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: Journal of Biological Chemistry

Article Title: Cell Type-specific Effect of Hypoxia and Platelet-derived Growth Factor-BB on Extracellular Matrix Turnover and Its Consequences for Lung Remodeling

doi: 10.1074/jbc.m602178200

Figure Lengend Snippet: FIGURE 1. A, representative gelatin-based zymograms of the effect of hypoxia (H) on MMP-2 and MMP-9 expression by fibroblasts and VSMCs compared with normoxia (N). Treatment of cell culture medium with 20 mMEDTA(24h)abolishedtheexpressionofpro-MMP-2andpro-MMP-9.Arrowsindicatethemigrationposition of purified pro-MMP-2 and pro-MMP-9. B, densitometric analysis of pro-MMP-2 expression by hypoxia in human primary lung fibroblast cell lines (n 8) and the effect of PDGF-BB and the underlying intracellular signaling inhibition. PD98059 inhibits Erk1/2 MAP kinase, SB203580 inhibits p38 MAP kinase, and SB202474 is a negative control for SB203580. C, densitometric analysis of pro-MMP-2 expression by hypoxia in human primary VSMC lines (n 8) and the effect of PDGF-BB and the underlying intracellular signaling pathways. Values are presented as the percentage of control (normoxia 24 h), and each bar represents the mean S.D. of six independent experiments.

Article Snippet: The effect of the MMP inhibitor OO-Hy (catalog number 44244; Calbiochem, Lucern, Switzerland) and of neutralizing polyclonal anti-PDGF-BB antibodies (AB-220-NA; R & D Systems) on proliferation (48 h) was also investigated.

Techniques: Expressing, Cell Culture, Purification, Inhibition, Negative Control, Protein-Protein interactions, Control

Journal: Journal of Biological Chemistry

Article Title: Cell Type-specific Effect of Hypoxia and Platelet-derived Growth Factor-BB on Extracellular Matrix Turnover and Its Consequences for Lung Remodeling

doi: 10.1074/jbc.m602178200

Figure Lengend Snippet: FIGURE 2. The effect of hypoxia and/or PDGF-BB on TIMP-1 (A) and TIMP-2 (B) by human lung fibroblasts and VSMCs within 48 h was assessed by ELISA. The role of Erk1/2 and p38 MAP kinase on the stimulatory effect of both TIMPs was also determined using PD98059-inhibiting Erk1/2 MAP kinase, SB203580-inhibiting p38 MAP kinase,andSB202474asanegativecontrolforSB203580.Eachbarrepresentsthemean S.D.ofsixindepend- ent experiments, each in triplicate. The control was defined as normoxia (24 h). C, aliquots of cell culture medium from human lung fibroblasts and VSMCs containing the same amount of protein were incubated in the absence () or in the presence () of 4-aminophenylmercuric acetate (APMA; 1 mM, 37 °C, 24 h) and subjected to gelatin zymography. Arrows indicate the migration position of purified pro-MMP-2 and active MMP-2. N, normoxia; H, hypoxia).

Article Snippet: The effect of the MMP inhibitor OO-Hy (catalog number 44244; Calbiochem, Lucern, Switzerland) and of neutralizing polyclonal anti-PDGF-BB antibodies (AB-220-NA; R & D Systems) on proliferation (48 h) was also investigated.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Cell Culture, Incubation, Zymography, Migration, Purification

Journal: Journal of Biological Chemistry

Article Title: Cell Type-specific Effect of Hypoxia and Platelet-derived Growth Factor-BB on Extracellular Matrix Turnover and Its Consequences for Lung Remodeling

doi: 10.1074/jbc.m602178200

Figure Lengend Snippet: FIGURE 3. A, the effect of hypoxia (black bars) and PDGF-BB (striped bars) on MMP-1 expression by fibroblasts and VSMC within 48 h was assessed by ELISA. Each bar represents the mean S.D. of triplicates in six inde- pendent experiments. B, a representative immunoblot demonstrates the role of Erk1/2 and p38 MAP kinase in thehypoxia-inducedexpressionofMMP-1,MMP-13,andhypoxia-induciblefactor-1(HIF-1)aswellasonthe antagonizing effect of PDGF-BB in human lung fibroblasts. PD98059 inhibits Erk1/2 MAP kinase, SB203580 inhibits p38 MAP kinase and SB202474 is a negative control for SB203580. Ab, neutralizing antibody; control t0, protein extract from untreated cells before stimulation. C, hypoxia- and PDGF-BB-induced MMP-13 expression by fibroblasts but not by VSMC within 48 h. Each bar represents the mean S.D. of triplicate determinations from six independent experiments. Similar results were obtained with two additional cell lines.

Article Snippet: The effect of the MMP inhibitor OO-Hy (catalog number 44244; Calbiochem, Lucern, Switzerland) and of neutralizing polyclonal anti-PDGF-BB antibodies (AB-220-NA; R & D Systems) on proliferation (48 h) was also investigated.

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

Journal: Journal of Biological Chemistry

Article Title: Cell Type-specific Effect of Hypoxia and Platelet-derived Growth Factor-BB on Extracellular Matrix Turnover and Its Consequences for Lung Remodeling

doi: 10.1074/jbc.m602178200

Figure Lengend Snippet: FIGURE 4. A, the effect of hypoxia and PDGF-BB on the release of soluble collagen type I within 48 h was determined by ELISA in the cell culture medium obtained from human lung fibroblasts (n 6). Each bar represents the mean S.D. Experiments in each cell line were performed in triplicates. B, the inducing effect of hypoxia and PDGF-BB on collagen type I 1 chain (COL1A1) was confirmed on the mRNA level in three cell lines and the effect of Erk1/2 and p38 MAP kinase was assessed by reverse transcriptase-PCR. -ac- tin gene expression was used as a housekeeping gene, and data are displayed as a representative PCR product analysis. PD98059 inhibits Erk1/2 MAP kinase,SB203580inhibitsp38MAPkinase,andSB202474isanegativecontrol for SB203580.

Article Snippet: The effect of the MMP inhibitor OO-Hy (catalog number 44244; Calbiochem, Lucern, Switzerland) and of neutralizing polyclonal anti-PDGF-BB antibodies (AB-220-NA; R & D Systems) on proliferation (48 h) was also investigated.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Reverse Transcription, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Cell Type-specific Effect of Hypoxia and Platelet-derived Growth Factor-BB on Extracellular Matrix Turnover and Its Consequences for Lung Remodeling

doi: 10.1074/jbc.m602178200

Figure Lengend Snippet: FIGURE 5. A, hypoxia and/or PDGF-BB increased fibroblast numbers significantly within 72 h. Under hypoxic culture condition, PDGF-BB mediates the mitogenic effect that is transmitted via Erk1/2. PD98059 inhibits Erk1/2 MAP kinase, SB203580 inhibits p38 MAP kinase, and SB202474 is a negative control for SB203580. The general inhibitor of MMP activity cis-9-octadecenoyl-N-hydroxylamide oleoyl-N-hydroxylamide (10 mM, OO-Hy) was added 3 h before cells were exposed to any other stimulus. B, hypoxia (black bar) and PDGF-BB up-regulated VSMC proliferation via Erk1/2 and p38 MAP kinase. Purified human soluble collagen type 1 (sol collagen type I (1 mg/ml)) was used to assess its contribution to hypoxia-mediated proliferation. Each bar represents the mean S.D. of triplicates performed in six independent experiments.

Article Snippet: The effect of the MMP inhibitor OO-Hy (catalog number 44244; Calbiochem, Lucern, Switzerland) and of neutralizing polyclonal anti-PDGF-BB antibodies (AB-220-NA; R & D Systems) on proliferation (48 h) was also investigated.

Techniques: Negative Control, Activity Assay, Purification

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: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pericytes contribute to airway remodeling in a mouse model of chronic allergic asthma

doi: 10.1152/ajplung.00286.2014

Figure Lengend Snippet: A : qPCR analysis of PDGF-BB and PDGFRβ mRNA expression in whole-lung lysates from saline and HDM-exposed mice (i.n. 5 days/wk for 5 wk); n = 5 representative of 2 independent experiments. ELISA analysis of PDGF-BB expression in BAL supernatants ( B ) and immunoblotting for PDGF-BB protein expression ( C ) in whole-lung lysates from saline and HDM-exposed mice (i.n. 5 days/wk for 5 wk); n = 5–6 (ELISA) and n = 3 (immunoblotting) per group, representative of 2 independent experiments. The immunoblot shown in C presents representative lanes for saline and HDM-exposed mice from 3 replicates per group run on the same gel. D : quantification of PDGF-BB expression assessed by immunoblotting in whole lung homogenates from saline and HDM-exposed mice. * P < 0.05, ** P < 0.01.

Article Snippet: The membranes were incubated in blocking buffer (5% skim milk) in Tris-buffered saline containing 0.5% Tween for 1 h at room temperature and probed with a polyclonal rabbit anti-human PDGF-BB antibody (Aviva Systems Biology, San Diego, CA) diluted 1:1,000 in blocking buffer.

Techniques: Expressing, Saline, Enzyme-linked Immunosorbent Assay, Western Blot