBS and stained for 24 h with 2 μg mL −1 of anti‐human procollagen I alpha one antibody (AF6220, R&D Systems) and 0.848 μg mL −1 anti‐LOX antibody (ab174316, abcam) or 4 μg mL −1 anti‐human PDGFR beta antibody (AF385, R&D Systems) and 10 μg mL −1 anti‐human/mouse/rat alpha‐smooth muscle actin antibody (MAB1420, abcam) in 0.1% BSA and 0.1% Triton X‐100 in PBS for 24 h in a gentle shaker.

Techniques: Cell Culture, Expressing, Membrane, Staining, Whisker Assay, Fluorescence

Journal: bioRxiv

Article Title: Interleukin-1 is overexpressed in injured muscles following spinal cord injury and promotes neurogenic heterotopic ossification

doi: 10.1101/2021.10.19.464906

Figure Lengend Snippet: IL-1β is expressed in human NHO biopsies and stimulates calcium mineralization of NHO-derived FAPs in vitro . (A) Representative immunohistochemistry staining of serial sections of a human NHO biopsy (SCI patient) demonstrating the colocalization of the macrophage marker CD68 (top panel left) and IL-1β protein expression (top panel right). Black rectangle shows IL-1β expressing CD68 + macrophages in the fibrotic tissue surrounding NHO while red rectangle shows IL-1β expressing multinucleated CD68 + osteoclasts. Mouse IgG3 (bottom left panel) and Rabbit IgG (bottom right panel) confirm the specificity of CD68 and IL-1β antibodies. 20X magnification and scale bar = 50μm. (B) IL-1β concentration in plasma of healthy volunteers and NHO patients. Each dot represents a different individual. Bars are means ± SD. Statistical difference was calculated using two-sided non-parametric Mann-Whitney test, *** p<0.001. (C) Dose-response of recombinant human IL-1α (0.01; 1 and 100 ng/mL) and (D) IL-1β (0.01; 0.5 and 10 ng/mL) on calcium mineralization of PDGFRα + CD56 - FAPs derived from 5 different NHO biopsies / patients cultured in osteogenic medium. (E) Osteogenic differentiation assay of FAPs cultured with LPS-stimulated CD14 + blood monocyte-conditioned medium (CM + ) and incubated with anti-IL-1α, anti-IL-1β neutralizing antibodies or isotype control (100 ng/mL). (F) Osteogenic differentiation assay of FAPs derived from 6 different biopsies / patients cultured with CM + and incubated with 10, 100 or 500 ng/mL of recombinant human IL1RA. In (C-E), each dot represents a result from a different NHO biopsy / patient. Boxes extend from the 25th to 75th percentiles, middle line and whiskers represent respectively median and minimum and maximum value. Statistical differences were analyzed using non-parametric repeated measure Friedman test with Dunn’s correction for multiple comparisons, * p < 0.05; ** p < 0.01. (G) RUNX2 protein expression level of FAPs evaluated by Western blot following 7 days of osteogenic differentiation with IL-1β (10 ng/mL), IL-1α (100 ng/mL), NHOmac CM + , control isotype antibody, anti-IL-1α and anti-IL-1β antibody (100 ng/mL) or IL-1RA (500 ng/mL). Statistical differences were analyzed using non-parametric repeated measure Friedman test with Dunn’s correction for multiple comparisons, * p < 0.05; ** p < 0.01.

Article Snippet: Human MPCs were trypsinized and incubated 30 min with biotinylated anti-human PDGFRα (BAF322 R&D Systems) goat polyclonal antibody and CD56-PE (clone B159, BD Pharmigen) monoclonal antibody in PBS 2% fetal bovine serum (FBS), 2mM EDTA or with control isotypes IgG1 PE (A07796, Beckman Coulter) and biotinylated goat IgG (BAF108, R&D Systems).

Techniques: Derivative Assay, In Vitro, Immunohistochemistry, Staining, Marker, Expressing, Concentration Assay, Clinical Proteomics, MANN-WHITNEY, Recombinant, Cell Culture, Differentiation Assay, Incubation, Control, Western Blot

Journal: Genome Medicine

Article Title: A dynamic single cell-based framework for digital twins to prioritize disease genes and drug targets

doi: 10.1186/s13073-022-01048-4

Figure Lengend Snippet: Construction of MNMs and prioritization of URs. A (1) Determination of DEGs (red) in a cell type (gray). (2) Bioinformatic identification of the predicted UR of the DEGs. (3) Identification of another cell type (blue) in which that UR is differentially expressed. (4) A directed interaction from the blue to the gray cell type is formed. B Example of one MNM (at 0 h) which was constructed based on directed interactions as described in A. C MNMs created at each time point. Node sizes correspond to the number of DEGs between cells isolated from patients compared to healthy controls. Edge width represents the number of predicted URs. Edge color corresponds to the source cell. D Heatmap of top 40 URs ranked based on the number of cell types that each UR is predicted to regulate at different time points. Color intensity boxes indicate the statistical significance of predictions. Grey boxes indicate non-significant predictions. A positive z-score indicates that the direction of the differential expression matches the predicted direction (orange), while a negative z-score indicates the opposite (blue). The inserted boxes on the right correspond to the top 20 URs in the left boxes. E , F Dynamic UR-target models showing predicted cell-type targets of PDGF-BB and IL-4 in allergen-stimulated patients. Node size denotes the significance of the association (z-score)

Article Snippet: For blocking experiments, PBMCs (1 × 10 6 cells) from eight SAR patients were stimulated for 5 days with 10 μg/mL BPE in the absence or presence of either a neutralizing anti-human IL-4 antibody (MAB204) or anti-human PDGF-BB antibody (AF-220-NA, both from R&D Systems, Minneapolis, USA) at a concentration of 5 μg/mL.

Techniques: Construct, Isolation, Quantitative Proteomics

Journal: Genome Medicine

Article Title: A dynamic single cell-based framework for digital twins to prioritize disease genes and drug targets

doi: 10.1186/s13073-022-01048-4

Figure Lengend Snippet: Protein expression patterns of predicted URs in supernatants of allergen-stimulated PBMC from SAR patients and controls. A PDGF-BB, B IFN-α, and C CCL5 levels in supernatants. * P -value < 0.05, ** P -value < 0.01, Wilcoxon signed rank test

Article Snippet: For blocking experiments, PBMCs (1 × 10 6 cells) from eight SAR patients were stimulated for 5 days with 10 μg/mL BPE in the absence or presence of either a neutralizing anti-human IL-4 antibody (MAB204) or anti-human PDGF-BB antibody (AF-220-NA, both from R&D Systems, Minneapolis, USA) at a concentration of 5 μg/mL.

Techniques: Expressing

Journal: Genome Medicine

Article Title: A dynamic single cell-based framework for digital twins to prioritize disease genes and drug targets

doi: 10.1186/s13073-022-01048-4

Figure Lengend Snippet: Effects of PDGF-BB neutralization on the release of IL-6, IL-13, and VEGF from allergen-stimulated PBMC. A IL-6, B IL-13, and C VEGF levels in supernatants harvested at day 5; D allergen-specific lymphoproliferation. Box plots are shown, red lines indicate median values, * P -value < 0.05, Wilcoxon signed ranks test. The different shapes of data points represent different SAR patients

Article Snippet: For blocking experiments, PBMCs (1 × 10 6 cells) from eight SAR patients were stimulated for 5 days with 10 μg/mL BPE in the absence or presence of either a neutralizing anti-human IL-4 antibody (MAB204) or anti-human PDGF-BB antibody (AF-220-NA, both from R&D Systems, Minneapolis, USA) at a concentration of 5 μg/mL.

Techniques: Neutralization

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: A novel population of subepithelial platelet-derived growth factor receptor ?-positive cells in the mouse and human colon

doi: 10.1152/ajpgi.00001.2013

Figure Lengend Snippet: The information of primary and secondary antibodies

Article Snippet: PDGFRα , Goat , AF-307-NA , 1:100 , R & D Systems.

Techniques:

Journal: PLoS ONE

Article Title: TMEFF2 Is a PDGF-AA Binding Protein with Methylation-Associated Gene Silencing in Multiple Cancer Types Including Glioma

doi: 10.1371/journal.pone.0018608

Figure Lengend Snippet: ( A ) Binding of dimeric PDGF ligands to TECD-FLAG coated wells. PDGF-AA, AB or BB were applied to TECD-FLAG coated wells (solid symbols) or blank wells (open symbols) and detected with biotinylated anti-PDGF-A (for PDGF-AA & AB) or PDGF-B (for PDGF-BB) antibodies followed by streptavidin-HRP. Anti-PDGF pAb coated wells were used as a positive control for PDGF-AA binding (x). ( B ) Binding of six recombinant Fc-tagged ECDs and an anti-FLAG mAb to TECD-FLAG coated wells. HRP-conjugated anti-mouse and anti-human Fcγ were used to detect anti-FLAG mAb and Fc-tagged proteins, respectively. ( C ) TECD-Fc was applied to wells coated with PDGF-AA, AB or BB and detected with HRP-conjugated anti-human Fcγ. ( D ) TECD-Fc and other Fc–tagged ECD of various transmembrane proteins were applied to PDGF-AA coated wells and detected with HRP-conjugated anti-human Fcγ. TNFR, tumor necrosis factor receptor; PDGFRβ, PDGF receptor β; mOX40, murine OX40. Error bars represent standard deviations between duplicates. Representative graphs of at least three independent experiments are shown.

Article Snippet: Recombinant human PDGF-AA, AB, BB, CC and DD, recombinant human PDGF receptor α extracellular domain (PDGF sRα), recombinant human PDGFRβ-Fc, goat anti-human PDGF, and biotinylated goat anti-human PDGF-A and PDGF-B antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Positive Control, Recombinant

Journal: PLoS ONE

Article Title: TMEFF2 Is a PDGF-AA Binding Protein with Methylation-Associated Gene Silencing in Multiple Cancer Types Including Glioma

doi: 10.1371/journal.pone.0018608

Figure Lengend Snippet: ( A ) & ( C ) Dose-dependent stimulation of BrdU incorporation by PDGF-AA and PDGF-AB in NR6 cells. ( B ) & ( D ) Effects of increasing concentrations of TECD-Fc (filled bars) or PDGF sRα (open bars) on 10 ng/ml PDGF-AA ( B ) or PDGF-AB ( D ) stimulated BrdU incorporation.

Article Snippet: Recombinant human PDGF-AA, AB, BB, CC and DD, recombinant human PDGF receptor α extracellular domain (PDGF sRα), recombinant human PDGFRβ-Fc, goat anti-human PDGF, and biotinylated goat anti-human PDGF-A and PDGF-B antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: BrdU Incorporation Assay

Journal: PLoS ONE

Article Title: TMEFF2 Is a PDGF-AA Binding Protein with Methylation-Associated Gene Silencing in Multiple Cancer Types Including Glioma

doi: 10.1371/journal.pone.0018608

Figure Lengend Snippet: ( A ) Affymetrix signal intensity of TMEFF2 expression in prostate cancer vs non-cancerous tissues based on GeneLogic data. ( B ) Affymetrix signal intensity of TMEFF2 expression in normal brain vs brain cancer tissues based on GeneLogic data. Each open circle in ( A ) & ( B ) represents one patient sample. Box-and Whisker plots are also included under the raw data to indicate the mean and the 25th and 75th percentile ranges. The whiskers are drawn at 1.5 times the interquartile range from the box. ( C ) & ( D ) Normalized signals of TMEFF2 ( C ) and PDGF-A ( D ) mRNA expression in Proneural (PN), Proliferative (Prolif), or Mesenchymal (MES) subtypes of 36 glioma samples. Mean signals for each subtype are shown as insets. * p ≤0.05; **, p ≤0.005. ( E ) TMEFF2 expression is negatively correlated with PDGF-A expression in 133 (76 MD Anderson and 57 UCSF) HGG samples (Pearson correlation coefficient r = −0.37). Each axis represents normalized signals of each gene. All expression data were obtained using Affymetrix HG-U133A and HG-U133B GeneChips from probe 223557_s_at for TMEFF2 and 205463_s_at for PDGF-A, respectively.

Article Snippet: Recombinant human PDGF-AA, AB, BB, CC and DD, recombinant human PDGF receptor α extracellular domain (PDGF sRα), recombinant human PDGFRβ-Fc, goat anti-human PDGF, and biotinylated goat anti-human PDGF-A and PDGF-B antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Whisker Assay

Journal: PLoS ONE

Article Title: TMEFF2 Is a PDGF-AA Binding Protein with Methylation-Associated Gene Silencing in Multiple Cancer Types Including Glioma

doi: 10.1371/journal.pone.0018608

Figure Lengend Snippet: (A) TMEFF2 methylation status vs. G-CIMP status. (B) TMEFF2 methylation status vs. IDH1 mutation status. (C) TMEFF2 methylation status vs. GBM molecular subtypes. (D) PDGF-A expression vs. GBM molecular subtypes. (E) TMEFF2 expression vs. PDGF-A expression [t-test p-value = 6.6×10 −13 between PDGF-A expression levels in samples with high TMEFF2 (expression value≥10) vs. those with low TMEFF2 (expression value<10)].

Article Snippet: Recombinant human PDGF-AA, AB, BB, CC and DD, recombinant human PDGF receptor α extracellular domain (PDGF sRα), recombinant human PDGFRβ-Fc, goat anti-human PDGF, and biotinylated goat anti-human PDGF-A and PDGF-B antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Methylation, Mutagenesis, Expressing

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Gain-of-function PDGFRA mutations, earlier reported in gastrointestinal stromal tumors, are common in small intestinal inflammatory fibroid polyps. A study of 60 cases.

doi: 10.1038/modpathol.2009.62

Figure Lengend Snippet: Figure 1 Inflammatory fibroid polyps can contain large numbers of inflammatory cells, especially eosinophils (a). These elements are highlighted as CD45 positive (b). (c–f) Histologically, inflammatory fibroid polyp is composed of polygonal to spindled cells in a highly vascular background. Collagenous (d) or edematous (e) matrix with capillary vessels (f) variably infiltrated by eosinophilic granulocytes and other inflammatory cells (c–f). Cytoplasmic expression of PDGFRA is common; immunohistochemistry with the polyclonal antibody, SC338 (g), and the monoclonal antibody, MAB322 (h).

Article Snippet: Immunohistochemical studies on PDGFRA expression were carried out with two antibodies, a rabbit polyclonal antibody, SC338 (Santa Cruz, Biotechnology Inc., Santa Cruz, CA, USA), and a monoclonal mouse antibody, MAB322 (R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Expressing, Immunohistochemistry

Journal: Oncogene

Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

doi: 10.1038/onc.2008.410

Figure Lengend Snippet: Figure 1 The basic stretch of amino acids in the conserved putative cleavage site is important for PDGF-DD processing and generation of a PDGFRb agonist. (a) An alignment of the identified cleavage site region in PDGF-C, -R231KSR, with the homologous stretch of amino acids in PDGF-D implies a potential role for the -R254KSK- site in uPA-mediated proteolysis. Conserved basic amino acids are highlighted. (b) An alanine scan mutagenesis assay performed in the conserved basic stretch of amino acids in PDGF-D. Serum-free conditioned media from cells transfected with constructs encoding the depicted mutant PDGF-D(fl) species were incubated with HMWuPA, TCA-precipitated and analysed for processed PDGF-D (arrowhead) by immunoblotting under reducing conditions. (c) In complement to the above experiment, the generated PDGF-DD species were analysed for their ability to induce PDGFRb tyrosine phosphorylation. Serum-free condi- tioned media from cells transfected with constructs encoding the different mutant PDGF-D(fl) species were incubated with HMWu- PA and thereafter applied on PDGFRb-expressing PAE cells. Following cell lysis and specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were determined by immunoblotting under reducing conditions.

Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

Techniques: Mutagenesis, Transfection, Construct, Incubation, Western Blot, Generated, Phospho-proteomics, Expressing, Lysis, Immunoprecipitation

Journal: Oncogene

Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

doi: 10.1038/onc.2008.410

Figure Lengend Snippet: Figure 2 PDGF-DD interacts directly with uPA. (a) A direct interaction between PDGF-DD and uPA was demonstrated in a Ni-NTA bead assay, where beads coated with PDGF-DD(fl) were incubated with serum-free conditioned medium from uPA-trans- fected cells. Uncoated beads served as negative control. Eluted proteins were analysed by immunoblotting under reducing condi- tions. (b) Specific domain–domain interactions between PDGF-DD and uPA were identified by an ELISA. Serum-free conditioned media from transfected cells expressing the three depicted PDGF- DD species were applied to an ELISA plate coated with HMWuPA and LMWuPA, respectively. Bound PDGF-DD was assayed with specific polyclonal antibodies and visualized using the alkaline phosphatase yellow liquid substrate system. Data are presented as means of triplicates±s.d. after subtraction of absorbance gener- ated by coating proteins and serum-free medium.

Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

Techniques: Incubation, Negative Control, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Expressing

Journal: Oncogene

Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

doi: 10.1038/onc.2008.410

Figure Lengend Snippet: Figure 3 PDGF-DD activation by uPA can be modulated by the presence of uPAR. (a) Immunoblotting under reducing conditions of generated PDGF-D species (arrowheads) after in vitro incubation of purified PDGF-DD(fl) with enzymatically comparable levels of LMWuPA and HMWuPA. (b) The PDGF-DD species generated by uPA-mediated proteolysis were analysed for their ability to induce PDGFRb tyrosine phosphorylation. Serum-free conditioned media from cells transfected with PDGF-D(fl) were incubated with LMWuPA or HMWuPA with or without the addition of soluble uPAR (suPAR) and thereafter applied on PDGFRb-expressing PAE cells. Following specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were analysed by immunoblotting under reducing conditions. (c) The ability of scuPA to process PDGF- DD(fl) into active species was explored by applying serum-free conditioned media from cells transfected with PDGF-D(fl) and/or uPA as well as uPAR on PDGFRb-expressing PAE cells. Following cell lysis and specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were analysed by immunoblotting under reducing conditions. To diminish undesired effects generated by plasmin activity induced by the presence of uPA, a2-antiplasmin was included in the serum-free cell culture media after transfection. Also protein levels of uPAR, uPA and PDGF-D(fl) in cells used as transfection host were monitored by immunoblotting under reduced conditions to control transfection efficacy and endogenous levels of protein of interest. Arrowheads indicate the presence of HMWuPA.

Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

Techniques: Activation Assay, Western Blot, Generated, In Vitro, Incubation, Phospho-proteomics, Transfection, Expressing, Immunoprecipitation, Lysis, Activity Assay, Cell Culture, Control

Journal: Oncogene

Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

doi: 10.1038/onc.2008.410

Figure Lengend Snippet: Figure 4 PDGFRb is regulated by uPAR and facilitates HMWuPA-induced PAE cell migration. (a) Immunoblotting under reduced conditions of cell lysates from semiconfluent cultures to monitor total PDGFRb protein levels in PAE cells with high uPAR expression in comparison to mock-transfected control cells. Calnexin was used as control to ensure equal protein loading. (b) Phosphotyrosine blotting under reducing conditions against immunoprecipitated PDGFRb, following PAE cell stimulation with purified proteins as illustrated. The phosphorylation capacity of PDGFRb in cells with high and low uPAR expression, was compared with mock- transfected PDGFRb-expressing control cells. Cells with low uPAR expression (specifically assigned low expression) were identified in the initial screen of uPAR-expressing clones (data not shown). PDGF-BB was used to monitor phosphorylation induced by another PDGFRb ligand. (c and d) Migration properties of the stably transfected PAE cell lines were determined in a chemotaxis assay. Serum- starved cells were seeded, treated as indicated and allowed to migrate through a porous membrane for 4 h. The response was quantified by cell counting from eight views/membrane and results from one representative experiment are presented as fold migration (migration index) relative to basal migration levels (untreated cells) for each cell line ±s.d. Statistical calculations were carried out with a two- sided Student’s t-test analysis (*Po0.05; ***Po0.001).

Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

Techniques: Migration, Western Blot, Expressing, Comparison, Transfection, Control, Immunoprecipitation, Cell Stimulation, Phospho-proteomics, Clone Assay, Stable Transfection, Chemotaxis Assay, Membrane, Cell Counting

Journal: Oncogene

Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

doi: 10.1038/onc.2008.410

Figure Lengend Snippet: Figure 5 Activated PDGF-DD induces cellular transformation of NIH/3T3 cells more effectively than latent PDGF-DD. (a) RT–PCR analysis of PDGF-DD expression in cells stably transfected with PDGF-D(fl) and PDGF-D(GFD), as compared with mock-trans- fected cells. Primers were designed for amplification of the C-terminal part of PDGF-D and b2-microglobulin served as an endogenous reference gene. (b) A cell growth analysis under serum-free conditions monitoring the differences in proliferation capacity of cells stably transfected with equal amounts of PDGF-D(fl) and PDGF-D(GFD), was carried out. Data are presented as means of triplicates±s.d. The experiment was repeated twice. (c) Microphotographs showing representative views of transfected NIH/3T3 cells grown in soft agar to monitor cell number and morphology of the foci generated from cells expressing PDGF-DD(fl) and PDGF-DD(GFD). Quantifications were made from five high magnification pictures (lower panel) and statistical calculations were carried out with a two-sided t-test analysis (**Po0.01). Data are presented as mean number of foci ±s.d. The experiment was repeated twice. Scale bar 0.5mm. (d) Immunofluor- escent staining of permeabilized, fixated NIH/3T3 cells expressing PDGF-DD(fl) and PDGF-DD(GFD), visualizing the presence of uPA (upper panel) and uPAR (lower panel) in transformed cells.

Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

Techniques: Transformation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Stable Transfection, Transfection, Generated, Staining

Journal: Oncogene

Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

doi: 10.1038/onc.2008.410

Figure Lengend Snippet: Figure 6 Latent PDGF-DD induces tumour growth in nude mice more effectively than activated PDGF-DD. (a) NIH/3T3 cells stably expressing PDGF-DD(fl) or PDGF-DD(GFD) were xenografted subcutaneously into the midline dorsum of nude mice. Tumour volume was monitored every second day and the experiment was repeated once. Data are presented as mean tumour volume±s.d (PDGF-DD(fl), N ¼ 10; PDGF-DD(GFD), N ¼ 8) starting from day 10 after the majority of the tumours were palpable. Statistical calculations were carried out with a two-sided t-test analysis (*Po0.05; **Po0.01; ***Po0.001). (b) Sections from PDGF-DD derived tumours were stained for PECAM-1/CD31 and detected vessels were quantified by counting nine representative sections/ tumour type and 10 randomly selected views per section. Sizes were categorized as follows: >30 mm large, 10–30 mm medium-sized and o10 mm small. Statistical calculations were carried out with a two- sided Student’s t-test analysis (*Po0.05). Data are presented as mean vessel size ±s.d. (c) Immunohistochemical analysis of sections from tumours expressing PDGF-DD(fl) and PDGF-DD(GFD), was per- formed to confirm abundant PDGF-DD expression and to investigate the expression of uPA and uPAR in both tumour types. Scale bar 25 mm.

Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

Techniques: Stable Transfection, Expressing, Derivative Assay, Staining, Immunohistochemical staining

Journal: Oncogene

Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

doi: 10.1038/onc.2008.410

Figure Lengend Snippet: Figure 7 The pericellular localization of PDGF-DD is controlled by proteolytic processing. (a) NIH/3T3 cells were transiently transfected with PDGF-D(fl) and PDGF-D(GFD), and the pericellular binding of secreted PDGF-DD species was visualized in non-permeabilized (NP) cells by immunofluorescent staining. Permeabilized (P) transfected cells served to ensure equal transfec- tion efficacy. (b) As a control for the experimental setting, NIH/ 3T3 cells were transiently transfected with the two isoforms of VEGF-B. VEGF-B186 is known to be freely diffusible, whereas VEGF-B167 is considered to be strictly matrix-bound. Permeabi- lized (P) transfected cells served to ensure equal transfection efficacy.

Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

Techniques: Transfection, Binding Assay, Staining, Control