pdgf bb  (R&D Systems)

 
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  • 99
    Name:
    Recombinant Equine PDGF BB Protein CF
    Description:
    The Recombinant Equine PDGF BB Protein from R D Systems is derived from E coli The Recombinant Equine PDGF BB Protein has been validated for the following applications Bioactivity
    Catalog Number:
    8585-bb-010/cf
    Price:
    259
    Applications:
    Bioactivity
    Purity:
    >95%, by SDS-PAGE with silver staining
    Conjugate:
    Unconjugated
    Size:
    10 ug
    Category:
    Proteins and Enzymes
    Source:
    E. coli-derived Recombinant Equine PDGF-BB Protein
    Buy from Supplier


    Structured Review

    R&D Systems pdgf bb
    VEGF-A associated with both PDGFRα and PDGFRβ. Binding of VEGF-A to either PDGFRα or PDGFRβ was examined using a cross-linking approach. MSCs were unstimulated (basal) or stimulated with either 10 ng/ml VEGF-A 165 (165) or <t>PDGF-BB</t> (BB) as a positive control, or 10 ng/ml <t>TGF-β</t> 1 as a negative control (not depicted), for 10 min at 37°C. To inhibit growth factor binding to the respective PDGFR, MSCs were also pretreated with either 10 μg/ml anti-PDGFRα (Rα) or -PDGFRβ (Rβ) cell surface neutralization antibodies for 30 min at 37°C, before growth factor stimulation. Growth factor binding to PDGFR was captured by adding 1 mM of a cell membrane–impermeable cross-linking agent (DTSSP), followed by immunoprecipitation (IP) analysis using anti-PDGFRα or anti-PDGFRβ, then growth factor association detected by immunoblot (IB) analysis using corresponding (A) anti–VEGF-A or (B) –PDGF-B. Membranes were reprobed with anti-PDGFRα or -PDGFRβ as loading controls. A representative of three independent experiments is shown for each analysis.
    The Recombinant Equine PDGF BB Protein from R D Systems is derived from E coli The Recombinant Equine PDGF BB Protein has been validated for the following applications Bioactivity
    https://www.bioz.com/result/pdgf bb/product/R&D Systems
    Average 99 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    pdgf bb - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Vascular endothelial growth factor can signal through platelet-derived growth factor receptors"

    Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200608093

    VEGF-A associated with both PDGFRα and PDGFRβ. Binding of VEGF-A to either PDGFRα or PDGFRβ was examined using a cross-linking approach. MSCs were unstimulated (basal) or stimulated with either 10 ng/ml VEGF-A 165 (165) or PDGF-BB (BB) as a positive control, or 10 ng/ml TGF-β 1 as a negative control (not depicted), for 10 min at 37°C. To inhibit growth factor binding to the respective PDGFR, MSCs were also pretreated with either 10 μg/ml anti-PDGFRα (Rα) or -PDGFRβ (Rβ) cell surface neutralization antibodies for 30 min at 37°C, before growth factor stimulation. Growth factor binding to PDGFR was captured by adding 1 mM of a cell membrane–impermeable cross-linking agent (DTSSP), followed by immunoprecipitation (IP) analysis using anti-PDGFRα or anti-PDGFRβ, then growth factor association detected by immunoblot (IB) analysis using corresponding (A) anti–VEGF-A or (B) –PDGF-B. Membranes were reprobed with anti-PDGFRα or -PDGFRβ as loading controls. A representative of three independent experiments is shown for each analysis.
    Figure Legend Snippet: VEGF-A associated with both PDGFRα and PDGFRβ. Binding of VEGF-A to either PDGFRα or PDGFRβ was examined using a cross-linking approach. MSCs were unstimulated (basal) or stimulated with either 10 ng/ml VEGF-A 165 (165) or PDGF-BB (BB) as a positive control, or 10 ng/ml TGF-β 1 as a negative control (not depicted), for 10 min at 37°C. To inhibit growth factor binding to the respective PDGFR, MSCs were also pretreated with either 10 μg/ml anti-PDGFRα (Rα) or -PDGFRβ (Rβ) cell surface neutralization antibodies for 30 min at 37°C, before growth factor stimulation. Growth factor binding to PDGFR was captured by adding 1 mM of a cell membrane–impermeable cross-linking agent (DTSSP), followed by immunoprecipitation (IP) analysis using anti-PDGFRα or anti-PDGFRβ, then growth factor association detected by immunoblot (IB) analysis using corresponding (A) anti–VEGF-A or (B) –PDGF-B. Membranes were reprobed with anti-PDGFRα or -PDGFRβ as loading controls. A representative of three independent experiments is shown for each analysis.

    Techniques Used: Binding Assay, Positive Control, Negative Control, Neutralization, Immunoprecipitation

    PDGF-induced MSC migration was inhibited by VEGF-A. The effects of VEGF-A on PDGF-induced MSC migration was examined using Boyden chamber migration assays. MSCs were preincubated with 10 ng/ml VEGF-A 165 for 10 min, before adding the cell suspension onto the upper chamber membrane surface and exposure to 10 ng/ml PDGF-AA or -BB in the lower half of a Boyden chamber for 5 h. MSCs not exposed to either growth factor represents a growth factor–independent migration control. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the membrane underside. Data shown are the mean number migratory cells ± the SD determined from 10 random fields from each of two independent experiments. *, P
    Figure Legend Snippet: PDGF-induced MSC migration was inhibited by VEGF-A. The effects of VEGF-A on PDGF-induced MSC migration was examined using Boyden chamber migration assays. MSCs were preincubated with 10 ng/ml VEGF-A 165 for 10 min, before adding the cell suspension onto the upper chamber membrane surface and exposure to 10 ng/ml PDGF-AA or -BB in the lower half of a Boyden chamber for 5 h. MSCs not exposed to either growth factor represents a growth factor–independent migration control. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the membrane underside. Data shown are the mean number migratory cells ± the SD determined from 10 random fields from each of two independent experiments. *, P

    Techniques Used: Migration

    VEGF-A induced a dose-dependent increase in PDGFR tyrosine phosphorylation. The effects of varying VEGF-A 165 concentration on induced PDGFR tyrosine phosphorylation levels was determined by specific ELISAs. MSCs in serum-free conditions were exposed to 0.5, 1, 2, 5, 10, 25, 50, 100, or 200 ng/ml VEGF-A 165 for 10 min at 37°C. As a control, cells were also exposed to identical concentrations of PDGF-BB. MSC lysates were assayed for either PDGFRα or PDGFRβ tyrosine phosphorylation using a corresponding ELISA. Increased tyrosine phosphorylation is represented by an increase in optical density (OD 450nm ). Data shown are mean OD 450nm ± the SD determined from two independent experiments performed in triplicate.
    Figure Legend Snippet: VEGF-A induced a dose-dependent increase in PDGFR tyrosine phosphorylation. The effects of varying VEGF-A 165 concentration on induced PDGFR tyrosine phosphorylation levels was determined by specific ELISAs. MSCs in serum-free conditions were exposed to 0.5, 1, 2, 5, 10, 25, 50, 100, or 200 ng/ml VEGF-A 165 for 10 min at 37°C. As a control, cells were also exposed to identical concentrations of PDGF-BB. MSC lysates were assayed for either PDGFRα or PDGFRβ tyrosine phosphorylation using a corresponding ELISA. Increased tyrosine phosphorylation is represented by an increase in optical density (OD 450nm ). Data shown are mean OD 450nm ± the SD determined from two independent experiments performed in triplicate.

    Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay

    Inhibiting PDGFRα or PDGFRβ attenuated VEGF-A–induced MSC migration. (A) MSCs, or HUVECs used as a VEGFR- positive cell, were pretreated with either 10 μg/ml anti-VEGFR1 or -VEGFR2 neutralization antibodies, 100 nM VEGFR2 tyrosine kinase inhibitor (VEGFR2-TK), 2 μM PDGFR tyrosine kinase inhibitor (PDGFR-TK), and 10 μg/ml anti-PDGFRα or -PDGFRβ neutralization antibodies; then, either 10 ng/ml VEGF-A 165 or 10 ng/ml VEGF-A 121 (not depicted) were added to the lower half of a Boyden chamber for 5 h. No inhibition represents control VEGF-A–induced migration. (B) As a control, MSCs were pretreated with either 10 μg/ml anti-PDGFRα or -PDGFRβ neutralization antibodies, and then 10 ng/ml PDGF-BB was added to the lower half of a Boyden chamber for 5 h. No inhibition represents control PDGF-BB–induced migration. (C) MSCs were transfected with either 3 μg siRNA PDGFRα, siRNA PDGFRβ, or scrambled siRNA used as a control. Transfected MSCs in serum-free conditions were either unstimulated as a control, or exposed to 10 ng/ml VEGF-A 165 in the lower half of a Boyden chamber for 5 h. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the membrane underside. Data shown are the mean number of migratory cells ± the SD, which were determined from 10 random fields from each of four (A) or two (B and C) independent experiments. *, P
    Figure Legend Snippet: Inhibiting PDGFRα or PDGFRβ attenuated VEGF-A–induced MSC migration. (A) MSCs, or HUVECs used as a VEGFR- positive cell, were pretreated with either 10 μg/ml anti-VEGFR1 or -VEGFR2 neutralization antibodies, 100 nM VEGFR2 tyrosine kinase inhibitor (VEGFR2-TK), 2 μM PDGFR tyrosine kinase inhibitor (PDGFR-TK), and 10 μg/ml anti-PDGFRα or -PDGFRβ neutralization antibodies; then, either 10 ng/ml VEGF-A 165 or 10 ng/ml VEGF-A 121 (not depicted) were added to the lower half of a Boyden chamber for 5 h. No inhibition represents control VEGF-A–induced migration. (B) As a control, MSCs were pretreated with either 10 μg/ml anti-PDGFRα or -PDGFRβ neutralization antibodies, and then 10 ng/ml PDGF-BB was added to the lower half of a Boyden chamber for 5 h. No inhibition represents control PDGF-BB–induced migration. (C) MSCs were transfected with either 3 μg siRNA PDGFRα, siRNA PDGFRβ, or scrambled siRNA used as a control. Transfected MSCs in serum-free conditions were either unstimulated as a control, or exposed to 10 ng/ml VEGF-A 165 in the lower half of a Boyden chamber for 5 h. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the membrane underside. Data shown are the mean number of migratory cells ± the SD, which were determined from 10 random fields from each of four (A) or two (B and C) independent experiments. *, P

    Techniques Used: Migration, Neutralization, Inhibition, Transfection

    VEGF-A–induced HDF migration was PDGFRα and PDGFRβ dependent. (A) The expression of cell surface PDGFRs on HDFs were determined by single-color flow cytometry. Analysis of PDGFRα and PDGFRβ was performed using anti–human PE-conjugated antibodies, using an IgG 1 -PE antibody as a control. (B) The effects of VEGF-A on HDF migration and the involvement of PDGFRs were examined using Boyden chamber migration assays. HDF migration was evaluated in serum-free conditions after 5-h exposure to growth factor; 20 ng/ml VEGF-A 165 , VEGF-A 121 , PDGF-AA, or PDGF-BB in the lower half of a Boyden chamber. Basal represents growth factor–independent migration. (C) HDFs were pretreated with either 10 μg/ml anti-PDGFRα or -PDGFRβ neutralization antibodies, before adding 20 ng/ml VEGF-A 165 to the lower half of a Boyden chamber for 5 h. No inhibition represents control VEGF-A 165 –induced migration. (D) HDFs were transfected with either 3 μg siRNA PDGFRα, siRNA PDGFRβ, or scrambled siRNA used as a control. Transfected HDFs in serum-free conditions were either unstimulated as a control, or exposed to 20 ng/ml VEGF-A 165 in the lower half of a Boyden chamber for 5 h. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the underside of the membrane. Data shown are the mean number of migratory cells ± the SD determined from 10 random fields from each of three independent experiments. *, P
    Figure Legend Snippet: VEGF-A–induced HDF migration was PDGFRα and PDGFRβ dependent. (A) The expression of cell surface PDGFRs on HDFs were determined by single-color flow cytometry. Analysis of PDGFRα and PDGFRβ was performed using anti–human PE-conjugated antibodies, using an IgG 1 -PE antibody as a control. (B) The effects of VEGF-A on HDF migration and the involvement of PDGFRs were examined using Boyden chamber migration assays. HDF migration was evaluated in serum-free conditions after 5-h exposure to growth factor; 20 ng/ml VEGF-A 165 , VEGF-A 121 , PDGF-AA, or PDGF-BB in the lower half of a Boyden chamber. Basal represents growth factor–independent migration. (C) HDFs were pretreated with either 10 μg/ml anti-PDGFRα or -PDGFRβ neutralization antibodies, before adding 20 ng/ml VEGF-A 165 to the lower half of a Boyden chamber for 5 h. No inhibition represents control VEGF-A 165 –induced migration. (D) HDFs were transfected with either 3 μg siRNA PDGFRα, siRNA PDGFRβ, or scrambled siRNA used as a control. Transfected HDFs in serum-free conditions were either unstimulated as a control, or exposed to 20 ng/ml VEGF-A 165 in the lower half of a Boyden chamber for 5 h. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the underside of the membrane. Data shown are the mean number of migratory cells ± the SD determined from 10 random fields from each of three independent experiments. *, P

    Techniques Used: Migration, Expressing, Flow Cytometry, Cytometry, Neutralization, Inhibition, Transfection

    Exposure to VEGF-A increased MSC migration and proliferation. (A) MSC migration was examined in serum-free conditions after a 5-h exposure to a growth factor; 10 ng/ml VEGF-A 165 , VEGF-A 121 , PDGF-AA, or PDGF-BB in the lower half of a Boyden chamber. Basal represents growth factor–independent migration. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the membrane underside. Data shown are the mean number of migratory cells ± the SD determined from 10 random fields from each of four independent experiments. *, P
    Figure Legend Snippet: Exposure to VEGF-A increased MSC migration and proliferation. (A) MSC migration was examined in serum-free conditions after a 5-h exposure to a growth factor; 10 ng/ml VEGF-A 165 , VEGF-A 121 , PDGF-AA, or PDGF-BB in the lower half of a Boyden chamber. Basal represents growth factor–independent migration. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the membrane underside. Data shown are the mean number of migratory cells ± the SD determined from 10 random fields from each of four independent experiments. *, P

    Techniques Used: Migration

    VEGF-A–induced PDGFR tyrosine phosphorylation was comparable to PDGF-BB–induced PDGFRα level. (A) RTK array analysis of lysates from MSCs transfected with 3 μg scrambled siRNA as a control, siRNA PDGFRα or siRNA PDGFRβ, stimulated using 20 ng/ml PDGF-BB in serum-free conditions for 10 min at 37°C. Each array was identically exposed to detection reagents and film. A representative example of two independent experiments is shown. (B) Bar graph comparing VEGF-A 165 – and PDGF-BB–induced PDGFR tyrosine phosphorylation levels. Data represent VEGF-A 165 and PDGF-BB–stimulated controls from RTK array analysis shown in Fig. 5 B and Fig. 6 A , respectively. Mean pixel density ± the SD of duplicate spots, normalized against duplicate phosphotyrosine-positive control spots = 100. (C) Immunoprecipitation (IP) analysis of PDGFR tyrosine phosphorylation levels. MSCs in serum-free conditions were unstimulated with growth factor (basal), or stimulated with either 20 ng/ml VEGF-A 165 or PDGF-BB as a control, for 10 min at 37°C. PDGFRs were isolated from MSC lysates by IP analysis using anti-PDGFRα or anti-PDGFRβ, and then tyrosine phosphorylation detected by immunoblot (IB) analysis using anti-phosphotyrosine (Tyr-P). Membranes were reprobed with corresponding anti-PDGFRα or anti-PDGFRβ as loading controls. A representative of two independent experiments is shown.
    Figure Legend Snippet: VEGF-A–induced PDGFR tyrosine phosphorylation was comparable to PDGF-BB–induced PDGFRα level. (A) RTK array analysis of lysates from MSCs transfected with 3 μg scrambled siRNA as a control, siRNA PDGFRα or siRNA PDGFRβ, stimulated using 20 ng/ml PDGF-BB in serum-free conditions for 10 min at 37°C. Each array was identically exposed to detection reagents and film. A representative example of two independent experiments is shown. (B) Bar graph comparing VEGF-A 165 – and PDGF-BB–induced PDGFR tyrosine phosphorylation levels. Data represent VEGF-A 165 and PDGF-BB–stimulated controls from RTK array analysis shown in Fig. 5 B and Fig. 6 A , respectively. Mean pixel density ± the SD of duplicate spots, normalized against duplicate phosphotyrosine-positive control spots = 100. (C) Immunoprecipitation (IP) analysis of PDGFR tyrosine phosphorylation levels. MSCs in serum-free conditions were unstimulated with growth factor (basal), or stimulated with either 20 ng/ml VEGF-A 165 or PDGF-BB as a control, for 10 min at 37°C. PDGFRs were isolated from MSC lysates by IP analysis using anti-PDGFRα or anti-PDGFRβ, and then tyrosine phosphorylation detected by immunoblot (IB) analysis using anti-phosphotyrosine (Tyr-P). Membranes were reprobed with corresponding anti-PDGFRα or anti-PDGFRβ as loading controls. A representative of two independent experiments is shown.

    Techniques Used: Transfection, Positive Control, Immunoprecipitation, Isolation

    2) Product Images from "Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-? and VEGFR-2 expression"

    Article Title: Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-? and VEGFR-2 expression

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00411.2011

    PDGF-BB and VEGF induce greater ERK1/2 activation in VSMC compared with ASMC under hypoxia. VSMC ( A ) and ASMC ( B ) were incubated with PDGF-BB (10 ng/ml) and VEGF-A (10 ng/ml) under hypoxia for 24 h. VEGF-A-neutralizing antibody (α VEGF IgG) and
    Figure Legend Snippet: PDGF-BB and VEGF induce greater ERK1/2 activation in VSMC compared with ASMC under hypoxia. VSMC ( A ) and ASMC ( B ) were incubated with PDGF-BB (10 ng/ml) and VEGF-A (10 ng/ml) under hypoxia for 24 h. VEGF-A-neutralizing antibody (α VEGF IgG) and

    Techniques Used: Activation Assay, Incubation

    3) Product Images from "Soluble receptor-mediated selective inhibition of VEGFR and PDGFRβ signaling during physiologic and tumor angiogenesis"

    Article Title: Soluble receptor-mediated selective inhibition of VEGFR and PDGFRβ signaling during physiologic and tumor angiogenesis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0803194105

    Characterization of PDGFRβ and Tie2-Fc adenoviruses. Tail vein i.v. administration of Ad sPDGFRβ ( A ) or Ad Tie2-Fc ( C ) (10 9 pfu) to adult mice produced plasma expression of their respective ectodomains for > 30 days as determined by Western blotting. Affinity-purified PDGFRβ-His (200 ng/ml) and Tie2-Fc (5 μg/ml) ectodomains from adenoviral supernatants inhibited PDGF-BB- (10 ng/ml) and angiopoietin-1- (Ang1, 200 ng/ml) induced Akt phosphorylation in NIH 3T3 and HUVEC cells, respectively ( B and D ). IDE,insulin-degrading enzyme.
    Figure Legend Snippet: Characterization of PDGFRβ and Tie2-Fc adenoviruses. Tail vein i.v. administration of Ad sPDGFRβ ( A ) or Ad Tie2-Fc ( C ) (10 9 pfu) to adult mice produced plasma expression of their respective ectodomains for > 30 days as determined by Western blotting. Affinity-purified PDGFRβ-His (200 ng/ml) and Tie2-Fc (5 μg/ml) ectodomains from adenoviral supernatants inhibited PDGF-BB- (10 ng/ml) and angiopoietin-1- (Ang1, 200 ng/ml) induced Akt phosphorylation in NIH 3T3 and HUVEC cells, respectively ( B and D ). IDE,insulin-degrading enzyme.

    Techniques Used: Mouse Assay, Produced, Expressing, Western Blot, Affinity Purification

    4) Product Images from "TGF-?1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels"

    Article Title: TGF-?1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels

    Journal: Respiratory Research

    doi: 10.1186/1465-9921-6-141

    TUNEL positivity in HFL-1 with FCS, TGF-β1 and PDGF-BB. After staining, TUNEL positive cells as a % of total cells were counted under the microscope in 5 high-power fields. Vertical axis: TUNEL positivity expressed as % of positive control (DNAse treated). Horizontal axis: condition. TGF-β1 increased TUNEL positivity. In contrast, FCS or PDGF-BB did not affect TUNEL positivity. *p
    Figure Legend Snippet: TUNEL positivity in HFL-1 with FCS, TGF-β1 and PDGF-BB. After staining, TUNEL positive cells as a % of total cells were counted under the microscope in 5 high-power fields. Vertical axis: TUNEL positivity expressed as % of positive control (DNAse treated). Horizontal axis: condition. TGF-β1 increased TUNEL positivity. In contrast, FCS or PDGF-BB did not affect TUNEL positivity. *p

    Techniques Used: TUNEL Assay, Staining, Microscopy, Positive Control

    5) Product Images from "Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway"

    Article Title: Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030503

    The role of PI3K/Akt pathway in PDGF-BB-induced proliferation, migration, and tube formation of EPCs. ( A, B, C ) Cells in the control group (bright bars, left half) and the pEGFP-N2-PDGFR-β group (dark bars, right half) were without pretreatment or pretreated with AG1295, LY294002, or sc-221226 for 1 h and then treated with 20 ng/mL PDGF-BB (for the control cells) or 80 ng/mL PDGF-BB (for the pEGFP-N2-PDGFR-β cells). PDGF-BB-induced proliferation ( A ), migration ( B ), and angiogenesis ( C ) of EPCs were significantly inhibited by pretreatment with AG1295, LY294002, or sc-221226. ** P
    Figure Legend Snippet: The role of PI3K/Akt pathway in PDGF-BB-induced proliferation, migration, and tube formation of EPCs. ( A, B, C ) Cells in the control group (bright bars, left half) and the pEGFP-N2-PDGFR-β group (dark bars, right half) were without pretreatment or pretreated with AG1295, LY294002, or sc-221226 for 1 h and then treated with 20 ng/mL PDGF-BB (for the control cells) or 80 ng/mL PDGF-BB (for the pEGFP-N2-PDGFR-β cells). PDGF-BB-induced proliferation ( A ), migration ( B ), and angiogenesis ( C ) of EPCs were significantly inhibited by pretreatment with AG1295, LY294002, or sc-221226. ** P

    Techniques Used: Migration

    Effects of PDGFR-β overexpression on PDGF-BB-induced EPC migration. EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations. EPC migration was examined by the Transwell system. * P
    Figure Legend Snippet: Effects of PDGFR-β overexpression on PDGF-BB-induced EPC migration. EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations. EPC migration was examined by the Transwell system. * P

    Techniques Used: Over Expression, Migration, Transfection, Incubation

    Effects of PDGFR-β overexpression on PDGF-BB-induced EPC proliferation. EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations.EPC proliferation was examined by the MTS assay. * P
    Figure Legend Snippet: Effects of PDGFR-β overexpression on PDGF-BB-induced EPC proliferation. EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations.EPC proliferation was examined by the MTS assay. * P

    Techniques Used: Over Expression, Transfection, Incubation, MTS Assay

    ELISA of PDGF-BB secreted by EPCs in response to PDGFR-β transfection. The concentration of PDGF-BB in the supernatant of culture medium was measured using ELISA. The concentration of PDGF-BB was significantly lower in the pEGFP-N2-PDGFR-β group than in the control or pEGFP-N2 group at 48 h and 72 h post-transfection. * P
    Figure Legend Snippet: ELISA of PDGF-BB secreted by EPCs in response to PDGFR-β transfection. The concentration of PDGF-BB in the supernatant of culture medium was measured using ELISA. The concentration of PDGF-BB was significantly lower in the pEGFP-N2-PDGFR-β group than in the control or pEGFP-N2 group at 48 h and 72 h post-transfection. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Transfection, Concentration Assay

    Effects of PDGFR-β overexpression on PDGF-BB-induced EPC tube formation. EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations. EPC tube formation was examined by the In Vitro Angiogenesis Assay Kit. * P
    Figure Legend Snippet: Effects of PDGFR-β overexpression on PDGF-BB-induced EPC tube formation. EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations. EPC tube formation was examined by the In Vitro Angiogenesis Assay Kit. * P

    Techniques Used: Over Expression, Transfection, Incubation, In Vitro, Angiogenesis Assay

    6) Product Images from "Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway"

    Article Title: Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030503

    The role of PI3K/Akt pathway in PDGF-BB-induced proliferation, migration, and tube formation of EPCs. ( A, B, C ) Cells in the control group (bright bars, left half) and the pEGFP-N2-PDGFR-β group (dark bars, right half) were without pretreatment or pretreated with AG1295, LY294002, or sc-221226 for 1 h and then treated with 20 ng/mL PDGF-BB (for the control cells) or 80 ng/mL PDGF-BB (for the pEGFP-N2-PDGFR-β cells). PDGF-BB-induced proliferation ( A ), migration ( B ), and angiogenesis ( C ) of EPCs were significantly inhibited by pretreatment with AG1295, LY294002, or sc-221226. ** P
    Figure Legend Snippet: The role of PI3K/Akt pathway in PDGF-BB-induced proliferation, migration, and tube formation of EPCs. ( A, B, C ) Cells in the control group (bright bars, left half) and the pEGFP-N2-PDGFR-β group (dark bars, right half) were without pretreatment or pretreated with AG1295, LY294002, or sc-221226 for 1 h and then treated with 20 ng/mL PDGF-BB (for the control cells) or 80 ng/mL PDGF-BB (for the pEGFP-N2-PDGFR-β cells). PDGF-BB-induced proliferation ( A ), migration ( B ), and angiogenesis ( C ) of EPCs were significantly inhibited by pretreatment with AG1295, LY294002, or sc-221226. ** P

    Techniques Used: Migration

    Effects of PDGFR-β overexpression on PDGF-BB-induced EPC migration. EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations. EPC migration was examined by the Transwell system. * P
    Figure Legend Snippet: Effects of PDGFR-β overexpression on PDGF-BB-induced EPC migration. EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations. EPC migration was examined by the Transwell system. * P

    Techniques Used: Over Expression, Migration, Transfection, Incubation

    Effects of PDGFR-β overexpression on PDGF-BB-induced EPC proliferation. EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations.EPC proliferation was examined by the MTS assay. * P
    Figure Legend Snippet: Effects of PDGFR-β overexpression on PDGF-BB-induced EPC proliferation. EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations.EPC proliferation was examined by the MTS assay. * P

    Techniques Used: Over Expression, Transfection, Incubation, MTS Assay

    ELISA of PDGF-BB secreted by EPCs in response to PDGFR-β transfection. The concentration of PDGF-BB in the supernatant of culture medium was measured using ELISA. The concentration of PDGF-BB was significantly lower in the pEGFP-N2-PDGFR-β group than in the control or pEGFP-N2 group at 48 h and 72 h post-transfection. * P
    Figure Legend Snippet: ELISA of PDGF-BB secreted by EPCs in response to PDGFR-β transfection. The concentration of PDGF-BB in the supernatant of culture medium was measured using ELISA. The concentration of PDGF-BB was significantly lower in the pEGFP-N2-PDGFR-β group than in the control or pEGFP-N2 group at 48 h and 72 h post-transfection. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Transfection, Concentration Assay

    Effects of PDGFR-β overexpression on PDGF-BB-induced EPC tube formation. EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations. EPC tube formation was examined by the In Vitro Angiogenesis Assay Kit. * P
    Figure Legend Snippet: Effects of PDGFR-β overexpression on PDGF-BB-induced EPC tube formation. EPCs (untransfected,or transfected with either pEGFP-N2 or pEGFP-N2-PDGFR-β) were incubated with PDGF-BB of different concentrations. EPC tube formation was examined by the In Vitro Angiogenesis Assay Kit. * P

    Techniques Used: Over Expression, Transfection, Incubation, In Vitro, Angiogenesis Assay

    7) Product Images from "C-reactive protein and vein graft disease: evidence for a direct effect on smooth muscle cell phenotype via modulation of PDGF receptor-?"

    Article Title: C-reactive protein and vein graft disease: evidence for a direct effect on smooth muscle cell phenotype via modulation of PDGF receptor-?

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00079.2008

    A : representative semiquantitative RT-PCR analysis of PDGF receptor (PDGFR)-α, PDGFRβ, PDGF-A, and PDGF-D gene expression in HSV SMCs treated with 5 and 50 μg/ml CRP for 24 h. No reproducible evidence of PDGF-B gene expression
    Figure Legend Snippet: A : representative semiquantitative RT-PCR analysis of PDGF receptor (PDGFR)-α, PDGFRβ, PDGF-A, and PDGF-D gene expression in HSV SMCs treated with 5 and 50 μg/ml CRP for 24 h. No reproducible evidence of PDGF-B gene expression

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    8) Product Images from "Interferon-γ blocks signalling through PDGFRβ in human brain pericytes"

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0722-4

    Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p
    Figure Legend Snippet: Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p

    Techniques Used:

    Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p
    Figure Legend Snippet: Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p

    Techniques Used: Staining

    Human brain pericytes respond to PDGF-BB signals in vitro . a Pericytes were serum starved for 2 h, then treated with vehicle (lanes 1 and 7) or PDGF-BB (100 ng/mL) (lanes 2–6, and 8) for the indicated times, and analysed by SDS-PAGE. Representative blots from three cases are shown. b Blots from a were analysed and quantified with Image Studio™. Phosphorylated PDGFRβ (Tyr751) (p-PDGFRβ) ( b ), PDGFRβ ( c ), and PDGF-BB ( f ) were normalized to GAPDH; phosphorylated Akt (Ser473) (p-Akt) ( d ) and phosphorylated ERK (Tyr204) (p-ERK) ( e ) were normalized to total Akt and ERK, respectively. **Bottom band in pERK blot is GAPDH
    Figure Legend Snippet: Human brain pericytes respond to PDGF-BB signals in vitro . a Pericytes were serum starved for 2 h, then treated with vehicle (lanes 1 and 7) or PDGF-BB (100 ng/mL) (lanes 2–6, and 8) for the indicated times, and analysed by SDS-PAGE. Representative blots from three cases are shown. b Blots from a were analysed and quantified with Image Studio™. Phosphorylated PDGFRβ (Tyr751) (p-PDGFRβ) ( b ), PDGFRβ ( c ), and PDGF-BB ( f ) were normalized to GAPDH; phosphorylated Akt (Ser473) (p-Akt) ( d ) and phosphorylated ERK (Tyr204) (p-ERK) ( e ) were normalized to total Akt and ERK, respectively. **Bottom band in pERK blot is GAPDH

    Techniques Used: In Vitro, SDS Page

    Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Expressing, Western Blot, Software, Staining

    Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Western Blot, Software

    9) Product Images from "Interferon-γ blocks signalling through PDGFRβ in human brain pericytes"

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0722-4

    Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p
    Figure Legend Snippet: Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p

    Techniques Used:

    IFNγ blocks pericyte migration independently of PDGF-BB. a , b Cell migration was measured by scratching wells after 48 h of IFNγ (1 ng/mL) treatment and measuring the number of cells that moved into the gap area after a further 48 h with vehicle or PDGF-BB (10 ng/mL) treatment. Representative images of Coomassie-stained pericytes are presented in a , and manual counts of cells in the gap area (normalized to vehicle-treated condition), plotted as mean ± s.e.m. ( n = 3) #### , ****( p
    Figure Legend Snippet: IFNγ blocks pericyte migration independently of PDGF-BB. a , b Cell migration was measured by scratching wells after 48 h of IFNγ (1 ng/mL) treatment and measuring the number of cells that moved into the gap area after a further 48 h with vehicle or PDGF-BB (10 ng/mL) treatment. Representative images of Coomassie-stained pericytes are presented in a , and manual counts of cells in the gap area (normalized to vehicle-treated condition), plotted as mean ± s.e.m. ( n = 3) #### , ****( p

    Techniques Used: Migration, Staining

    Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p
    Figure Legend Snippet: Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p

    Techniques Used: Staining

    Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Expressing, Western Blot, Software, Staining

    Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Western Blot, Software

    Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p
    Figure Legend Snippet: Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p

    Techniques Used:

    Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p
    Figure Legend Snippet: Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p

    Techniques Used: Staining

    Human brain pericytes respond to PDGF-BB signals in vitro . a Pericytes were serum starved for 2 h, then treated with vehicle (lanes 1 and 7) or PDGF-BB (100 ng/mL) (lanes 2–6, and 8) for the indicated times, and analysed by SDS-PAGE. Representative blots from three cases are shown. b Blots from a were analysed and quantified with Image Studio™. Phosphorylated PDGFRβ (Tyr751) (p-PDGFRβ) ( b ), PDGFRβ ( c ), and PDGF-BB ( f ) were normalized to GAPDH; phosphorylated Akt (Ser473) (p-Akt) ( d ) and phosphorylated ERK (Tyr204) (p-ERK) ( e ) were normalized to total Akt and ERK, respectively. **Bottom band in pERK blot is GAPDH
    Figure Legend Snippet: Human brain pericytes respond to PDGF-BB signals in vitro . a Pericytes were serum starved for 2 h, then treated with vehicle (lanes 1 and 7) or PDGF-BB (100 ng/mL) (lanes 2–6, and 8) for the indicated times, and analysed by SDS-PAGE. Representative blots from three cases are shown. b Blots from a were analysed and quantified with Image Studio™. Phosphorylated PDGFRβ (Tyr751) (p-PDGFRβ) ( b ), PDGFRβ ( c ), and PDGF-BB ( f ) were normalized to GAPDH; phosphorylated Akt (Ser473) (p-Akt) ( d ) and phosphorylated ERK (Tyr204) (p-ERK) ( e ) were normalized to total Akt and ERK, respectively. **Bottom band in pERK blot is GAPDH

    Techniques Used: In Vitro, SDS Page

    Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Expressing, Western Blot, Software, Staining

    Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Western Blot, Software

    10) Product Images from "Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-? and VEGFR-2 expression"

    Article Title: Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-? and VEGFR-2 expression

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00411.2011

    PDGF-BB and VEGF induce greater ERK1/2 activation in VSMC compared with ASMC under hypoxia. VSMC ( A ) and ASMC ( B ) were incubated with PDGF-BB (10 ng/ml) and VEGF-A (10 ng/ml) under hypoxia for 24 h. VEGF-A-neutralizing antibody (α VEGF IgG) and
    Figure Legend Snippet: PDGF-BB and VEGF induce greater ERK1/2 activation in VSMC compared with ASMC under hypoxia. VSMC ( A ) and ASMC ( B ) were incubated with PDGF-BB (10 ng/ml) and VEGF-A (10 ng/ml) under hypoxia for 24 h. VEGF-A-neutralizing antibody (α VEGF IgG) and

    Techniques Used: Activation Assay, Incubation

    11) Product Images from "Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts *"

    Article Title: Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.673566

    Up-regulation of HAS-mediated HA synthesis by TGF-β1, bFGF, EGF, and PDGF-BB in normal human skin fibroblasts. A , HA content in the culture media of Detroit 551 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 or 48 h. The HA concentrations in the media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis by TGF-β1, bFGF, EGF, and PDGF-BB in normal human skin fibroblasts. A , HA content in the culture media of Detroit 551 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 or 48 h. The HA concentrations in the media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in HS27 cells. A , HA content in the culture media of HS27 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in HS27 cells. A , HA content in the culture media of HS27 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    Effects of TGF-β1, bFGF, EGF, and PDGF-BB, and knockdown of HYBID on the size distribution of newly produced HA. A , Detroit 551 cells were metabolically labeled with 10 μCi/ml [ 3 H]glucosamine in the absence ( Control ; open circle ) or presence of 1 ( gray triangle ) or 10 ( closed circle ) ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 48 h. The radiolabeled HA was isolated from the conditioned media and subjected to size-exclusion chromatography. B , knockdown efficiency and specificity for HYBID was evaluated by quantitative real-time PCR. The expression levels of HYBID , HAS1 , and HAS2 mRNAs in HYBID knocked-down cells at 24 h after treatment with siRNA were measured by quantitative real-time PCR. As for controls, the cells were transfected with control non-silencing siRNA ( Control siRNA ). Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to control siRNA-treated cells. Student's t test was used for statistical analysis. ***, p
    Figure Legend Snippet: Effects of TGF-β1, bFGF, EGF, and PDGF-BB, and knockdown of HYBID on the size distribution of newly produced HA. A , Detroit 551 cells were metabolically labeled with 10 μCi/ml [ 3 H]glucosamine in the absence ( Control ; open circle ) or presence of 1 ( gray triangle ) or 10 ( closed circle ) ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 48 h. The radiolabeled HA was isolated from the conditioned media and subjected to size-exclusion chromatography. B , knockdown efficiency and specificity for HYBID was evaluated by quantitative real-time PCR. The expression levels of HYBID , HAS1 , and HAS2 mRNAs in HYBID knocked-down cells at 24 h after treatment with siRNA were measured by quantitative real-time PCR. As for controls, the cells were transfected with control non-silencing siRNA ( Control siRNA ). Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to control siRNA-treated cells. Student's t test was used for statistical analysis. ***, p

    Techniques Used: Produced, Metabolic Labelling, Labeling, Isolation, Size-exclusion Chromatography, Real-time Polymerase Chain Reaction, Expressing, Transfection

    Down-regulation of HYBID-mediated HA depolymerization by TGF-β1, bFGF, EGF, and PDGF-BB. A , HA depolymerization by Detroit 551 cells treated with growth factors. Cells treated without ( Control ; open circle ) or with 1 ( gray triangle ) or 10 ng/ml ( closed circle ) TGF-β1, bFGF, EGF, or PDGF-BB were cultured with [ 3 H]HA for 48 h, and HA fragments in the culture media were examined by size-exclusion chromatography. B and C , the expression levels of HYBID mRNA ( B ) and protein ( C ) in cells treated without ( Control ) or with 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB. The mRNA levels at 8 and 24 h and the protein expression at 24 h after the treatment were measured by quantitative real-time PCR and immunoblotting, respectively. Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to 8 h control cells. Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Down-regulation of HYBID-mediated HA depolymerization by TGF-β1, bFGF, EGF, and PDGF-BB. A , HA depolymerization by Detroit 551 cells treated with growth factors. Cells treated without ( Control ; open circle ) or with 1 ( gray triangle ) or 10 ng/ml ( closed circle ) TGF-β1, bFGF, EGF, or PDGF-BB were cultured with [ 3 H]HA for 48 h, and HA fragments in the culture media were examined by size-exclusion chromatography. B and C , the expression levels of HYBID mRNA ( B ) and protein ( C ) in cells treated without ( Control ) or with 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB. The mRNA levels at 8 and 24 h and the protein expression at 24 h after the treatment were measured by quantitative real-time PCR and immunoblotting, respectively. Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to 8 h control cells. Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Cell Culture, Size-exclusion Chromatography, Expressing, Real-time Polymerase Chain Reaction

    Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in NHDF-Ad cells. A , HA content in the culture media of NHDF-Ad skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in NHDF-Ad cells. A , HA content in the culture media of NHDF-Ad skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    12) Product Images from "Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture -- primary culture cells markedly differ from fourth-passage cells"

    Article Title: Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture -- primary culture cells markedly differ from fourth-passage cells

    Journal: Arthritis Research

    doi:

    Proliferation rates, as assessed by incorporation of [ 3 H]-thymidine (counts per minute [cpm]) in: first-passage (white bars) and conventional (conv.) fourth-passage (dark gray bars) normal skin-FB ( n = 3 each) (A) isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) OA-SFB ( n = 3, 3, and 4, respectively) (B) and isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) RA-SFB ( n = 4, 3, and 5, respectively) (C) , at rest (control) or following stimulation with IL-1β (50, 100, or 150 U/ml) or PDGF-BB (2.5, 5, or 10 U/ml). See Results for details. @ P ≤ 0.05 for the comparison between cytokine-stimulated FB and non-stimulated control FB within the same FB preparation. * P ≤ 0.05 for the comparison between OA-SFB and RA-SFB.
    Figure Legend Snippet: Proliferation rates, as assessed by incorporation of [ 3 H]-thymidine (counts per minute [cpm]) in: first-passage (white bars) and conventional (conv.) fourth-passage (dark gray bars) normal skin-FB ( n = 3 each) (A) isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) OA-SFB ( n = 3, 3, and 4, respectively) (B) and isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) RA-SFB ( n = 4, 3, and 5, respectively) (C) , at rest (control) or following stimulation with IL-1β (50, 100, or 150 U/ml) or PDGF-BB (2.5, 5, or 10 U/ml). See Results for details. @ P ≤ 0.05 for the comparison between cytokine-stimulated FB and non-stimulated control FB within the same FB preparation. * P ≤ 0.05 for the comparison between OA-SFB and RA-SFB.

    Techniques Used: Isolation

    13) Product Images from "β-catenin-activated autocrine PDGF/Src signaling is a therapeutic target in pancreatic cancer"

    Article Title: β-catenin-activated autocrine PDGF/Src signaling is a therapeutic target in pancreatic cancer

    Journal: Theranostics

    doi: 10.7150/thno.28201

    The PDGF signaling pathway is significantly upregulated in the KPA tumors. ( A ) The differentially activated pathways were analyzed by Ingenuity Pathway Analysis and the genes with activation Z-score > 2 were highlighted by red color. ( B ) Expression of the PDGF family genes in the KPC and KPA cancer cells was investigated by real-time RT-PCR. The experiments were repeated three times and the data represent mean ± SEM (n=3). ( C ) Intracellular protein level of PDGF-A and PDGF-B was investigated by Western blotting and secreted PDGF-A and PDGF-B was determined by ELISA assay. Error bars are Means ± SEM. (n=3), * p
    Figure Legend Snippet: The PDGF signaling pathway is significantly upregulated in the KPA tumors. ( A ) The differentially activated pathways were analyzed by Ingenuity Pathway Analysis and the genes with activation Z-score > 2 were highlighted by red color. ( B ) Expression of the PDGF family genes in the KPC and KPA cancer cells was investigated by real-time RT-PCR. The experiments were repeated three times and the data represent mean ± SEM (n=3). ( C ) Intracellular protein level of PDGF-A and PDGF-B was investigated by Western blotting and secreted PDGF-A and PDGF-B was determined by ELISA assay. Error bars are Means ± SEM. (n=3), * p

    Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

    Activation of PDGF/Src signaling predicts poor clinical outcome in pancreatic cancer patients. (A ) Representative images show various expressions of PDGF-B and p-Src in pancreatic tumor tissues. Scale bars represent 50 μm. ( B ) Serum PDGF level (Mean ± SD.) of 20 pancreatic cancer patients with high co-expression of tumor PDGF/p-Src (+/+) was determined by ELISA assay and was compared to the other patients without co-expression (-/+, +/- or -/-). ( C ) The associations between serum PDGF level with the expression of PDGF and p-Src in tumor tissues were tested. Each dot represents an individual patient. ( D ) Overall survival of a cohort of pancreatic cancer patients (n=77) with high tumor PDGF/p-Src (+/+, median survival=12.2 months) was compared to the patients with low tumor PDGF/p-Src (-/-, median survival=25.8 months) or with high PDGF or high p-Src alone (+/- or -/+, median survival=26 months). ( E ) Ten and twelve tumor tissues of the high or low PDGF/p-Src (+/+ and -/-) groups were subjected to genomic DNA isolation and sequencing. The mutation rates of RNF43 and AXIN1 genes in these two groups were shown separately. The total mutation rates were also included.
    Figure Legend Snippet: Activation of PDGF/Src signaling predicts poor clinical outcome in pancreatic cancer patients. (A ) Representative images show various expressions of PDGF-B and p-Src in pancreatic tumor tissues. Scale bars represent 50 μm. ( B ) Serum PDGF level (Mean ± SD.) of 20 pancreatic cancer patients with high co-expression of tumor PDGF/p-Src (+/+) was determined by ELISA assay and was compared to the other patients without co-expression (-/+, +/- or -/-). ( C ) The associations between serum PDGF level with the expression of PDGF and p-Src in tumor tissues were tested. Each dot represents an individual patient. ( D ) Overall survival of a cohort of pancreatic cancer patients (n=77) with high tumor PDGF/p-Src (+/+, median survival=12.2 months) was compared to the patients with low tumor PDGF/p-Src (-/-, median survival=25.8 months) or with high PDGF or high p-Src alone (+/- or -/+, median survival=26 months). ( E ) Ten and twelve tumor tissues of the high or low PDGF/p-Src (+/+ and -/-) groups were subjected to genomic DNA isolation and sequencing. The mutation rates of RNF43 and AXIN1 genes in these two groups were shown separately. The total mutation rates were also included.

    Techniques Used: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, DNA Extraction, Sequencing, Mutagenesis

    14) Product Images from "Platelet counts predict prognosis in IPF, but are not the main source of pulmonary TGFβ1"

    Article Title: Platelet counts predict prognosis in IPF, but are not the main source of pulmonary TGFβ1

    Journal: bioRxiv

    doi: 10.1101/2020.03.06.978874

    Human blood platelet counts predict mortality in IPF and levels of TGFβ1 correlate with an activated blood platelet signature. a) Kaplan-Meier survival curve of IPF patients ( n =214) divided into three strata according to platelet count. The three strata were defined bias-free by dividing into three equal groups: Group 1=lowest third (blue), Group 2=middle third (red) and Group 3=highest third (green). Censored patients are indicated on the graph as ticks. Number of patients at risk for each time-point are shown below the Kaplan-Meier curve. b) Concentrations of total or active TGFβ1, PDGF-BB, CCL5, CXCL4 and MMP7 were measured in non-ILD and ILD patients’ plasma ( n =11 and 47 respectively) by ELISA or MLEC bioassay. c) Concentration of mediators in ILD patients’ plasma were correlated with paired blood platelet counts ( n =47). d) Concentration of mediators in ILD patients’ plasma were correlated with paired CXCL4 levels ( n =47). Any statistical differences were determined using log rank test for trend, Mann Whitney-U test or linear regression and any significant differences are indicated (n/s = not significant, * p
    Figure Legend Snippet: Human blood platelet counts predict mortality in IPF and levels of TGFβ1 correlate with an activated blood platelet signature. a) Kaplan-Meier survival curve of IPF patients ( n =214) divided into three strata according to platelet count. The three strata were defined bias-free by dividing into three equal groups: Group 1=lowest third (blue), Group 2=middle third (red) and Group 3=highest third (green). Censored patients are indicated on the graph as ticks. Number of patients at risk for each time-point are shown below the Kaplan-Meier curve. b) Concentrations of total or active TGFβ1, PDGF-BB, CCL5, CXCL4 and MMP7 were measured in non-ILD and ILD patients’ plasma ( n =11 and 47 respectively) by ELISA or MLEC bioassay. c) Concentration of mediators in ILD patients’ plasma were correlated with paired blood platelet counts ( n =47). d) Concentration of mediators in ILD patients’ plasma were correlated with paired CXCL4 levels ( n =47). Any statistical differences were determined using log rank test for trend, Mann Whitney-U test or linear regression and any significant differences are indicated (n/s = not significant, * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, MANN-WHITNEY

    Platelet-derived mediators may contribute to neutrophil recruitment in IPF lung and disease severity a) Representative flow cytometric plot showing platelet population in non-ILD and ILD BALF. Quantification of percentage of BALF platelets was done by flow cytometric analysis ( n =3 non-ILD and n =6 ILD). b) Total or active TGFβ1, PDGF-BB, CCL5, CXCL4 and MMP7 concentrations were measured in non-ILD and ILD patients’ BALF ( n =6 and 19 respectively) by ELISA or MLEC bioassay. c) Concentration of mediators in ILD patients’ BALF were correlated with paired CXCL4 ( n =19). d) Concentration of CCL5 in ILD patients’ BALF were correlated with paired CXCL4 or MMP7 ( n =19). Statistical differences were determined using Mann Whitney-U tests or linear regression and any significant differences are indicated (n/s = not significant, * p
    Figure Legend Snippet: Platelet-derived mediators may contribute to neutrophil recruitment in IPF lung and disease severity a) Representative flow cytometric plot showing platelet population in non-ILD and ILD BALF. Quantification of percentage of BALF platelets was done by flow cytometric analysis ( n =3 non-ILD and n =6 ILD). b) Total or active TGFβ1, PDGF-BB, CCL5, CXCL4 and MMP7 concentrations were measured in non-ILD and ILD patients’ BALF ( n =6 and 19 respectively) by ELISA or MLEC bioassay. c) Concentration of mediators in ILD patients’ BALF were correlated with paired CXCL4 ( n =19). d) Concentration of CCL5 in ILD patients’ BALF were correlated with paired CXCL4 or MMP7 ( n =19). Statistical differences were determined using Mann Whitney-U tests or linear regression and any significant differences are indicated (n/s = not significant, * p

    Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, MANN-WHITNEY

    15) Product Images from "In vitro immunological and biological evaluations of the angiogenic potential of platelet-rich fibrin preparations: a standardized comparison with PRP preparations"

    Article Title: In vitro immunological and biological evaluations of the angiogenic potential of platelet-rich fibrin preparations: a standardized comparison with PRP preparations

    Journal: International Journal of Implant Dentistry

    doi: 10.1186/s40729-015-0032-0

    The concentration of platelets, VEGF, PDGF-BB, and DLL1 in PPP, PRP, PRF exu, and PRF ext preparations. n = 5
    Figure Legend Snippet: The concentration of platelets, VEGF, PDGF-BB, and DLL1 in PPP, PRP, PRF exu, and PRF ext preparations. n = 5

    Techniques Used: Concentration Assay

    The concentration of platelets, VEGF, PDGF-BB, and DLL1 in PPP, PRP, PRF exu, and PRF ext preparations. n = 5
    Figure Legend Snippet: The concentration of platelets, VEGF, PDGF-BB, and DLL1 in PPP, PRP, PRF exu, and PRF ext preparations. n = 5

    Techniques Used: Concentration Assay

    16) Product Images from "In vitro immunological and biological evaluations of the angiogenic potential of platelet-rich fibrin preparations: a standardized comparison with PRP preparations"

    Article Title: In vitro immunological and biological evaluations of the angiogenic potential of platelet-rich fibrin preparations: a standardized comparison with PRP preparations

    Journal: International Journal of Implant Dentistry

    doi: 10.1186/s40729-015-0032-0

    The concentration of platelets, VEGF, PDGF-BB, and DLL1 in PPP, PRP, PRF exu, and PRF ext preparations. n = 5
    Figure Legend Snippet: The concentration of platelets, VEGF, PDGF-BB, and DLL1 in PPP, PRP, PRF exu, and PRF ext preparations. n = 5

    Techniques Used: Concentration Assay

    The concentration of platelets, VEGF, PDGF-BB, and DLL1 in PPP, PRP, PRF exu, and PRF ext preparations. n = 5
    Figure Legend Snippet: The concentration of platelets, VEGF, PDGF-BB, and DLL1 in PPP, PRP, PRF exu, and PRF ext preparations. n = 5

    Techniques Used: Concentration Assay

    17) Product Images from "PDGF-BB secreted by preosteoclasts induces CD31hiEmcnhi vessel subtype in coupling osteogenesis"

    Article Title: PDGF-BB secreted by preosteoclasts induces CD31hiEmcnhi vessel subtype in coupling osteogenesis

    Journal: Nature medicine

    doi: 10.1038/nm.3668

    Preosteoclasts secrete PDGF-BB to induce migration of MSCs and EPCs. ( a ) Transwell assays for the migration of MSCs using conditioned medium (CM) collected from different cell cultures with (+) or without (−) bone slices. ( b ) Transwell assays for the migration of MSCs using conditioned medium of preosteoclasts + bone slices (POC CM) with addition of individual neutralizing antibody (Ab), IgG or Noggin, as indicated; or using conditioned medium of osteoclasts + bone slices (OC CM) with addition of individual neutralizing Ab or IgG. ( c,d ) ELISA analysis of concentrations of PDGF-BB ( c ) and CTX ( d ) in different conditioned media. ( e ) Immunoprecipitation and immunoblotting analysis of PDGF-BB levels in different conditioned media. Platelet: mouse platelet lysate (positive control). ( f ) Transwell assays for the migration of EPC using conditioned media from different cell cultures as indicated or conditioned medium of preosteoclasts + bone slices (POC CM) with addition of IgG or PDGF-BB neutralizing Ab. FV: field of view (×200 magnification). Medium Ctrl: serum free α-MEM. Mo/Mac: monocytes/macrophages; POC: preosteoclasts; OC: osteoclasts. n = 4. Data are shown as the mean ± s.d. * P
    Figure Legend Snippet: Preosteoclasts secrete PDGF-BB to induce migration of MSCs and EPCs. ( a ) Transwell assays for the migration of MSCs using conditioned medium (CM) collected from different cell cultures with (+) or without (−) bone slices. ( b ) Transwell assays for the migration of MSCs using conditioned medium of preosteoclasts + bone slices (POC CM) with addition of individual neutralizing antibody (Ab), IgG or Noggin, as indicated; or using conditioned medium of osteoclasts + bone slices (OC CM) with addition of individual neutralizing Ab or IgG. ( c,d ) ELISA analysis of concentrations of PDGF-BB ( c ) and CTX ( d ) in different conditioned media. ( e ) Immunoprecipitation and immunoblotting analysis of PDGF-BB levels in different conditioned media. Platelet: mouse platelet lysate (positive control). ( f ) Transwell assays for the migration of EPC using conditioned media from different cell cultures as indicated or conditioned medium of preosteoclasts + bone slices (POC CM) with addition of IgG or PDGF-BB neutralizing Ab. FV: field of view (×200 magnification). Medium Ctrl: serum free α-MEM. Mo/Mac: monocytes/macrophages; POC: preosteoclasts; OC: osteoclasts. n = 4. Data are shown as the mean ± s.d. * P

    Techniques Used: Migration, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Positive Control

    Knockout of PDGF-BB in preosteoclasts reduces CD31 hi Emcn hi vessels and bone formation. ( a ) μCT images and quantification of trabecular bone fraction (Tb. BV/TV), trabecular thickness (Tb. Th), cortical thickness (Ct. Th), and periosteal perimeter (Ps. Pm). Scale bars, 1 mm. ( b ) Calcein double labeling of trabecular (TB) and periosteal bone (PB) with quantification of mineral apposition rate (MAR) and bone formation rate (BFR). Scale bar, 20 μm. n = 5. ( c ) Immunostaining of TRAP (red) and PDGF-BB (green) with quantification of number of TRAP positive cells (N. TRAP + ) per respective bone surface (BS). Scale bar, 50 μm. ( d , e ) ELISA for serum CTX ( d ), serum or bone marrow (BM) PDGF-BB and VEGF concentrations ( e ). ( f ) Microphil-perfused angiography with quantification of vessel volume and surface. Scale bar, 1 mm. n = 5. ( g ) Immunostaining of CD31 (red) and Endomucin (green) with quantification of CD31 hi Emcn hi (yellow) cells in BM and periosteum (P) (bottom). Scale bar, 50 μm. ( h ) Flow cytometry plots (left) with percentage (right) of CD31 hi Emcn hi cells in total bone marrow cells (BMCs). n = 4 . ( i ) Immunostaining of Endomucin (red) and Ki67 (green). ( j ) Immunostaining of Osteocalcin (green) (left) with quantification of osteocalcin + cell numbers (middle) on TB and PB surface. Serum osteocalcin concentrations by ELISA (right). Scale bar, 20 μm. Dashed lines outline bone surface. DAPI stains (blue) nuclei. CB, cortical bone; BM, bone marrow. Data are shown as the mean ± s.d. n = 7−8, unless otherwise noted. * P
    Figure Legend Snippet: Knockout of PDGF-BB in preosteoclasts reduces CD31 hi Emcn hi vessels and bone formation. ( a ) μCT images and quantification of trabecular bone fraction (Tb. BV/TV), trabecular thickness (Tb. Th), cortical thickness (Ct. Th), and periosteal perimeter (Ps. Pm). Scale bars, 1 mm. ( b ) Calcein double labeling of trabecular (TB) and periosteal bone (PB) with quantification of mineral apposition rate (MAR) and bone formation rate (BFR). Scale bar, 20 μm. n = 5. ( c ) Immunostaining of TRAP (red) and PDGF-BB (green) with quantification of number of TRAP positive cells (N. TRAP + ) per respective bone surface (BS). Scale bar, 50 μm. ( d , e ) ELISA for serum CTX ( d ), serum or bone marrow (BM) PDGF-BB and VEGF concentrations ( e ). ( f ) Microphil-perfused angiography with quantification of vessel volume and surface. Scale bar, 1 mm. n = 5. ( g ) Immunostaining of CD31 (red) and Endomucin (green) with quantification of CD31 hi Emcn hi (yellow) cells in BM and periosteum (P) (bottom). Scale bar, 50 μm. ( h ) Flow cytometry plots (left) with percentage (right) of CD31 hi Emcn hi cells in total bone marrow cells (BMCs). n = 4 . ( i ) Immunostaining of Endomucin (red) and Ki67 (green). ( j ) Immunostaining of Osteocalcin (green) (left) with quantification of osteocalcin + cell numbers (middle) on TB and PB surface. Serum osteocalcin concentrations by ELISA (right). Scale bar, 20 μm. Dashed lines outline bone surface. DAPI stains (blue) nuclei. CB, cortical bone; BM, bone marrow. Data are shown as the mean ± s.d. n = 7−8, unless otherwise noted. * P

    Techniques Used: Knock-Out, Labeling, Immunostaining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    CTSK inhibitor increases TRAP + cell PDGF-BB secretion to couple CD31 hi Emcn hi vessels with bone formation. ( a ) Quantification of cells positive immunostaining for TRAP and PDGF-BB (N. TRAP + PDGF-BB + ) on trabecular (TB) and periosteal (P) bone in wild-type ( Ctsk +/+ ) with or without cathepsin K inhibitor (L-235) or knockout ( Ctsk –/– ) mice. n = 8. ( b ) PDGF-BB and VEGF concentrations by ELISA in serum and bone marrow (BM). n = 8. ( c ) Quantification of vessel volume and surface. n = 6. ( d ) CD31 (red) and Endomucin (green) immunostaining with quantification of CD31 hi Emcn hi (yellow) cells. Scale bar, 50 μm. n = 8. ( e ) Percentage of CD31 hi Emcn hi cells in total BM cells (BMCs) by flow cytometry. n = 3. ( f ) PDGF-BB and VEGF concentrations by ELISA in serum and BM of Pdgfb –/– or Pdgfb +/+ littermates treated with vehicle or L-235. n = 5. ( g ) Quantification of vessel volume and surface. n = 5. ( h ) Quantification of CD31 hi Emcn hi immunostaining in TB (left) and P (middle). n = 5. Endomucin (red) and Ki67 (green) immunostaining of proliferating endothelial cells (right). ( i ) μCT quantification of cortical thickness (Ct. Th), periosteal perimeter (Ps. Pm), trabecular bone fraction (Tb. BV/TV) and trabecular thickness (Tb. Th). n = 5. ( j ) Serum osteocalcin and CTX concentrations by ELISA. n = 5. Dashed lines outline bone surface. DAPI stains (blue) nuclei. CB: cortical bone. Data shown as mean ± s.d. * P
    Figure Legend Snippet: CTSK inhibitor increases TRAP + cell PDGF-BB secretion to couple CD31 hi Emcn hi vessels with bone formation. ( a ) Quantification of cells positive immunostaining for TRAP and PDGF-BB (N. TRAP + PDGF-BB + ) on trabecular (TB) and periosteal (P) bone in wild-type ( Ctsk +/+ ) with or without cathepsin K inhibitor (L-235) or knockout ( Ctsk –/– ) mice. n = 8. ( b ) PDGF-BB and VEGF concentrations by ELISA in serum and bone marrow (BM). n = 8. ( c ) Quantification of vessel volume and surface. n = 6. ( d ) CD31 (red) and Endomucin (green) immunostaining with quantification of CD31 hi Emcn hi (yellow) cells. Scale bar, 50 μm. n = 8. ( e ) Percentage of CD31 hi Emcn hi cells in total BM cells (BMCs) by flow cytometry. n = 3. ( f ) PDGF-BB and VEGF concentrations by ELISA in serum and BM of Pdgfb –/– or Pdgfb +/+ littermates treated with vehicle or L-235. n = 5. ( g ) Quantification of vessel volume and surface. n = 5. ( h ) Quantification of CD31 hi Emcn hi immunostaining in TB (left) and P (middle). n = 5. Endomucin (red) and Ki67 (green) immunostaining of proliferating endothelial cells (right). ( i ) μCT quantification of cortical thickness (Ct. Th), periosteal perimeter (Ps. Pm), trabecular bone fraction (Tb. BV/TV) and trabecular thickness (Tb. Th). n = 5. ( j ) Serum osteocalcin and CTX concentrations by ELISA. n = 5. Dashed lines outline bone surface. DAPI stains (blue) nuclei. CB: cortical bone. Data shown as mean ± s.d. * P

    Techniques Used: Immunostaining, Knock-Out, Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    Increasing PDGF-BB stimulates CD31 hi Emcn hi vessels and bone formation in OVX osteoporotic mice. ( a ) PDGF-BB and VEGF concentrations by ELISA in serum and bone marrow (BM) in sham-operated (SHAM) or ovariectomized mice (OVX). ( b ) Quantification of vessel volume and surface. ( c ) TRAP (red) and PDGF-BB (green) immunostaining and quantification of TRAP + and TRAP + PDGF-BB + cells on trabecular (TB) and periosteal bone (PB) surfaces, respectively. ( d ) CD31 (red) and Endomucin (green) immunostaining and quantification of CD31 hi Emcn hi (yellow) cells in BM and periosteum. Scale bar, 50 μm. ( e ) PDGF-BB and VEGF concentrations by ELISA in serum and bone marrow of OVX mice treated with vehicle, PDGF-BB, or cathepsin K inhibitor (L-235). ( f,g ) Quantification of vessel volume and surface ( f ) and immunostaining of CD31 hi Emcn hi cells ( g ). ( h ) Quantificaion of mineral apposition rate (MAR) and bone formation rate (BFR) in TB and periosteal bone (PB). ( i ) Serum osteocalcin and CTX concentrations by ELISA. ( j ) Model of PDGF-BB secreted by preosteoclasts to couple angiogenesis and osteogenesis. In periosteal bone modeling, preosteoclast secretion of PDGF-BB induces formation of CD31 hi Emcn hi vessels and stimulates secretion of S1P to promote osteoblast differentiation. In trabecular bone remodeling, CD31 hi Emcn hi vessels induced by preosteoclast secretion of PDGF-BB improves transport of nutrients, oxygen, minerals and metabolic wastes during bone remodeling. Dashed lines outline bone surface. Scale bar, 50 μm. Data shown as mean ± s.d. For a−d and h n = 5; For e − g and i n = 10. * P
    Figure Legend Snippet: Increasing PDGF-BB stimulates CD31 hi Emcn hi vessels and bone formation in OVX osteoporotic mice. ( a ) PDGF-BB and VEGF concentrations by ELISA in serum and bone marrow (BM) in sham-operated (SHAM) or ovariectomized mice (OVX). ( b ) Quantification of vessel volume and surface. ( c ) TRAP (red) and PDGF-BB (green) immunostaining and quantification of TRAP + and TRAP + PDGF-BB + cells on trabecular (TB) and periosteal bone (PB) surfaces, respectively. ( d ) CD31 (red) and Endomucin (green) immunostaining and quantification of CD31 hi Emcn hi (yellow) cells in BM and periosteum. Scale bar, 50 μm. ( e ) PDGF-BB and VEGF concentrations by ELISA in serum and bone marrow of OVX mice treated with vehicle, PDGF-BB, or cathepsin K inhibitor (L-235). ( f,g ) Quantification of vessel volume and surface ( f ) and immunostaining of CD31 hi Emcn hi cells ( g ). ( h ) Quantificaion of mineral apposition rate (MAR) and bone formation rate (BFR) in TB and periosteal bone (PB). ( i ) Serum osteocalcin and CTX concentrations by ELISA. ( j ) Model of PDGF-BB secreted by preosteoclasts to couple angiogenesis and osteogenesis. In periosteal bone modeling, preosteoclast secretion of PDGF-BB induces formation of CD31 hi Emcn hi vessels and stimulates secretion of S1P to promote osteoblast differentiation. In trabecular bone remodeling, CD31 hi Emcn hi vessels induced by preosteoclast secretion of PDGF-BB improves transport of nutrients, oxygen, minerals and metabolic wastes during bone remodeling. Dashed lines outline bone surface. Scale bar, 50 μm. Data shown as mean ± s.d. For a−d and h n = 5; For e − g and i n = 10. * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunostaining

    18) Product Images from "Ethyl Acetate Fraction of Amomum xanthioides Exerts Antihepatofibrotic Actions via the Regulation of Fibrogenic Cytokines in a Dimethylnitrosamine-Induced Rat Model"

    Article Title: Ethyl Acetate Fraction of Amomum xanthioides Exerts Antihepatofibrotic Actions via the Regulation of Fibrogenic Cytokines in a Dimethylnitrosamine-Induced Rat Model

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2016/6014380

    The mRNA expression levels of liver fibrosis-related genes in liver tissues. The analyses of mRNA expression levels were performed to determine the mRNA levels of (a) collagen type 1a1, α -SMA, TGF- β , PDGF- β , and CTGF and (b) TIMP-1, MMP-2, BAMBI, and Smad7 using real-time PCR. Gene expression is presented with the level in the naive group set as 1 after normalization to β -actin. The data are expressed as the mean ± SD ( n = 6). ## p
    Figure Legend Snippet: The mRNA expression levels of liver fibrosis-related genes in liver tissues. The analyses of mRNA expression levels were performed to determine the mRNA levels of (a) collagen type 1a1, α -SMA, TGF- β , PDGF- β , and CTGF and (b) TIMP-1, MMP-2, BAMBI, and Smad7 using real-time PCR. Gene expression is presented with the level in the naive group set as 1 after normalization to β -actin. The data are expressed as the mean ± SD ( n = 6). ## p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Determination of fibrogenic cytokines and TIMP-1 levels in liver tissues. The quantitative analysis of (a) TGF- β 1, (b) PDGF-BB, (c) CTGF, and (d) TIMP-1 was performed in liver tissues using ELISA kits. The data are expressed as the mean ± SD ( n = 6). ## p
    Figure Legend Snippet: Determination of fibrogenic cytokines and TIMP-1 levels in liver tissues. The quantitative analysis of (a) TGF- β 1, (b) PDGF-BB, (c) CTGF, and (d) TIMP-1 was performed in liver tissues using ELISA kits. The data are expressed as the mean ± SD ( n = 6). ## p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    19) Product Images from "Interferon-γ blocks signalling through PDGFRβ in human brain pericytes"

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0722-4

    Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p
    Figure Legend Snippet: Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p

    Techniques Used:

    Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p
    Figure Legend Snippet: Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p

    Techniques Used: Staining

    Human brain pericytes respond to PDGF-BB signals in vitro . a Pericytes were serum starved for 2 h, then treated with vehicle (lanes 1 and 7) or PDGF-BB (100 ng/mL) (lanes 2–6, and 8) for the indicated times, and analysed by SDS-PAGE. Representative blots from three cases are shown. b Blots from a were analysed and quantified with Image Studio™. Phosphorylated PDGFRβ (Tyr751) (p-PDGFRβ) ( b ), PDGFRβ ( c ), and PDGF-BB ( f ) were normalized to GAPDH; phosphorylated Akt (Ser473) (p-Akt) ( d ) and phosphorylated ERK (Tyr204) (p-ERK) ( e ) were normalized to total Akt and ERK, respectively. **Bottom band in pERK blot is GAPDH
    Figure Legend Snippet: Human brain pericytes respond to PDGF-BB signals in vitro . a Pericytes were serum starved for 2 h, then treated with vehicle (lanes 1 and 7) or PDGF-BB (100 ng/mL) (lanes 2–6, and 8) for the indicated times, and analysed by SDS-PAGE. Representative blots from three cases are shown. b Blots from a were analysed and quantified with Image Studio™. Phosphorylated PDGFRβ (Tyr751) (p-PDGFRβ) ( b ), PDGFRβ ( c ), and PDGF-BB ( f ) were normalized to GAPDH; phosphorylated Akt (Ser473) (p-Akt) ( d ) and phosphorylated ERK (Tyr204) (p-ERK) ( e ) were normalized to total Akt and ERK, respectively. **Bottom band in pERK blot is GAPDH

    Techniques Used: In Vitro, SDS Page

    Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Expressing, Western Blot, Software, Staining

    Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Western Blot, Software

    20) Product Images from "Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts *"

    Article Title: Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.673566

    Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in HS27 cells. A , HA content in the culture media of HS27 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in HS27 cells. A , HA content in the culture media of HS27 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    Effects of TGF-β1, bFGF, EGF, and PDGF-BB, and knockdown of HYBID on the size distribution of newly produced HA. A , Detroit 551 cells were metabolically labeled with 10 μCi/ml [ 3 H]glucosamine in the absence ( Control ; open circle ) or presence of 1 ( gray triangle ) or 10 ( closed circle ) ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 48 h. The radiolabeled HA was isolated from the conditioned media and subjected to size-exclusion chromatography. B , knockdown efficiency and specificity for HYBID was evaluated by quantitative real-time PCR. The expression levels of HYBID , HAS1 , and HAS2 mRNAs in HYBID knocked-down cells at 24 h after treatment with siRNA were measured by quantitative real-time PCR. As for controls, the cells were transfected with control non-silencing siRNA ( Control siRNA ). Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to control siRNA-treated cells. Student's t test was used for statistical analysis. ***, p
    Figure Legend Snippet: Effects of TGF-β1, bFGF, EGF, and PDGF-BB, and knockdown of HYBID on the size distribution of newly produced HA. A , Detroit 551 cells were metabolically labeled with 10 μCi/ml [ 3 H]glucosamine in the absence ( Control ; open circle ) or presence of 1 ( gray triangle ) or 10 ( closed circle ) ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 48 h. The radiolabeled HA was isolated from the conditioned media and subjected to size-exclusion chromatography. B , knockdown efficiency and specificity for HYBID was evaluated by quantitative real-time PCR. The expression levels of HYBID , HAS1 , and HAS2 mRNAs in HYBID knocked-down cells at 24 h after treatment with siRNA were measured by quantitative real-time PCR. As for controls, the cells were transfected with control non-silencing siRNA ( Control siRNA ). Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to control siRNA-treated cells. Student's t test was used for statistical analysis. ***, p

    Techniques Used: Produced, Metabolic Labelling, Labeling, Isolation, Size-exclusion Chromatography, Real-time Polymerase Chain Reaction, Expressing, Transfection

    Down-regulation of HYBID-mediated HA depolymerization by TGF-β1, bFGF, EGF, and PDGF-BB. A , HA depolymerization by Detroit 551 cells treated with growth factors. Cells treated without ( Control ; open circle ) or with 1 ( gray triangle ) or 10 ng/ml ( closed circle ) TGF-β1, bFGF, EGF, or PDGF-BB were cultured with [ 3 H]HA for 48 h, and HA fragments in the culture media were examined by size-exclusion chromatography. B and C , the expression levels of HYBID mRNA ( B ) and protein ( C ) in cells treated without ( Control ) or with 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB. The mRNA levels at 8 and 24 h and the protein expression at 24 h after the treatment were measured by quantitative real-time PCR and immunoblotting, respectively. Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to 8 h control cells. Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Down-regulation of HYBID-mediated HA depolymerization by TGF-β1, bFGF, EGF, and PDGF-BB. A , HA depolymerization by Detroit 551 cells treated with growth factors. Cells treated without ( Control ; open circle ) or with 1 ( gray triangle ) or 10 ng/ml ( closed circle ) TGF-β1, bFGF, EGF, or PDGF-BB were cultured with [ 3 H]HA for 48 h, and HA fragments in the culture media were examined by size-exclusion chromatography. B and C , the expression levels of HYBID mRNA ( B ) and protein ( C ) in cells treated without ( Control ) or with 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB. The mRNA levels at 8 and 24 h and the protein expression at 24 h after the treatment were measured by quantitative real-time PCR and immunoblotting, respectively. Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to 8 h control cells. Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Cell Culture, Size-exclusion Chromatography, Expressing, Real-time Polymerase Chain Reaction

    Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in NHDF-Ad cells. A , HA content in the culture media of NHDF-Ad skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in NHDF-Ad cells. A , HA content in the culture media of NHDF-Ad skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    21) Product Images from "Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts *"

    Article Title: Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.673566

    Up-regulation of HAS-mediated HA synthesis by TGF-β1, bFGF, EGF, and PDGF-BB in normal human skin fibroblasts. A , HA content in the culture media of Detroit 551 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 or 48 h. The HA concentrations in the media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis by TGF-β1, bFGF, EGF, and PDGF-BB in normal human skin fibroblasts. A , HA content in the culture media of Detroit 551 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 or 48 h. The HA concentrations in the media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in HS27 cells. A , HA content in the culture media of HS27 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in HS27 cells. A , HA content in the culture media of HS27 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    Effects of TGF-β1, bFGF, EGF, and PDGF-BB, and knockdown of HYBID on the size distribution of newly produced HA. A , Detroit 551 cells were metabolically labeled with 10 μCi/ml [ 3 H]glucosamine in the absence ( Control ; open circle ) or presence of 1 ( gray triangle ) or 10 ( closed circle ) ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 48 h. The radiolabeled HA was isolated from the conditioned media and subjected to size-exclusion chromatography. B , knockdown efficiency and specificity for HYBID was evaluated by quantitative real-time PCR. The expression levels of HYBID , HAS1 , and HAS2 mRNAs in HYBID knocked-down cells at 24 h after treatment with siRNA were measured by quantitative real-time PCR. As for controls, the cells were transfected with control non-silencing siRNA ( Control siRNA ). Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to control siRNA-treated cells. Student's t test was used for statistical analysis. ***, p
    Figure Legend Snippet: Effects of TGF-β1, bFGF, EGF, and PDGF-BB, and knockdown of HYBID on the size distribution of newly produced HA. A , Detroit 551 cells were metabolically labeled with 10 μCi/ml [ 3 H]glucosamine in the absence ( Control ; open circle ) or presence of 1 ( gray triangle ) or 10 ( closed circle ) ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 48 h. The radiolabeled HA was isolated from the conditioned media and subjected to size-exclusion chromatography. B , knockdown efficiency and specificity for HYBID was evaluated by quantitative real-time PCR. The expression levels of HYBID , HAS1 , and HAS2 mRNAs in HYBID knocked-down cells at 24 h after treatment with siRNA were measured by quantitative real-time PCR. As for controls, the cells were transfected with control non-silencing siRNA ( Control siRNA ). Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to control siRNA-treated cells. Student's t test was used for statistical analysis. ***, p

    Techniques Used: Produced, Metabolic Labelling, Labeling, Isolation, Size-exclusion Chromatography, Real-time Polymerase Chain Reaction, Expressing, Transfection

    Down-regulation of HYBID-mediated HA depolymerization by TGF-β1, bFGF, EGF, and PDGF-BB. A , HA depolymerization by Detroit 551 cells treated with growth factors. Cells treated without ( Control ; open circle ) or with 1 ( gray triangle ) or 10 ng/ml ( closed circle ) TGF-β1, bFGF, EGF, or PDGF-BB were cultured with [ 3 H]HA for 48 h, and HA fragments in the culture media were examined by size-exclusion chromatography. B and C , the expression levels of HYBID mRNA ( B ) and protein ( C ) in cells treated without ( Control ) or with 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB. The mRNA levels at 8 and 24 h and the protein expression at 24 h after the treatment were measured by quantitative real-time PCR and immunoblotting, respectively. Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to 8 h control cells. Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Down-regulation of HYBID-mediated HA depolymerization by TGF-β1, bFGF, EGF, and PDGF-BB. A , HA depolymerization by Detroit 551 cells treated with growth factors. Cells treated without ( Control ; open circle ) or with 1 ( gray triangle ) or 10 ng/ml ( closed circle ) TGF-β1, bFGF, EGF, or PDGF-BB were cultured with [ 3 H]HA for 48 h, and HA fragments in the culture media were examined by size-exclusion chromatography. B and C , the expression levels of HYBID mRNA ( B ) and protein ( C ) in cells treated without ( Control ) or with 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB. The mRNA levels at 8 and 24 h and the protein expression at 24 h after the treatment were measured by quantitative real-time PCR and immunoblotting, respectively. Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to 8 h control cells. Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Cell Culture, Size-exclusion Chromatography, Expressing, Real-time Polymerase Chain Reaction

    Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in NHDF-Ad cells. A , HA content in the culture media of NHDF-Ad skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in NHDF-Ad cells. A , HA content in the culture media of NHDF-Ad skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    22) Product Images from "Interferon-γ blocks signalling through PDGFRβ in human brain pericytes"

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0722-4

    Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p
    Figure Legend Snippet: Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p

    Techniques Used:

    IFNγ blocks pericyte migration independently of PDGF-BB. a , b Cell migration was measured by scratching wells after 48 h of IFNγ (1 ng/mL) treatment and measuring the number of cells that moved into the gap area after a further 48 h with vehicle or PDGF-BB (10 ng/mL) treatment. Representative images of Coomassie-stained pericytes are presented in a , and manual counts of cells in the gap area (normalized to vehicle-treated condition), plotted as mean ± s.e.m. ( n = 3) #### , ****( p
    Figure Legend Snippet: IFNγ blocks pericyte migration independently of PDGF-BB. a , b Cell migration was measured by scratching wells after 48 h of IFNγ (1 ng/mL) treatment and measuring the number of cells that moved into the gap area after a further 48 h with vehicle or PDGF-BB (10 ng/mL) treatment. Representative images of Coomassie-stained pericytes are presented in a , and manual counts of cells in the gap area (normalized to vehicle-treated condition), plotted as mean ± s.e.m. ( n = 3) #### , ****( p

    Techniques Used: Migration, Staining

    Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p
    Figure Legend Snippet: Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p

    Techniques Used: Staining

    Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Expressing, Western Blot, Software, Staining

    Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Western Blot, Software

    23) Product Images from "Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-? and VEGFR-2 expression"

    Article Title: Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-? and VEGFR-2 expression

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00411.2011

    PDGF-BB and VEGF induce greater ERK1/2 activation in VSMC compared with ASMC under hypoxia. VSMC ( A ) and ASMC ( B ) were incubated with PDGF-BB (10 ng/ml) and VEGF-A (10 ng/ml) under hypoxia for 24 h. VEGF-A-neutralizing antibody (α VEGF IgG) and
    Figure Legend Snippet: PDGF-BB and VEGF induce greater ERK1/2 activation in VSMC compared with ASMC under hypoxia. VSMC ( A ) and ASMC ( B ) were incubated with PDGF-BB (10 ng/ml) and VEGF-A (10 ng/ml) under hypoxia for 24 h. VEGF-A-neutralizing antibody (α VEGF IgG) and

    Techniques Used: Activation Assay, Incubation

    24) Product Images from "Neuropilin-1 Signaling through p130Cas Tyrosine Phosphorylation Is Essential for Growth Factor-Dependent Migration of Glioma and Endothelial Cells ▿"

    Article Title: Neuropilin-1 Signaling through p130Cas Tyrosine Phosphorylation Is Essential for Growth Factor-Dependent Migration of Glioma and Endothelial Cells ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00903-10

    Requirement of NRP1 and p130 Cas for HGF, PDGF, and VEGF stimulation of chemotactic migration of U87MG cells and HUVECs. (A) U87MG cells were transfected with 25 nM siNRP1, 25 nM sip130 Cas , or 25 nM siScr for 48 h. Cell migration in response to 25 ng/ml HGF or 50 ng/ml PDGF-BB was then determined in a Transwell migration assay as described in Materials and Methods. (B) HUVECs were transfected with 200 nM siNRP1, 200 nM sip130 Cas , or 200 nM siScr for 48 h, and cell migration in response to 25 ng/ml VEGF-A was determined as described for panel A. Values ( n ≥ 3) are means ± SEM, expressed as the number of cells migrating per field; *, P
    Figure Legend Snippet: Requirement of NRP1 and p130 Cas for HGF, PDGF, and VEGF stimulation of chemotactic migration of U87MG cells and HUVECs. (A) U87MG cells were transfected with 25 nM siNRP1, 25 nM sip130 Cas , or 25 nM siScr for 48 h. Cell migration in response to 25 ng/ml HGF or 50 ng/ml PDGF-BB was then determined in a Transwell migration assay as described in Materials and Methods. (B) HUVECs were transfected with 200 nM siNRP1, 200 nM sip130 Cas , or 200 nM siScr for 48 h, and cell migration in response to 25 ng/ml VEGF-A was determined as described for panel A. Values ( n ≥ 3) are means ± SEM, expressed as the number of cells migrating per field; *, P

    Techniques Used: Migration, Transfection, Transwell Migration Assay

    p130 Cas tyrosine phosphorylation is required for chemotactic migration of U87MG cells and HUVECs. (A and B) U87MG cells and HUVECs were infected with Ad.LacZ, Ad.p130 Cas , and Ad.p130 Cas15F and treated 48 h later with HGF, PDGF, and VEGF as described in the legend to Fig. 2. Data from three independent experiments are presented as relative units (RU) of p130 Cas phosphorylation (means ± SEM) normalized to GAPDH. *, P
    Figure Legend Snippet: p130 Cas tyrosine phosphorylation is required for chemotactic migration of U87MG cells and HUVECs. (A and B) U87MG cells and HUVECs were infected with Ad.LacZ, Ad.p130 Cas , and Ad.p130 Cas15F and treated 48 h later with HGF, PDGF, and VEGF as described in the legend to Fig. 2. Data from three independent experiments are presented as relative units (RU) of p130 Cas phosphorylation (means ± SEM) normalized to GAPDH. *, P

    Techniques Used: Migration, Infection

    Growth factor-stimulated tyrosine phosphorylation of p130 Cas is mediated via NRP1 in glioma and endothelial cells. (A) U87MG cells were transfected with 25 nM siRNA targeting NRP1 (siNRP1) or 25 nM control scrambled siRNA (siScr). At 48 h posttransfection, cells were incubated in serum-free medium (SFM) for ∼18 h prior to treatment with the SFM vehicle control (C) or treated with 25 ng/ml HGF (H) or 50 ng/ml PDGF-BB (P) for 5 min. Cell lysates were then prepared, blotted, and probed with the indicated antibodies. The positions of chondroitin sulfate-glycosylated NRP1 (NRP1-CS; 250 kDa) and unmodified NRP1 (130 kDa) in U87MG cells are indicated. (B) HUVECs were transfected with 200 nM siNRP1 or 200 nM siScr. At 48 h after transfection, cells were switched to EBM containing 0.5% (vol/vol) serum and incubated overnight. Cells were then treated for 10 min with the SFM vehicle control (C) or with 25 ng/ml VEGF-A (V). Cell lysates were then prepared, blotted, and probed with the indicated antibodies. (C) Confluent HUVECs were preincubated for 30 min with SFM plus 100 μM EG0086 (peptide antagonist), 5 μg/ml NRP1 blocking antibody (Ab), or the vehicle (control) and then stimulated with 25 ng/ml VEGF-A (V) or with no further treatment (C) for 10 min. Cell lysates were then prepared, blotted, and probed with the indicated antibodies. (D and E) U87MG cells and HUVECs were treated as described for panels A and B and probed with the indicated antibodies. The blots shown here and in all subsequent figures are representative of at least three separate experiments. In panels A, B, and C, quantitation of p130 Cas phosphorylation was performed by densitometry using ImageJ. In each panel, data from three independent experiments are presented as relative units (RU) of p130 Cas phosphorylation (means ± SEM) normalized to total p130 Cas . *, P
    Figure Legend Snippet: Growth factor-stimulated tyrosine phosphorylation of p130 Cas is mediated via NRP1 in glioma and endothelial cells. (A) U87MG cells were transfected with 25 nM siRNA targeting NRP1 (siNRP1) or 25 nM control scrambled siRNA (siScr). At 48 h posttransfection, cells were incubated in serum-free medium (SFM) for ∼18 h prior to treatment with the SFM vehicle control (C) or treated with 25 ng/ml HGF (H) or 50 ng/ml PDGF-BB (P) for 5 min. Cell lysates were then prepared, blotted, and probed with the indicated antibodies. The positions of chondroitin sulfate-glycosylated NRP1 (NRP1-CS; 250 kDa) and unmodified NRP1 (130 kDa) in U87MG cells are indicated. (B) HUVECs were transfected with 200 nM siNRP1 or 200 nM siScr. At 48 h after transfection, cells were switched to EBM containing 0.5% (vol/vol) serum and incubated overnight. Cells were then treated for 10 min with the SFM vehicle control (C) or with 25 ng/ml VEGF-A (V). Cell lysates were then prepared, blotted, and probed with the indicated antibodies. (C) Confluent HUVECs were preincubated for 30 min with SFM plus 100 μM EG0086 (peptide antagonist), 5 μg/ml NRP1 blocking antibody (Ab), or the vehicle (control) and then stimulated with 25 ng/ml VEGF-A (V) or with no further treatment (C) for 10 min. Cell lysates were then prepared, blotted, and probed with the indicated antibodies. (D and E) U87MG cells and HUVECs were treated as described for panels A and B and probed with the indicated antibodies. The blots shown here and in all subsequent figures are representative of at least three separate experiments. In panels A, B, and C, quantitation of p130 Cas phosphorylation was performed by densitometry using ImageJ. In each panel, data from three independent experiments are presented as relative units (RU) of p130 Cas phosphorylation (means ± SEM) normalized to total p130 Cas . *, P

    Techniques Used: Transfection, Incubation, Blocking Assay, Quantitation Assay

    Requirement of NRP1 intracellular domain for HGF, PDGF, and VEGF stimulation of chemotactic migration of U87MG cells and HUVECs. (A) U87MG cells were infected with Ad.GFP, Ad.NRP1, or Ad.NRP1ΔC for 48 h. Cell migration in response to 25 ng/ml HGF or 50 ng/ml PDGF was then determined. (B) Confluent HUVECs were infected with Ad.GFP, Ad.NRP1, or Ad.NRP1ΔC for 48 h, and cell migration in response to 25 ng/ml VEGF was determined. The inset shows levels of NRP1 and NRP1ΔC expression. Values ( n ≥ 3) are means ± SEM, expressed as the number of cells migrating per field. *, P
    Figure Legend Snippet: Requirement of NRP1 intracellular domain for HGF, PDGF, and VEGF stimulation of chemotactic migration of U87MG cells and HUVECs. (A) U87MG cells were infected with Ad.GFP, Ad.NRP1, or Ad.NRP1ΔC for 48 h. Cell migration in response to 25 ng/ml HGF or 50 ng/ml PDGF was then determined. (B) Confluent HUVECs were infected with Ad.GFP, Ad.NRP1, or Ad.NRP1ΔC for 48 h, and cell migration in response to 25 ng/ml VEGF was determined. The inset shows levels of NRP1 and NRP1ΔC expression. Values ( n ≥ 3) are means ± SEM, expressed as the number of cells migrating per field. *, P

    Techniques Used: Migration, Infection, Expressing

    Growth factor-mediated increase in colocalization of NRP1 and tyrosine-phosphorylated p130 Cas . (A) HUVECs were seeded onto glass coverslips and incubated in 0.5% FCS in EBM for ∼18 h prior to treatment with the SFM vehicle control or with 25 ng/ml VEGF. (B) U87MG cells were seeded onto glass coverslips and incubated in SFM for ∼18 h prior to treatment with the SFM vehicle control or with 25 ng/ml HGF or 50 ng/ml PDGF-BB for 5 min. Confocal imaging was performed as described in Materials and Methods, with NRP1 staining in green and phospho-p130 Cas (Y410) staining in red. Merged images show costaining of NRP1 and phospho-p130 Cas Y410 in yellow and are representative of at least three separate experiments. Quantification of NRP1 and phospho-p130 Cas colocalization (as described in Materials and Methods) is shown in the graphs and represents data from three independent experiments expressed as colocalization in the number of voxels (means ± SEM). *, P
    Figure Legend Snippet: Growth factor-mediated increase in colocalization of NRP1 and tyrosine-phosphorylated p130 Cas . (A) HUVECs were seeded onto glass coverslips and incubated in 0.5% FCS in EBM for ∼18 h prior to treatment with the SFM vehicle control or with 25 ng/ml VEGF. (B) U87MG cells were seeded onto glass coverslips and incubated in SFM for ∼18 h prior to treatment with the SFM vehicle control or with 25 ng/ml HGF or 50 ng/ml PDGF-BB for 5 min. Confocal imaging was performed as described in Materials and Methods, with NRP1 staining in green and phospho-p130 Cas (Y410) staining in red. Merged images show costaining of NRP1 and phospho-p130 Cas Y410 in yellow and are representative of at least three separate experiments. Quantification of NRP1 and phospho-p130 Cas colocalization (as described in Materials and Methods) is shown in the graphs and represents data from three independent experiments expressed as colocalization in the number of voxels (means ± SEM). *, P

    Techniques Used: Incubation, Imaging, Staining

    25) Product Images from "Saikosaponin a and Saikosaponin d Inhibit Proliferation and Migratory Activity of Rat HSC-T6 Cells"

    Article Title: Saikosaponin a and Saikosaponin d Inhibit Proliferation and Migratory Activity of Rat HSC-T6 Cells

    Journal: Journal of Medicinal Food

    doi: 10.1089/jmf.2013.2762

    SSa and SSd blocked PDGF-BB and TGF-β1-induced proliferative and migratory activities. The cell migratory activity of HSC-T6 was enhanced by PDGF-BB; however, the enhancing effects were blocked by pretreatment of SSa (A) and SSd (B) . SSa and SSd
    Figure Legend Snippet: SSa and SSd blocked PDGF-BB and TGF-β1-induced proliferative and migratory activities. The cell migratory activity of HSC-T6 was enhanced by PDGF-BB; however, the enhancing effects were blocked by pretreatment of SSa (A) and SSd (B) . SSa and SSd

    Techniques Used: Activity Assay

    26) Product Images from "microRNA-29a inhibits cardiac fibrosis in Sprague-Dawley rats by downregulating the expression of DNMT3A"

    Article Title: microRNA-29a inhibits cardiac fibrosis in Sprague-Dawley rats by downregulating the expression of DNMT3A

    Journal: Anatolian Journal of Cardiology

    doi: 10.14744/AnatolJCardiol.2018.98511

    Pathological changes of myocardial tissue and serum biochemical indices in rats after ISO treatment (a) H E and Mason staining results show that myocardial collagen deposition appears in myocardial tissues. (b) ELISA results show that PDGF-BB, PICP, and PIIINP concentrations are significantly higher in ISO-treated rats than in normal control rats (* P
    Figure Legend Snippet: Pathological changes of myocardial tissue and serum biochemical indices in rats after ISO treatment (a) H E and Mason staining results show that myocardial collagen deposition appears in myocardial tissues. (b) ELISA results show that PDGF-BB, PICP, and PIIINP concentrations are significantly higher in ISO-treated rats than in normal control rats (* P

    Techniques Used: Staining, Enzyme-linked Immunosorbent Assay

    27) Product Images from "Hematopoietic Stem Cell Cytokines and Fibroblast Growth factor-2 Stimulate Human Endothelial Cell-Pericyte Tube Co-Assembly in 3D Fibrin Matrices under Serum-Free Defined Conditions"

    Article Title: Hematopoietic Stem Cell Cytokines and Fibroblast Growth factor-2 Stimulate Human Endothelial Cell-Pericyte Tube Co-Assembly in 3D Fibrin Matrices under Serum-Free Defined Conditions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085147

    Neutralizing antibodies directed to the hematopoietic cytokines, SCF, IL-3 and SDF-1α markedly block EC tube formation, while antibodies to PDGF-BB and HB-EGF interfere with pericyte assembly on EC-lined tubes in 3D fibrin matrices under serum-free defined conditions. GFP-ECs were primed with hematopoietic cytokines (factors) or VEGF/FGF-2 overnight and then were seeded into fibrin matrices with hematopoietic factors and FGF-2 in the presence of Cherry-pericytes and in the presence of the indicated neutralizing antibodies or chemical inhibitors. (A) The antibodies were added at 50 µg/ml (IL-6, SCF, IL-3, SDF-1α) or 100 µg/ml (VEGF) versus controls and after 72 hr, cultures were fixed, photographed and quantitated for EC tube formation (n=10, values are averaged + SD, asterisks indicate significance compared to control, p
    Figure Legend Snippet: Neutralizing antibodies directed to the hematopoietic cytokines, SCF, IL-3 and SDF-1α markedly block EC tube formation, while antibodies to PDGF-BB and HB-EGF interfere with pericyte assembly on EC-lined tubes in 3D fibrin matrices under serum-free defined conditions. GFP-ECs were primed with hematopoietic cytokines (factors) or VEGF/FGF-2 overnight and then were seeded into fibrin matrices with hematopoietic factors and FGF-2 in the presence of Cherry-pericytes and in the presence of the indicated neutralizing antibodies or chemical inhibitors. (A) The antibodies were added at 50 µg/ml (IL-6, SCF, IL-3, SDF-1α) or 100 µg/ml (VEGF) versus controls and after 72 hr, cultures were fixed, photographed and quantitated for EC tube formation (n=10, values are averaged + SD, asterisks indicate significance compared to control, p

    Techniques Used: Blocking Assay

    28) Product Images from "Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts *"

    Article Title: Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.673566

    Up-regulation of HAS-mediated HA synthesis by TGF-β1, bFGF, EGF, and PDGF-BB in normal human skin fibroblasts. A , HA content in the culture media of Detroit 551 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 or 48 h. The HA concentrations in the media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis by TGF-β1, bFGF, EGF, and PDGF-BB in normal human skin fibroblasts. A , HA content in the culture media of Detroit 551 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 or 48 h. The HA concentrations in the media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in HS27 cells. A , HA content in the culture media of HS27 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in HS27 cells. A , HA content in the culture media of HS27 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    Effects of TGF-β1, bFGF, EGF, and PDGF-BB, and knockdown of HYBID on the size distribution of newly produced HA. A , Detroit 551 cells were metabolically labeled with 10 μCi/ml [ 3 H]glucosamine in the absence ( Control ; open circle ) or presence of 1 ( gray triangle ) or 10 ( closed circle ) ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 48 h. The radiolabeled HA was isolated from the conditioned media and subjected to size-exclusion chromatography. B , knockdown efficiency and specificity for HYBID was evaluated by quantitative real-time PCR. The expression levels of HYBID , HAS1 , and HAS2 mRNAs in HYBID knocked-down cells at 24 h after treatment with siRNA were measured by quantitative real-time PCR. As for controls, the cells were transfected with control non-silencing siRNA ( Control siRNA ). Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to control siRNA-treated cells. Student's t test was used for statistical analysis. ***, p
    Figure Legend Snippet: Effects of TGF-β1, bFGF, EGF, and PDGF-BB, and knockdown of HYBID on the size distribution of newly produced HA. A , Detroit 551 cells were metabolically labeled with 10 μCi/ml [ 3 H]glucosamine in the absence ( Control ; open circle ) or presence of 1 ( gray triangle ) or 10 ( closed circle ) ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 48 h. The radiolabeled HA was isolated from the conditioned media and subjected to size-exclusion chromatography. B , knockdown efficiency and specificity for HYBID was evaluated by quantitative real-time PCR. The expression levels of HYBID , HAS1 , and HAS2 mRNAs in HYBID knocked-down cells at 24 h after treatment with siRNA were measured by quantitative real-time PCR. As for controls, the cells were transfected with control non-silencing siRNA ( Control siRNA ). Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to control siRNA-treated cells. Student's t test was used for statistical analysis. ***, p

    Techniques Used: Produced, Metabolic Labelling, Labeling, Isolation, Size-exclusion Chromatography, Real-time Polymerase Chain Reaction, Expressing, Transfection

    Down-regulation of HYBID-mediated HA depolymerization by TGF-β1, bFGF, EGF, and PDGF-BB. A , HA depolymerization by Detroit 551 cells treated with growth factors. Cells treated without ( Control ; open circle ) or with 1 ( gray triangle ) or 10 ng/ml ( closed circle ) TGF-β1, bFGF, EGF, or PDGF-BB were cultured with [ 3 H]HA for 48 h, and HA fragments in the culture media were examined by size-exclusion chromatography. B and C , the expression levels of HYBID mRNA ( B ) and protein ( C ) in cells treated without ( Control ) or with 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB. The mRNA levels at 8 and 24 h and the protein expression at 24 h after the treatment were measured by quantitative real-time PCR and immunoblotting, respectively. Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to 8 h control cells. Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Down-regulation of HYBID-mediated HA depolymerization by TGF-β1, bFGF, EGF, and PDGF-BB. A , HA depolymerization by Detroit 551 cells treated with growth factors. Cells treated without ( Control ; open circle ) or with 1 ( gray triangle ) or 10 ng/ml ( closed circle ) TGF-β1, bFGF, EGF, or PDGF-BB were cultured with [ 3 H]HA for 48 h, and HA fragments in the culture media were examined by size-exclusion chromatography. B and C , the expression levels of HYBID mRNA ( B ) and protein ( C ) in cells treated without ( Control ) or with 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB. The mRNA levels at 8 and 24 h and the protein expression at 24 h after the treatment were measured by quantitative real-time PCR and immunoblotting, respectively. Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to 8 h control cells. Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Cell Culture, Size-exclusion Chromatography, Expressing, Real-time Polymerase Chain Reaction

    Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in NHDF-Ad cells. A , HA content in the culture media of NHDF-Ad skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in NHDF-Ad cells. A , HA content in the culture media of NHDF-Ad skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    29) Product Images from "Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts *"

    Article Title: Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.673566

    Up-regulation of HAS-mediated HA synthesis by TGF-β1, bFGF, EGF, and PDGF-BB in normal human skin fibroblasts. A , HA content in the culture media of Detroit 551 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 or 48 h. The HA concentrations in the media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis by TGF-β1, bFGF, EGF, and PDGF-BB in normal human skin fibroblasts. A , HA content in the culture media of Detroit 551 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 or 48 h. The HA concentrations in the media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in HS27 cells. A , HA content in the culture media of HS27 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in HS27 cells. A , HA content in the culture media of HS27 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    Effects of TGF-β1, bFGF, EGF, and PDGF-BB, and knockdown of HYBID on the size distribution of newly produced HA. A , Detroit 551 cells were metabolically labeled with 10 μCi/ml [ 3 H]glucosamine in the absence ( Control ; open circle ) or presence of 1 ( gray triangle ) or 10 ( closed circle ) ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 48 h. The radiolabeled HA was isolated from the conditioned media and subjected to size-exclusion chromatography. B , knockdown efficiency and specificity for HYBID was evaluated by quantitative real-time PCR. The expression levels of HYBID , HAS1 , and HAS2 mRNAs in HYBID knocked-down cells at 24 h after treatment with siRNA were measured by quantitative real-time PCR. As for controls, the cells were transfected with control non-silencing siRNA ( Control siRNA ). Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to control siRNA-treated cells. Student's t test was used for statistical analysis. ***, p
    Figure Legend Snippet: Effects of TGF-β1, bFGF, EGF, and PDGF-BB, and knockdown of HYBID on the size distribution of newly produced HA. A , Detroit 551 cells were metabolically labeled with 10 μCi/ml [ 3 H]glucosamine in the absence ( Control ; open circle ) or presence of 1 ( gray triangle ) or 10 ( closed circle ) ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 48 h. The radiolabeled HA was isolated from the conditioned media and subjected to size-exclusion chromatography. B , knockdown efficiency and specificity for HYBID was evaluated by quantitative real-time PCR. The expression levels of HYBID , HAS1 , and HAS2 mRNAs in HYBID knocked-down cells at 24 h after treatment with siRNA were measured by quantitative real-time PCR. As for controls, the cells were transfected with control non-silencing siRNA ( Control siRNA ). Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to control siRNA-treated cells. Student's t test was used for statistical analysis. ***, p

    Techniques Used: Produced, Metabolic Labelling, Labeling, Isolation, Size-exclusion Chromatography, Real-time Polymerase Chain Reaction, Expressing, Transfection

    Down-regulation of HYBID-mediated HA depolymerization by TGF-β1, bFGF, EGF, and PDGF-BB. A , HA depolymerization by Detroit 551 cells treated with growth factors. Cells treated without ( Control ; open circle ) or with 1 ( gray triangle ) or 10 ng/ml ( closed circle ) TGF-β1, bFGF, EGF, or PDGF-BB were cultured with [ 3 H]HA for 48 h, and HA fragments in the culture media were examined by size-exclusion chromatography. B and C , the expression levels of HYBID mRNA ( B ) and protein ( C ) in cells treated without ( Control ) or with 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB. The mRNA levels at 8 and 24 h and the protein expression at 24 h after the treatment were measured by quantitative real-time PCR and immunoblotting, respectively. Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to 8 h control cells. Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Down-regulation of HYBID-mediated HA depolymerization by TGF-β1, bFGF, EGF, and PDGF-BB. A , HA depolymerization by Detroit 551 cells treated with growth factors. Cells treated without ( Control ; open circle ) or with 1 ( gray triangle ) or 10 ng/ml ( closed circle ) TGF-β1, bFGF, EGF, or PDGF-BB were cultured with [ 3 H]HA for 48 h, and HA fragments in the culture media were examined by size-exclusion chromatography. B and C , the expression levels of HYBID mRNA ( B ) and protein ( C ) in cells treated without ( Control ) or with 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB. The mRNA levels at 8 and 24 h and the protein expression at 24 h after the treatment were measured by quantitative real-time PCR and immunoblotting, respectively. Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to 8 h control cells. Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Cell Culture, Size-exclusion Chromatography, Expressing, Real-time Polymerase Chain Reaction

    Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in NHDF-Ad cells. A , HA content in the culture media of NHDF-Ad skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in NHDF-Ad cells. A , HA content in the culture media of NHDF-Ad skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    30) Product Images from "Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts *"

    Article Title: Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.673566

    Up-regulation of HAS-mediated HA synthesis by TGF-β1, bFGF, EGF, and PDGF-BB in normal human skin fibroblasts. A , HA content in the culture media of Detroit 551 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 or 48 h. The HA concentrations in the media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis by TGF-β1, bFGF, EGF, and PDGF-BB in normal human skin fibroblasts. A , HA content in the culture media of Detroit 551 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 or 48 h. The HA concentrations in the media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in HS27 cells. A , HA content in the culture media of HS27 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in HS27 cells. A , HA content in the culture media of HS27 skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    Effects of TGF-β1, bFGF, EGF, and PDGF-BB, and knockdown of HYBID on the size distribution of newly produced HA. A , Detroit 551 cells were metabolically labeled with 10 μCi/ml [ 3 H]glucosamine in the absence ( Control ; open circle ) or presence of 1 ( gray triangle ) or 10 ( closed circle ) ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 48 h. The radiolabeled HA was isolated from the conditioned media and subjected to size-exclusion chromatography. B , knockdown efficiency and specificity for HYBID was evaluated by quantitative real-time PCR. The expression levels of HYBID , HAS1 , and HAS2 mRNAs in HYBID knocked-down cells at 24 h after treatment with siRNA were measured by quantitative real-time PCR. As for controls, the cells were transfected with control non-silencing siRNA ( Control siRNA ). Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to control siRNA-treated cells. Student's t test was used for statistical analysis. ***, p
    Figure Legend Snippet: Effects of TGF-β1, bFGF, EGF, and PDGF-BB, and knockdown of HYBID on the size distribution of newly produced HA. A , Detroit 551 cells were metabolically labeled with 10 μCi/ml [ 3 H]glucosamine in the absence ( Control ; open circle ) or presence of 1 ( gray triangle ) or 10 ( closed circle ) ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 48 h. The radiolabeled HA was isolated from the conditioned media and subjected to size-exclusion chromatography. B , knockdown efficiency and specificity for HYBID was evaluated by quantitative real-time PCR. The expression levels of HYBID , HAS1 , and HAS2 mRNAs in HYBID knocked-down cells at 24 h after treatment with siRNA were measured by quantitative real-time PCR. As for controls, the cells were transfected with control non-silencing siRNA ( Control siRNA ). Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to control siRNA-treated cells. Student's t test was used for statistical analysis. ***, p

    Techniques Used: Produced, Metabolic Labelling, Labeling, Isolation, Size-exclusion Chromatography, Real-time Polymerase Chain Reaction, Expressing, Transfection

    Down-regulation of HYBID-mediated HA depolymerization by TGF-β1, bFGF, EGF, and PDGF-BB. A , HA depolymerization by Detroit 551 cells treated with growth factors. Cells treated without ( Control ; open circle ) or with 1 ( gray triangle ) or 10 ng/ml ( closed circle ) TGF-β1, bFGF, EGF, or PDGF-BB were cultured with [ 3 H]HA for 48 h, and HA fragments in the culture media were examined by size-exclusion chromatography. B and C , the expression levels of HYBID mRNA ( B ) and protein ( C ) in cells treated without ( Control ) or with 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB. The mRNA levels at 8 and 24 h and the protein expression at 24 h after the treatment were measured by quantitative real-time PCR and immunoblotting, respectively. Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to 8 h control cells. Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Down-regulation of HYBID-mediated HA depolymerization by TGF-β1, bFGF, EGF, and PDGF-BB. A , HA depolymerization by Detroit 551 cells treated with growth factors. Cells treated without ( Control ; open circle ) or with 1 ( gray triangle ) or 10 ng/ml ( closed circle ) TGF-β1, bFGF, EGF, or PDGF-BB were cultured with [ 3 H]HA for 48 h, and HA fragments in the culture media were examined by size-exclusion chromatography. B and C , the expression levels of HYBID mRNA ( B ) and protein ( C ) in cells treated without ( Control ) or with 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB. The mRNA levels at 8 and 24 h and the protein expression at 24 h after the treatment were measured by quantitative real-time PCR and immunoblotting, respectively. Values are expressed as the mean ± S.D. ( n = 3) and shown as -fold increases in mRNA expression relative to 8 h control cells. Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Cell Culture, Size-exclusion Chromatography, Expressing, Real-time Polymerase Chain Reaction

    Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in NHDF-Ad cells. A , HA content in the culture media of NHDF-Ad skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p
    Figure Legend Snippet: Up-regulation of HAS-mediated HA synthesis and down-regulation of HYBID mRNA expression by TGF-β1, bFGF, EGF, and PDGF-BB in NHDF-Ad cells. A , HA content in the culture media of NHDF-Ad skin fibroblasts stimulated with growth factors. Cells were cultured in the absence ( Control ) or presence of 1 or 10 ng/ml TGF-β1, bFGF, EGF, or PDGF-BB for 24 h. The HA concentrations in the conditioned media were quantified by ELISA. Values represent the mean ± S.D. ( n = 3). Dunnett's test was used for statistical analysis. ***, p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    31) Product Images from "Intrinsic regulation of hemangioma involution by platelet-derived growth factor"

    Article Title: Intrinsic regulation of hemangioma involution by platelet-derived growth factor

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2012.58

    Effect of PDGF-BB on hemSC phenotype and cellular proliferation. ( a ) Fluorescence immunostaining for mesenchymal markers α -SMA, calponin and desmin in hemSCs. Umbilical artery SMCs were used as control for the mural markers. Effect of exogenous PDGF-BB on hemSC migration ( b ) and proliferation ( c ) (data presented as relative to control media treated cells; n =3)
    Figure Legend Snippet: Effect of PDGF-BB on hemSC phenotype and cellular proliferation. ( a ) Fluorescence immunostaining for mesenchymal markers α -SMA, calponin and desmin in hemSCs. Umbilical artery SMCs were used as control for the mural markers. Effect of exogenous PDGF-BB on hemSC migration ( b ) and proliferation ( c ) (data presented as relative to control media treated cells; n =3)

    Techniques Used: Fluorescence, Immunostaining, Migration

    Expression of PDGF signaling axis in hemangioma-derived CD133+ cells. ( a ) Expression of PDGFRs in hemSCs and bm-MPCs (data normalized to 18 S rRNA and presented as relative to bm-MPCs; two different hemSC preparations run in triplicates were averaged). ( b ) Expression of PDGF transcripts in hemSCs (data presented as in ( a ); * P
    Figure Legend Snippet: Expression of PDGF signaling axis in hemangioma-derived CD133+ cells. ( a ) Expression of PDGFRs in hemSCs and bm-MPCs (data normalized to 18 S rRNA and presented as relative to bm-MPCs; two different hemSC preparations run in triplicates were averaged). ( b ) Expression of PDGF transcripts in hemSCs (data presented as in ( a ); * P

    Techniques Used: Expressing, Derivative Assay

    32) Product Images from "Interferon-γ blocks signalling through PDGFRβ in human brain pericytes"

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0722-4

    Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p
    Figure Legend Snippet: Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p

    Techniques Used:

    IFNγ blocks pericyte migration independently of PDGF-BB. a , b Cell migration was measured by scratching wells after 48 h of IFNγ (1 ng/mL) treatment and measuring the number of cells that moved into the gap area after a further 48 h with vehicle or PDGF-BB (10 ng/mL) treatment. Representative images of Coomassie-stained pericytes are presented in a , and manual counts of cells in the gap area (normalized to vehicle-treated condition), plotted as mean ± s.e.m. ( n = 3) #### , ****( p
    Figure Legend Snippet: IFNγ blocks pericyte migration independently of PDGF-BB. a , b Cell migration was measured by scratching wells after 48 h of IFNγ (1 ng/mL) treatment and measuring the number of cells that moved into the gap area after a further 48 h with vehicle or PDGF-BB (10 ng/mL) treatment. Representative images of Coomassie-stained pericytes are presented in a , and manual counts of cells in the gap area (normalized to vehicle-treated condition), plotted as mean ± s.e.m. ( n = 3) #### , ****( p

    Techniques Used: Migration, Staining

    Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p
    Figure Legend Snippet: Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p

    Techniques Used: Staining

    Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Expressing, Western Blot, Software, Staining

    Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Western Blot, Software

    33) Product Images from "Interferon-γ blocks signalling through PDGFRβ in human brain pericytes"

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0722-4

    Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p
    Figure Legend Snippet: Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p

    Techniques Used:

    IFNγ blocks pericyte migration independently of PDGF-BB. a , b Cell migration was measured by scratching wells after 48 h of IFNγ (1 ng/mL) treatment and measuring the number of cells that moved into the gap area after a further 48 h with vehicle or PDGF-BB (10 ng/mL) treatment. Representative images of Coomassie-stained pericytes are presented in a , and manual counts of cells in the gap area (normalized to vehicle-treated condition), plotted as mean ± s.e.m. ( n = 3) #### , ****( p
    Figure Legend Snippet: IFNγ blocks pericyte migration independently of PDGF-BB. a , b Cell migration was measured by scratching wells after 48 h of IFNγ (1 ng/mL) treatment and measuring the number of cells that moved into the gap area after a further 48 h with vehicle or PDGF-BB (10 ng/mL) treatment. Representative images of Coomassie-stained pericytes are presented in a , and manual counts of cells in the gap area (normalized to vehicle-treated condition), plotted as mean ± s.e.m. ( n = 3) #### , ****( p

    Techniques Used: Migration, Staining

    Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p
    Figure Legend Snippet: Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p

    Techniques Used: Staining

    Human brain pericytes respond to PDGF-BB signals in vitro . a Pericytes were serum starved for 2 h, then treated with vehicle (lanes 1 and 7) or PDGF-BB (100 ng/mL) (lanes 2–6, and 8) for the indicated times, and analysed by SDS-PAGE. Representative blots from three cases are shown. b Blots from a were analysed and quantified with Image Studio™. Phosphorylated PDGFRβ (Tyr751) (p-PDGFRβ) ( b ), PDGFRβ ( c ), and PDGF-BB ( f ) were normalized to GAPDH; phosphorylated Akt (Ser473) (p-Akt) ( d ) and phosphorylated ERK (Tyr204) (p-ERK) ( e ) were normalized to total Akt and ERK, respectively. **Bottom band in pERK blot is GAPDH
    Figure Legend Snippet: Human brain pericytes respond to PDGF-BB signals in vitro . a Pericytes were serum starved for 2 h, then treated with vehicle (lanes 1 and 7) or PDGF-BB (100 ng/mL) (lanes 2–6, and 8) for the indicated times, and analysed by SDS-PAGE. Representative blots from three cases are shown. b Blots from a were analysed and quantified with Image Studio™. Phosphorylated PDGFRβ (Tyr751) (p-PDGFRβ) ( b ), PDGFRβ ( c ), and PDGF-BB ( f ) were normalized to GAPDH; phosphorylated Akt (Ser473) (p-Akt) ( d ) and phosphorylated ERK (Tyr204) (p-ERK) ( e ) were normalized to total Akt and ERK, respectively. **Bottom band in pERK blot is GAPDH

    Techniques Used: In Vitro, SDS Page

    Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Expressing, Western Blot, Software, Staining

    Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p
    Figure Legend Snippet: Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p

    Techniques Used: Western Blot, Software

    34) Product Images from "Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-? and VEGFR-2 expression"

    Article Title: Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-? and VEGFR-2 expression

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00411.2011

    PDGF-BB and VEGF induce greater ERK1/2 activation in VSMC compared with ASMC under hypoxia. VSMC ( A ) and ASMC ( B ) were incubated with PDGF-BB (10 ng/ml) and VEGF-A (10 ng/ml) under hypoxia for 24 h. VEGF-A-neutralizing antibody (α VEGF IgG) and
    Figure Legend Snippet: PDGF-BB and VEGF induce greater ERK1/2 activation in VSMC compared with ASMC under hypoxia. VSMC ( A ) and ASMC ( B ) were incubated with PDGF-BB (10 ng/ml) and VEGF-A (10 ng/ml) under hypoxia for 24 h. VEGF-A-neutralizing antibody (α VEGF IgG) and

    Techniques Used: Activation Assay, Incubation

    35) Product Images from "The FGF-2-Derived Peptide FREG Inhibits Melanoma Growth In Vitro and In Vivo"

    Article Title: The FGF-2-Derived Peptide FREG Inhibits Melanoma Growth In Vitro and In Vivo

    Journal: Molecular Therapy

    doi: 10.1038/mt.2010.211

    FREG interacts with PDGF-Rα . ( a ) FREG binding to plastic-immobilized PDGF-Rα (7 µg/ml) was measured in solid-phase assays. Biotinylated FREG (form 0 to 2 mmol/l) was incubated for 4 hours onto immobilized-PDGF-Rα. Unbound material was washed away and a colorimetric assay was performed. Biotinylated FREG bound PDGF-Rα in a saturable and concentration-dependent manner. ( b ) SK-MEL-110 untransfected or overexpressing PDGF-Rα were seeded on glass coverslips and treated with biotinylated FREG (100 ng/ml) for 10 minutes at 4 °C and stained with Fluorescein Avidin D. Bar = 40 µm. Biotinylated FREG clearly bound to PDGF-Rα-transfected cells more than pCDNA 3 -transfected cells or other controls. ( c ) Endogenous PDGF-Rα phosphorylation was evaluated in SK-MEL-110 treated for 5 minutes with PDGF-AA (10 ng/ml) or with increasing dose of FREG (1, 10, and 100 ng/ml). PDGF-AA, used as positive control, induced phosphorylation of its selective receptor; FREG induced PDGF-Rα phosphorylation with a rate comparable to PDGF-AA. ( d ) Correlation between peptide effect and PDGF-Rα expression in four melanoma cell lines (namely: mouse B16F10, human SK-MEL-110, human SK-MEL-28, human MEWO). FREG was used at 100 ng/ml and its inhibitory effect on cell proliferation was correlated to PDGF-Rα expression measured by western blot analysis. Spearman correlation index was computed with PRISM 5 software for Windows. PDGF-Rα, platelet-derived growth factor-receptor-α.
    Figure Legend Snippet: FREG interacts with PDGF-Rα . ( a ) FREG binding to plastic-immobilized PDGF-Rα (7 µg/ml) was measured in solid-phase assays. Biotinylated FREG (form 0 to 2 mmol/l) was incubated for 4 hours onto immobilized-PDGF-Rα. Unbound material was washed away and a colorimetric assay was performed. Biotinylated FREG bound PDGF-Rα in a saturable and concentration-dependent manner. ( b ) SK-MEL-110 untransfected or overexpressing PDGF-Rα were seeded on glass coverslips and treated with biotinylated FREG (100 ng/ml) for 10 minutes at 4 °C and stained with Fluorescein Avidin D. Bar = 40 µm. Biotinylated FREG clearly bound to PDGF-Rα-transfected cells more than pCDNA 3 -transfected cells or other controls. ( c ) Endogenous PDGF-Rα phosphorylation was evaluated in SK-MEL-110 treated for 5 minutes with PDGF-AA (10 ng/ml) or with increasing dose of FREG (1, 10, and 100 ng/ml). PDGF-AA, used as positive control, induced phosphorylation of its selective receptor; FREG induced PDGF-Rα phosphorylation with a rate comparable to PDGF-AA. ( d ) Correlation between peptide effect and PDGF-Rα expression in four melanoma cell lines (namely: mouse B16F10, human SK-MEL-110, human SK-MEL-28, human MEWO). FREG was used at 100 ng/ml and its inhibitory effect on cell proliferation was correlated to PDGF-Rα expression measured by western blot analysis. Spearman correlation index was computed with PRISM 5 software for Windows. PDGF-Rα, platelet-derived growth factor-receptor-α.

    Techniques Used: Binding Assay, Incubation, Colorimetric Assay, Concentration Assay, Staining, Avidin-Biotin Assay, Transfection, Positive Control, Expressing, Western Blot, Software, Derivative Assay

    36) Product Images from "Reduced adult endothelial cell EphB4 function promotes venous remodeling"

    Article Title: Reduced adult endothelial cell EphB4 function promotes venous remodeling

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00333.2012

    Increased smooth muscle cell (SMC) mitogens and chemoattractants secreted by EphB4 +/− EC. A – C : bar graphs showing amounts of VEGF-A ( A ), PDGF-BB ( B ), and TGF-β1 ( C ) protein detected in EphB4 +/+ or EphB4 +/− EC-conditioned
    Figure Legend Snippet: Increased smooth muscle cell (SMC) mitogens and chemoattractants secreted by EphB4 +/− EC. A – C : bar graphs showing amounts of VEGF-A ( A ), PDGF-BB ( B ), and TGF-β1 ( C ) protein detected in EphB4 +/+ or EphB4 +/− EC-conditioned

    Techniques Used:

    37) Product Images from "Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair"

    Article Title: Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.02.016

    MesoT Cells Efficiently Differentiate to Smooth Muscle and ECs (A) MesoT cells treated with PDGF-BB (50 ng/ml) in CDM (–Activin A) for 12 days were probed with antibodies for alpha smooth muscle actin (αSMA), calponin, and myosin heavy chain 11 (MYH11). Scale bars, 100 μm (left) and 50 μm (right). (B) MesoT cells grown in CDM (–A) supplemented with PDGF-BB or VEGF-A 165 and SB431542 were quantified based on expression of lineage specific markers for SMCs and ECs. All experiments in biological triplicate. (C) 3,3’-dioctadecyloxacarbocyanine perchlorate (DiO)-labeled SMCs as shown in (A) were treated with 100 μM carbachol or 50 mM KCl to stimulate functional contraction. Scale bar, 200 μm. (D) SMC surface area was measured after treatment (C). Contraction is shown as the % change in cell surface area for individual cells. Each treatment group was compared to corresponding control time point to determine statistical significance. b = 20. (E and F) MesoT cells treated with CDM (–A), VEGF-A 165 , and SB431542 for 12 days were fixed and probed with antibodies for CD31, vWF, and DAPI (E) or characterized by flow cytometry with VE-cadherin (blue) or isotype control (red) (F). Scale bar, 50 μm. (G) Trans -endothelial electrical resistance (TEER) was measured after culturing MesoT cells in CDM (–A) supplemented with VEGF-A 165 alone (+VEGF) or with SB431542 (+VEGF +SB) after 14, 21, and 28 days and compared against primary dermal microvascular endothelium (1° ECs). n = 3. (H) Barrier integrity was tested by measuring FITC-dextran (40 kDa) perfusion from the apical to basolateral side. Control represents the absence of cells. No statistical significance was determined when comparing cells to 1° ECs. (I) Schematic of the bioreactor culture system used in (G) and (H). (J) Immunofluorescence of cell monolayer as in (I) showing expression of tight junction marker ZO1 (top) and H E staining (bottom). Scale bars, 100 μm. (K) Transmission electron microscopy image of MesoT-derived endothelium. Red arrows depict tight cell junctions. Inset (top) is depicted on bottom. Scale bars, 500 nm and 200 nm, respectively. .
    Figure Legend Snippet: MesoT Cells Efficiently Differentiate to Smooth Muscle and ECs (A) MesoT cells treated with PDGF-BB (50 ng/ml) in CDM (–Activin A) for 12 days were probed with antibodies for alpha smooth muscle actin (αSMA), calponin, and myosin heavy chain 11 (MYH11). Scale bars, 100 μm (left) and 50 μm (right). (B) MesoT cells grown in CDM (–A) supplemented with PDGF-BB or VEGF-A 165 and SB431542 were quantified based on expression of lineage specific markers for SMCs and ECs. All experiments in biological triplicate. (C) 3,3’-dioctadecyloxacarbocyanine perchlorate (DiO)-labeled SMCs as shown in (A) were treated with 100 μM carbachol or 50 mM KCl to stimulate functional contraction. Scale bar, 200 μm. (D) SMC surface area was measured after treatment (C). Contraction is shown as the % change in cell surface area for individual cells. Each treatment group was compared to corresponding control time point to determine statistical significance. b = 20. (E and F) MesoT cells treated with CDM (–A), VEGF-A 165 , and SB431542 for 12 days were fixed and probed with antibodies for CD31, vWF, and DAPI (E) or characterized by flow cytometry with VE-cadherin (blue) or isotype control (red) (F). Scale bar, 50 μm. (G) Trans -endothelial electrical resistance (TEER) was measured after culturing MesoT cells in CDM (–A) supplemented with VEGF-A 165 alone (+VEGF) or with SB431542 (+VEGF +SB) after 14, 21, and 28 days and compared against primary dermal microvascular endothelium (1° ECs). n = 3. (H) Barrier integrity was tested by measuring FITC-dextran (40 kDa) perfusion from the apical to basolateral side. Control represents the absence of cells. No statistical significance was determined when comparing cells to 1° ECs. (I) Schematic of the bioreactor culture system used in (G) and (H). (J) Immunofluorescence of cell monolayer as in (I) showing expression of tight junction marker ZO1 (top) and H E staining (bottom). Scale bars, 100 μm. (K) Transmission electron microscopy image of MesoT-derived endothelium. Red arrows depict tight cell junctions. Inset (top) is depicted on bottom. Scale bars, 500 nm and 200 nm, respectively. .

    Techniques Used: Expressing, Labeling, Functional Assay, Flow Cytometry, Cytometry, Immunofluorescence, Marker, Staining, Transmission Assay, Electron Microscopy, Derivative Assay

    38) Product Images from "In vitro effects of imatinib mesylate on radiosensitivity and chemosensitivity of breast cancer cells"

    Article Title: In vitro effects of imatinib mesylate on radiosensitivity and chemosensitivity of breast cancer cells

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-10-412

    Imatinib inhibits PDGF BB dependent cell migration in breast cancer cells . Migration assays were performed in breast cancer cell lines. Cells were incubated with either imatinib, PDGF BB or their combination for up to 72 hours. Cell migration was measured and student's t-test was performed. * indicates p-values
    Figure Legend Snippet: Imatinib inhibits PDGF BB dependent cell migration in breast cancer cells . Migration assays were performed in breast cancer cell lines. Cells were incubated with either imatinib, PDGF BB or their combination for up to 72 hours. Cell migration was measured and student's t-test was performed. * indicates p-values

    Techniques Used: Migration, Incubation

    Imatinib inhibits PDGF BB dependent cell proliferation in breast cancer cell lines . Cells were incubated with increasing concentrations of PDGF BB and imatinib (IC 50). Cell growth was measured by MTT-assay.
    Figure Legend Snippet: Imatinib inhibits PDGF BB dependent cell proliferation in breast cancer cell lines . Cells were incubated with increasing concentrations of PDGF BB and imatinib (IC 50). Cell growth was measured by MTT-assay.

    Techniques Used: Incubation, MTT Assay

    39) Product Images from "Saikosaponin a and Saikosaponin d Inhibit Proliferation and Migratory Activity of Rat HSC-T6 Cells"

    Article Title: Saikosaponin a and Saikosaponin d Inhibit Proliferation and Migratory Activity of Rat HSC-T6 Cells

    Journal: Journal of Medicinal Food

    doi: 10.1089/jmf.2013.2762

    SSa and SSd blocked PDGF-BB and TGF-β1-induced proliferative and migratory activities. The cell migratory activity of HSC-T6 was enhanced by PDGF-BB; however, the enhancing effects were blocked by pretreatment of SSa (A) and SSd (B) . SSa and SSd
    Figure Legend Snippet: SSa and SSd blocked PDGF-BB and TGF-β1-induced proliferative and migratory activities. The cell migratory activity of HSC-T6 was enhanced by PDGF-BB; however, the enhancing effects were blocked by pretreatment of SSa (A) and SSd (B) . SSa and SSd

    Techniques Used: Activity Assay

    40) Product Images from "Saikosaponin a and Saikosaponin d Inhibit Proliferation and Migratory Activity of Rat HSC-T6 Cells"

    Article Title: Saikosaponin a and Saikosaponin d Inhibit Proliferation and Migratory Activity of Rat HSC-T6 Cells

    Journal: Journal of Medicinal Food

    doi: 10.1089/jmf.2013.2762

    SSa and SSd blocked PDGF-BB and TGF-β1-induced proliferative and migratory activities. The cell migratory activity of HSC-T6 was enhanced by PDGF-BB; however, the enhancing effects were blocked by pretreatment of SSa (A) and SSd (B) . SSa and SSd
    Figure Legend Snippet: SSa and SSd blocked PDGF-BB and TGF-β1-induced proliferative and migratory activities. The cell migratory activity of HSC-T6 was enhanced by PDGF-BB; however, the enhancing effects were blocked by pretreatment of SSa (A) and SSd (B) . SSa and SSd

    Techniques Used: Activity Assay

    SSa and SSd blocked PDGF-BB and TGF-β1-induced proliferative and migratory activities. The cell migratory activity of HSC-T6 was enhanced by PDGF-BB; however, the enhancing effects were blocked by pretreatment of SSa (A) and SSd (B) . SSa and SSd
    Figure Legend Snippet: SSa and SSd blocked PDGF-BB and TGF-β1-induced proliferative and migratory activities. The cell migratory activity of HSC-T6 was enhanced by PDGF-BB; however, the enhancing effects were blocked by pretreatment of SSa (A) and SSd (B) . SSa and SSd

    Techniques Used: Activity Assay

    Related Articles

    Transfection:

    Article Title: Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway
    Article Snippet: .. To compare the expression level of PDGF-BB between the untransfected and transfected EPCs, we measured the concentration of PDGF-BB in the supernatant of the culture medium using ELISA (R & D Systems) according to the manufacturer's instructions. ..

    Quantitative RT-PCR:

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes
    Article Snippet: .. Although PDGFRβ expression remained relatively stable over time in the absence of the PDGF-BB, qRT-PCR revealed an increase in PDGFRβ gene expression at 48 and 72 h post-PDGF-BB treatment in the presence of IFNγ, although this was not statistically significant (Fig. ). αSMA mRNA expression closely followed results from protein analysis. αSMA expression was reduced by both PDGF-BB and IFNγ treatment at all time points (Fig. ). .. Ki67 was increased in response to PDGF-BB at both 24 and 48 h after stimulation, and this effect was blocked by the presence of IFNγ (Fig. ).

    Enzyme-linked Immunosorbent Assay:

    Article Title: Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway
    Article Snippet: .. To compare the expression level of PDGF-BB between the untransfected and transfected EPCs, we measured the concentration of PDGF-BB in the supernatant of the culture medium using ELISA (R & D Systems) according to the manufacturer's instructions. ..

    Concentration Assay:

    Article Title: Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway
    Article Snippet: .. To compare the expression level of PDGF-BB between the untransfected and transfected EPCs, we measured the concentration of PDGF-BB in the supernatant of the culture medium using ELISA (R & D Systems) according to the manufacturer's instructions. ..

    Incubation:

    Article Title: Soluble receptor-mediated selective inhibition of VEGFR and PDGFRβ signaling during physiologic and tumor angiogenesis
    Article Snippet: .. NIH 3T3 cells were incubated in the presence or absence of 200 ng/ml soluble PDGFRβ ectodomain for 10 min before stimulation with PDGF-BB (10 ng/ml, R & D Systems). .. For HUVECs, cells were incubated with 150 ng/ml Ang1 in the presence or absence of excess soluble Tie2 protein (5 μg/ml) for 15 min. Inhibition of Akt phosphorylation was determined by Western blotting of whole-cell extracts with primary antibody specific to phosphorylated Ser-473 Akt antibody (Cell Signaling Technology).

    other:

    Article Title: Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-? and VEGFR-2 expression
    Article Snippet: We further established that VSMC proliferation under hypoxia is initiated predominantly by VEGF-A and PDGF-BB via VEGFR-2 and PDGFR-β, respectively.

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes
    Article Snippet: As shown in Fig. , levels of PDGFRβ declined significantly with exposure to the PDGF-BB (maximally at 24 h after ligand addition), suggesting that this mechanism of receptor degradation might also be operating in human brain pericytes.

    Chemotaxis Assay:

    Article Title: C-reactive protein and vein graft disease: evidence for a direct effect on smooth muscle cell phenotype via modulation of PDGF receptor-?
    Article Snippet: .. As shown in , we observed a maximal threefold enhancement of chemotaxis to PDGF-BB ( P < 0.05) in cells pretreated with 20 μg/ml CRP for 48 h vs. control; this enhancement was effectively blocked by a PDGF-neutralizing antibody. .. Activation of PDGFR tyrosine kinase function stimulates a series of cell signaling events leading to multiple cellular activities, including membrane ruffling and chemotaxis.

    Expressing:

    Article Title: Over-Expression of PDGFR-? Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway
    Article Snippet: .. To compare the expression level of PDGF-BB between the untransfected and transfected EPCs, we measured the concentration of PDGF-BB in the supernatant of the culture medium using ELISA (R & D Systems) according to the manufacturer's instructions. ..

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes
    Article Snippet: .. Although PDGFRβ expression remained relatively stable over time in the absence of the PDGF-BB, qRT-PCR revealed an increase in PDGFRβ gene expression at 48 and 72 h post-PDGF-BB treatment in the presence of IFNγ, although this was not statistically significant (Fig. ). αSMA mRNA expression closely followed results from protein analysis. αSMA expression was reduced by both PDGF-BB and IFNγ treatment at all time points (Fig. ). .. Ki67 was increased in response to PDGF-BB at both 24 and 48 h after stimulation, and this effect was blocked by the presence of IFNγ (Fig. ).

    Recombinant:

    Article Title: TGF-?1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels
    Article Snippet: .. Goat anti-caspase 3 antibody (CRP32), which reacts with both precursor and active forms of human caspase 3, and goat anti-PARP, which reacts with both intact and cleaved forms of human PARP, rabbit anti-cIAP-1 antibody, mouse anti-XIAP antibody, recombinant human TGF-β1, PDGF-BB and anti-TGF-β1 antibody were purchased from R & D Systems (Minneapolis, MN). .. Mouse anti-Bcl-2 antibody and mouse anti-Bax antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

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    R&D Systems pdgf bb
    VEGF-A associated with both PDGFRα and PDGFRβ. Binding of VEGF-A to either PDGFRα or PDGFRβ was examined using a cross-linking approach. MSCs were unstimulated (basal) or stimulated with either 10 ng/ml VEGF-A 165 (165) or <t>PDGF-BB</t> (BB) as a positive control, or 10 ng/ml <t>TGF-β</t> 1 as a negative control (not depicted), for 10 min at 37°C. To inhibit growth factor binding to the respective PDGFR, MSCs were also pretreated with either 10 μg/ml anti-PDGFRα (Rα) or -PDGFRβ (Rβ) cell surface neutralization antibodies for 30 min at 37°C, before growth factor stimulation. Growth factor binding to PDGFR was captured by adding 1 mM of a cell membrane–impermeable cross-linking agent (DTSSP), followed by immunoprecipitation (IP) analysis using anti-PDGFRα or anti-PDGFRβ, then growth factor association detected by immunoblot (IB) analysis using corresponding (A) anti–VEGF-A or (B) –PDGF-B. Membranes were reprobed with anti-PDGFRα or -PDGFRβ as loading controls. A representative of three independent experiments is shown for each analysis.
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    VEGF-A associated with both PDGFRα and PDGFRβ. Binding of VEGF-A to either PDGFRα or PDGFRβ was examined using a cross-linking approach. MSCs were unstimulated (basal) or stimulated with either 10 ng/ml VEGF-A 165 (165) or PDGF-BB (BB) as a positive control, or 10 ng/ml TGF-β 1 as a negative control (not depicted), for 10 min at 37°C. To inhibit growth factor binding to the respective PDGFR, MSCs were also pretreated with either 10 μg/ml anti-PDGFRα (Rα) or -PDGFRβ (Rβ) cell surface neutralization antibodies for 30 min at 37°C, before growth factor stimulation. Growth factor binding to PDGFR was captured by adding 1 mM of a cell membrane–impermeable cross-linking agent (DTSSP), followed by immunoprecipitation (IP) analysis using anti-PDGFRα or anti-PDGFRβ, then growth factor association detected by immunoblot (IB) analysis using corresponding (A) anti–VEGF-A or (B) –PDGF-B. Membranes were reprobed with anti-PDGFRα or -PDGFRβ as loading controls. A representative of three independent experiments is shown for each analysis.

    Journal: The Journal of Cell Biology

    Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

    doi: 10.1083/jcb.200608093

    Figure Lengend Snippet: VEGF-A associated with both PDGFRα and PDGFRβ. Binding of VEGF-A to either PDGFRα or PDGFRβ was examined using a cross-linking approach. MSCs were unstimulated (basal) or stimulated with either 10 ng/ml VEGF-A 165 (165) or PDGF-BB (BB) as a positive control, or 10 ng/ml TGF-β 1 as a negative control (not depicted), for 10 min at 37°C. To inhibit growth factor binding to the respective PDGFR, MSCs were also pretreated with either 10 μg/ml anti-PDGFRα (Rα) or -PDGFRβ (Rβ) cell surface neutralization antibodies for 30 min at 37°C, before growth factor stimulation. Growth factor binding to PDGFR was captured by adding 1 mM of a cell membrane–impermeable cross-linking agent (DTSSP), followed by immunoprecipitation (IP) analysis using anti-PDGFRα or anti-PDGFRβ, then growth factor association detected by immunoblot (IB) analysis using corresponding (A) anti–VEGF-A or (B) –PDGF-B. Membranes were reprobed with anti-PDGFRα or -PDGFRβ as loading controls. A representative of three independent experiments is shown for each analysis.

    Article Snippet: Growth factors and inhibitors All growth factors, PDGF-AA (221-AA), PDGF-BB (220-BB), TGF-β1 (240-B), VEGF-A165 (293-VE), and VEGF-A121 (298-VS), were obtained from R & D Systems.

    Techniques: Binding Assay, Positive Control, Negative Control, Neutralization, Immunoprecipitation

    PDGF-induced MSC migration was inhibited by VEGF-A. The effects of VEGF-A on PDGF-induced MSC migration was examined using Boyden chamber migration assays. MSCs were preincubated with 10 ng/ml VEGF-A 165 for 10 min, before adding the cell suspension onto the upper chamber membrane surface and exposure to 10 ng/ml PDGF-AA or -BB in the lower half of a Boyden chamber for 5 h. MSCs not exposed to either growth factor represents a growth factor–independent migration control. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the membrane underside. Data shown are the mean number migratory cells ± the SD determined from 10 random fields from each of two independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

    doi: 10.1083/jcb.200608093

    Figure Lengend Snippet: PDGF-induced MSC migration was inhibited by VEGF-A. The effects of VEGF-A on PDGF-induced MSC migration was examined using Boyden chamber migration assays. MSCs were preincubated with 10 ng/ml VEGF-A 165 for 10 min, before adding the cell suspension onto the upper chamber membrane surface and exposure to 10 ng/ml PDGF-AA or -BB in the lower half of a Boyden chamber for 5 h. MSCs not exposed to either growth factor represents a growth factor–independent migration control. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the membrane underside. Data shown are the mean number migratory cells ± the SD determined from 10 random fields from each of two independent experiments. *, P

    Article Snippet: Growth factors and inhibitors All growth factors, PDGF-AA (221-AA), PDGF-BB (220-BB), TGF-β1 (240-B), VEGF-A165 (293-VE), and VEGF-A121 (298-VS), were obtained from R & D Systems.

    Techniques: Migration

    VEGF-A induced a dose-dependent increase in PDGFR tyrosine phosphorylation. The effects of varying VEGF-A 165 concentration on induced PDGFR tyrosine phosphorylation levels was determined by specific ELISAs. MSCs in serum-free conditions were exposed to 0.5, 1, 2, 5, 10, 25, 50, 100, or 200 ng/ml VEGF-A 165 for 10 min at 37°C. As a control, cells were also exposed to identical concentrations of PDGF-BB. MSC lysates were assayed for either PDGFRα or PDGFRβ tyrosine phosphorylation using a corresponding ELISA. Increased tyrosine phosphorylation is represented by an increase in optical density (OD 450nm ). Data shown are mean OD 450nm ± the SD determined from two independent experiments performed in triplicate.

    Journal: The Journal of Cell Biology

    Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

    doi: 10.1083/jcb.200608093

    Figure Lengend Snippet: VEGF-A induced a dose-dependent increase in PDGFR tyrosine phosphorylation. The effects of varying VEGF-A 165 concentration on induced PDGFR tyrosine phosphorylation levels was determined by specific ELISAs. MSCs in serum-free conditions were exposed to 0.5, 1, 2, 5, 10, 25, 50, 100, or 200 ng/ml VEGF-A 165 for 10 min at 37°C. As a control, cells were also exposed to identical concentrations of PDGF-BB. MSC lysates were assayed for either PDGFRα or PDGFRβ tyrosine phosphorylation using a corresponding ELISA. Increased tyrosine phosphorylation is represented by an increase in optical density (OD 450nm ). Data shown are mean OD 450nm ± the SD determined from two independent experiments performed in triplicate.

    Article Snippet: Growth factors and inhibitors All growth factors, PDGF-AA (221-AA), PDGF-BB (220-BB), TGF-β1 (240-B), VEGF-A165 (293-VE), and VEGF-A121 (298-VS), were obtained from R & D Systems.

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

    Inhibiting PDGFRα or PDGFRβ attenuated VEGF-A–induced MSC migration. (A) MSCs, or HUVECs used as a VEGFR- positive cell, were pretreated with either 10 μg/ml anti-VEGFR1 or -VEGFR2 neutralization antibodies, 100 nM VEGFR2 tyrosine kinase inhibitor (VEGFR2-TK), 2 μM PDGFR tyrosine kinase inhibitor (PDGFR-TK), and 10 μg/ml anti-PDGFRα or -PDGFRβ neutralization antibodies; then, either 10 ng/ml VEGF-A 165 or 10 ng/ml VEGF-A 121 (not depicted) were added to the lower half of a Boyden chamber for 5 h. No inhibition represents control VEGF-A–induced migration. (B) As a control, MSCs were pretreated with either 10 μg/ml anti-PDGFRα or -PDGFRβ neutralization antibodies, and then 10 ng/ml PDGF-BB was added to the lower half of a Boyden chamber for 5 h. No inhibition represents control PDGF-BB–induced migration. (C) MSCs were transfected with either 3 μg siRNA PDGFRα, siRNA PDGFRβ, or scrambled siRNA used as a control. Transfected MSCs in serum-free conditions were either unstimulated as a control, or exposed to 10 ng/ml VEGF-A 165 in the lower half of a Boyden chamber for 5 h. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the membrane underside. Data shown are the mean number of migratory cells ± the SD, which were determined from 10 random fields from each of four (A) or two (B and C) independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

    doi: 10.1083/jcb.200608093

    Figure Lengend Snippet: Inhibiting PDGFRα or PDGFRβ attenuated VEGF-A–induced MSC migration. (A) MSCs, or HUVECs used as a VEGFR- positive cell, were pretreated with either 10 μg/ml anti-VEGFR1 or -VEGFR2 neutralization antibodies, 100 nM VEGFR2 tyrosine kinase inhibitor (VEGFR2-TK), 2 μM PDGFR tyrosine kinase inhibitor (PDGFR-TK), and 10 μg/ml anti-PDGFRα or -PDGFRβ neutralization antibodies; then, either 10 ng/ml VEGF-A 165 or 10 ng/ml VEGF-A 121 (not depicted) were added to the lower half of a Boyden chamber for 5 h. No inhibition represents control VEGF-A–induced migration. (B) As a control, MSCs were pretreated with either 10 μg/ml anti-PDGFRα or -PDGFRβ neutralization antibodies, and then 10 ng/ml PDGF-BB was added to the lower half of a Boyden chamber for 5 h. No inhibition represents control PDGF-BB–induced migration. (C) MSCs were transfected with either 3 μg siRNA PDGFRα, siRNA PDGFRβ, or scrambled siRNA used as a control. Transfected MSCs in serum-free conditions were either unstimulated as a control, or exposed to 10 ng/ml VEGF-A 165 in the lower half of a Boyden chamber for 5 h. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the membrane underside. Data shown are the mean number of migratory cells ± the SD, which were determined from 10 random fields from each of four (A) or two (B and C) independent experiments. *, P

    Article Snippet: Growth factors and inhibitors All growth factors, PDGF-AA (221-AA), PDGF-BB (220-BB), TGF-β1 (240-B), VEGF-A165 (293-VE), and VEGF-A121 (298-VS), were obtained from R & D Systems.

    Techniques: Migration, Neutralization, Inhibition, Transfection

    VEGF-A–induced HDF migration was PDGFRα and PDGFRβ dependent. (A) The expression of cell surface PDGFRs on HDFs were determined by single-color flow cytometry. Analysis of PDGFRα and PDGFRβ was performed using anti–human PE-conjugated antibodies, using an IgG 1 -PE antibody as a control. (B) The effects of VEGF-A on HDF migration and the involvement of PDGFRs were examined using Boyden chamber migration assays. HDF migration was evaluated in serum-free conditions after 5-h exposure to growth factor; 20 ng/ml VEGF-A 165 , VEGF-A 121 , PDGF-AA, or PDGF-BB in the lower half of a Boyden chamber. Basal represents growth factor–independent migration. (C) HDFs were pretreated with either 10 μg/ml anti-PDGFRα or -PDGFRβ neutralization antibodies, before adding 20 ng/ml VEGF-A 165 to the lower half of a Boyden chamber for 5 h. No inhibition represents control VEGF-A 165 –induced migration. (D) HDFs were transfected with either 3 μg siRNA PDGFRα, siRNA PDGFRβ, or scrambled siRNA used as a control. Transfected HDFs in serum-free conditions were either unstimulated as a control, or exposed to 20 ng/ml VEGF-A 165 in the lower half of a Boyden chamber for 5 h. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the underside of the membrane. Data shown are the mean number of migratory cells ± the SD determined from 10 random fields from each of three independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

    doi: 10.1083/jcb.200608093

    Figure Lengend Snippet: VEGF-A–induced HDF migration was PDGFRα and PDGFRβ dependent. (A) The expression of cell surface PDGFRs on HDFs were determined by single-color flow cytometry. Analysis of PDGFRα and PDGFRβ was performed using anti–human PE-conjugated antibodies, using an IgG 1 -PE antibody as a control. (B) The effects of VEGF-A on HDF migration and the involvement of PDGFRs were examined using Boyden chamber migration assays. HDF migration was evaluated in serum-free conditions after 5-h exposure to growth factor; 20 ng/ml VEGF-A 165 , VEGF-A 121 , PDGF-AA, or PDGF-BB in the lower half of a Boyden chamber. Basal represents growth factor–independent migration. (C) HDFs were pretreated with either 10 μg/ml anti-PDGFRα or -PDGFRβ neutralization antibodies, before adding 20 ng/ml VEGF-A 165 to the lower half of a Boyden chamber for 5 h. No inhibition represents control VEGF-A 165 –induced migration. (D) HDFs were transfected with either 3 μg siRNA PDGFRα, siRNA PDGFRβ, or scrambled siRNA used as a control. Transfected HDFs in serum-free conditions were either unstimulated as a control, or exposed to 20 ng/ml VEGF-A 165 in the lower half of a Boyden chamber for 5 h. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the underside of the membrane. Data shown are the mean number of migratory cells ± the SD determined from 10 random fields from each of three independent experiments. *, P

    Article Snippet: Growth factors and inhibitors All growth factors, PDGF-AA (221-AA), PDGF-BB (220-BB), TGF-β1 (240-B), VEGF-A165 (293-VE), and VEGF-A121 (298-VS), were obtained from R & D Systems.

    Techniques: Migration, Expressing, Flow Cytometry, Cytometry, Neutralization, Inhibition, Transfection

    Exposure to VEGF-A increased MSC migration and proliferation. (A) MSC migration was examined in serum-free conditions after a 5-h exposure to a growth factor; 10 ng/ml VEGF-A 165 , VEGF-A 121 , PDGF-AA, or PDGF-BB in the lower half of a Boyden chamber. Basal represents growth factor–independent migration. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the membrane underside. Data shown are the mean number of migratory cells ± the SD determined from 10 random fields from each of four independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

    doi: 10.1083/jcb.200608093

    Figure Lengend Snippet: Exposure to VEGF-A increased MSC migration and proliferation. (A) MSC migration was examined in serum-free conditions after a 5-h exposure to a growth factor; 10 ng/ml VEGF-A 165 , VEGF-A 121 , PDGF-AA, or PDGF-BB in the lower half of a Boyden chamber. Basal represents growth factor–independent migration. Images below each bar graph are representative of migratory cells/field (using a 10× objective lens) on the membrane underside. Data shown are the mean number of migratory cells ± the SD determined from 10 random fields from each of four independent experiments. *, P

    Article Snippet: Growth factors and inhibitors All growth factors, PDGF-AA (221-AA), PDGF-BB (220-BB), TGF-β1 (240-B), VEGF-A165 (293-VE), and VEGF-A121 (298-VS), were obtained from R & D Systems.

    Techniques: Migration

    VEGF-A–induced PDGFR tyrosine phosphorylation was comparable to PDGF-BB–induced PDGFRα level. (A) RTK array analysis of lysates from MSCs transfected with 3 μg scrambled siRNA as a control, siRNA PDGFRα or siRNA PDGFRβ, stimulated using 20 ng/ml PDGF-BB in serum-free conditions for 10 min at 37°C. Each array was identically exposed to detection reagents and film. A representative example of two independent experiments is shown. (B) Bar graph comparing VEGF-A 165 – and PDGF-BB–induced PDGFR tyrosine phosphorylation levels. Data represent VEGF-A 165 and PDGF-BB–stimulated controls from RTK array analysis shown in Fig. 5 B and Fig. 6 A , respectively. Mean pixel density ± the SD of duplicate spots, normalized against duplicate phosphotyrosine-positive control spots = 100. (C) Immunoprecipitation (IP) analysis of PDGFR tyrosine phosphorylation levels. MSCs in serum-free conditions were unstimulated with growth factor (basal), or stimulated with either 20 ng/ml VEGF-A 165 or PDGF-BB as a control, for 10 min at 37°C. PDGFRs were isolated from MSC lysates by IP analysis using anti-PDGFRα or anti-PDGFRβ, and then tyrosine phosphorylation detected by immunoblot (IB) analysis using anti-phosphotyrosine (Tyr-P). Membranes were reprobed with corresponding anti-PDGFRα or anti-PDGFRβ as loading controls. A representative of two independent experiments is shown.

    Journal: The Journal of Cell Biology

    Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

    doi: 10.1083/jcb.200608093

    Figure Lengend Snippet: VEGF-A–induced PDGFR tyrosine phosphorylation was comparable to PDGF-BB–induced PDGFRα level. (A) RTK array analysis of lysates from MSCs transfected with 3 μg scrambled siRNA as a control, siRNA PDGFRα or siRNA PDGFRβ, stimulated using 20 ng/ml PDGF-BB in serum-free conditions for 10 min at 37°C. Each array was identically exposed to detection reagents and film. A representative example of two independent experiments is shown. (B) Bar graph comparing VEGF-A 165 – and PDGF-BB–induced PDGFR tyrosine phosphorylation levels. Data represent VEGF-A 165 and PDGF-BB–stimulated controls from RTK array analysis shown in Fig. 5 B and Fig. 6 A , respectively. Mean pixel density ± the SD of duplicate spots, normalized against duplicate phosphotyrosine-positive control spots = 100. (C) Immunoprecipitation (IP) analysis of PDGFR tyrosine phosphorylation levels. MSCs in serum-free conditions were unstimulated with growth factor (basal), or stimulated with either 20 ng/ml VEGF-A 165 or PDGF-BB as a control, for 10 min at 37°C. PDGFRs were isolated from MSC lysates by IP analysis using anti-PDGFRα or anti-PDGFRβ, and then tyrosine phosphorylation detected by immunoblot (IB) analysis using anti-phosphotyrosine (Tyr-P). Membranes were reprobed with corresponding anti-PDGFRα or anti-PDGFRβ as loading controls. A representative of two independent experiments is shown.

    Article Snippet: Growth factors and inhibitors All growth factors, PDGF-AA (221-AA), PDGF-BB (220-BB), TGF-β1 (240-B), VEGF-A165 (293-VE), and VEGF-A121 (298-VS), were obtained from R & D Systems.

    Techniques: Transfection, Positive Control, Immunoprecipitation, Isolation

    Flow cytometric analysis of surface antigens expressed by hASCs. (a) hASCs expressed CD29, CD44, and CD90 but did not express detectable levels of CD11b, CD31, and CD45. (b) The levels of human VEGF, HGF, FGF-2, IGF-1, EGF, PDGF-BB, and KGF were determined using quantitative ELISA kits. The values shown represent the mean ± SD.

    Journal: Regenerative Therapy

    Article Title: Xenogeneic transplantation of human adipose-derived stem cell sheets accelerate angiogenesis and the healing of skin wounds in a Zucker Diabetic Fatty rat model of obese diabetes

    doi: 10.1016/j.reth.2017.02.002

    Figure Lengend Snippet: Flow cytometric analysis of surface antigens expressed by hASCs. (a) hASCs expressed CD29, CD44, and CD90 but did not express detectable levels of CD11b, CD31, and CD45. (b) The levels of human VEGF, HGF, FGF-2, IGF-1, EGF, PDGF-BB, and KGF were determined using quantitative ELISA kits. The values shown represent the mean ± SD.

    Article Snippet: The levels of vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), fibroblast growth factor-2 (FGF-2), platelet-derived growth factor-BB (PDGF-BB), and epidermal growth factor (EGF) were determined using ELISA kits (R & D Systems).

    Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay

    Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p

    Journal: Journal of Neuroinflammation

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

    doi: 10.1186/s12974-016-0722-4

    Figure Lengend Snippet: Chronic IFNγ treatment and PDGFRβ knockdown blocks PDGF-BB-induced proliferation in pericytes. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). b , c After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-induced proliferative response. This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst ( b ); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment ( c ). Positive cells of the total cells measured by Hoechst ( d ) were quantified and plotted as mean ± s.e.m. ( n = 3), ****, #### ( p

    Article Snippet: Although PDGFRβ expression remained relatively stable over time in the absence of the PDGF-BB, qRT-PCR revealed an increase in PDGFRβ gene expression at 48 and 72 h post-PDGF-BB treatment in the presence of IFNγ, although this was not statistically significant (Fig. ). αSMA mRNA expression closely followed results from protein analysis. αSMA expression was reduced by both PDGF-BB and IFNγ treatment at all time points (Fig. ).

    Techniques:

    IFNγ blocks pericyte migration independently of PDGF-BB. a , b Cell migration was measured by scratching wells after 48 h of IFNγ (1 ng/mL) treatment and measuring the number of cells that moved into the gap area after a further 48 h with vehicle or PDGF-BB (10 ng/mL) treatment. Representative images of Coomassie-stained pericytes are presented in a , and manual counts of cells in the gap area (normalized to vehicle-treated condition), plotted as mean ± s.e.m. ( n = 3) #### , ****( p

    Journal: Journal of Neuroinflammation

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

    doi: 10.1186/s12974-016-0722-4

    Figure Lengend Snippet: IFNγ blocks pericyte migration independently of PDGF-BB. a , b Cell migration was measured by scratching wells after 48 h of IFNγ (1 ng/mL) treatment and measuring the number of cells that moved into the gap area after a further 48 h with vehicle or PDGF-BB (10 ng/mL) treatment. Representative images of Coomassie-stained pericytes are presented in a , and manual counts of cells in the gap area (normalized to vehicle-treated condition), plotted as mean ± s.e.m. ( n = 3) #### , ****( p

    Article Snippet: Although PDGFRβ expression remained relatively stable over time in the absence of the PDGF-BB, qRT-PCR revealed an increase in PDGFRβ gene expression at 48 and 72 h post-PDGF-BB treatment in the presence of IFNγ, although this was not statistically significant (Fig. ). αSMA mRNA expression closely followed results from protein analysis. αSMA expression was reduced by both PDGF-BB and IFNγ treatment at all time points (Fig. ).

    Techniques: Migration, Staining

    Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p

    Journal: Journal of Neuroinflammation

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

    doi: 10.1186/s12974-016-0722-4

    Figure Lengend Snippet: Chronic IFNγ treatment reduces PDGFRβ re-synthesis following ligand-stimulated degradation. a – c Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL); the final 48 h was in the presence of either vehicle or PDGF-BB (10 ng/mL), with representative images of PDGFRβ ( green ), αSMA ( red ), and Hoechst ( blue ) ( a ) Scale bar , 100 μm. Quantification of PDGFRβ ( b ) and αSMA ( c ) staining, mean ± s.e.m. ( n = 3), #### , ****( p

    Article Snippet: Although PDGFRβ expression remained relatively stable over time in the absence of the PDGF-BB, qRT-PCR revealed an increase in PDGFRβ gene expression at 48 and 72 h post-PDGF-BB treatment in the presence of IFNγ, although this was not statistically significant (Fig. ). αSMA mRNA expression closely followed results from protein analysis. αSMA expression was reduced by both PDGF-BB and IFNγ treatment at all time points (Fig. ).

    Techniques: Staining

    Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p

    Journal: Journal of Neuroinflammation

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

    doi: 10.1186/s12974-016-0722-4

    Figure Lengend Snippet: Chronic IFNγ increases PDGFRβ membrane expression. a , b Detection of PDGF-BB in pericyte conditioned media. Human brain pericytes at 90 % confluence were left untreated (control ( Con )) or serum starved for 2 h and then treated with vehicle, IFNγ (1 ng/mL), PDGF-BB (100 ng/mL), or both IFNγ and PDGF-BB for 24 h. Lysates were collected for western blot analysis with the indicated antibodies, and a representative blot is shown ( n = 2) ( a ) Bands were quantified with Image Studio™ software and intensity was normalized to vehicle control ( b ); PDGF-BB was normalized to GAPDH. c – e Pericytes were treated for 0, 24, 48, 72, or 96 h (more cytokines added once every 24 h to appropriate wells) with vehicle ( Veh ) or IFNγ (1 ng/mL). c Grey value intensities of PDGFRβ membrane staining are depicted in a pseudo-colour image according to the legend ( right ). d Representative images of membrane PDGFRβ ( red ) and Hoechst ( blue ) in pericytes after 96 h of vehicle or IFNγ treatment. Scale bar , 100 μm. e Quantification of total PDGFRβ ( white bars ) and membrane PDGFRβ ( black bars ) staining intensity per cell was normalized to 0 h time point, plotted as mean ± s.e.m. ( n = 3), ****( p

    Article Snippet: Although PDGFRβ expression remained relatively stable over time in the absence of the PDGF-BB, qRT-PCR revealed an increase in PDGFRβ gene expression at 48 and 72 h post-PDGF-BB treatment in the presence of IFNγ, although this was not statistically significant (Fig. ). αSMA mRNA expression closely followed results from protein analysis. αSMA expression was reduced by both PDGF-BB and IFNγ treatment at all time points (Fig. ).

    Techniques: Expressing, Western Blot, Software, Staining

    Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p

    Journal: Journal of Neuroinflammation

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

    doi: 10.1186/s12974-016-0722-4

    Figure Lengend Snippet: Chronic IFNγ treatment increases PDGFRβ phosphorylation and internalization. a Pericytes were treated for four consecutive days (once every 24 h) with either vehicle ( Veh ) or IFNγ (1 ng/mL). After 96 h total treatment, cells were serum starved for 2 h and then treated with vehicle (−) or PDGF-BB (100 ng/mL) for 30 min as depicted. b Representative western blots of treated pericytes ( n = 3). c – e Bands were quantified with Image Studio™ and normalized to vehicle control. p-PDGFRβ ( c ) was normalized to total PDGFRβ, and p-Akt ( d ) and p-ERK ( e ) were normalized to total Akt and ERK, respectively. f Representative images of pericytes treated as in a showing PDGFRβ ( green ) and Hoechst ( blue ), scale bar , 10 μm. g PDGFRβ puncta in f were quantified using MetaXpress™ software and normalized to cell number and vehicle control and plotted as mean ± s.e.m. ( n = 3), ****( p

    Article Snippet: Although PDGFRβ expression remained relatively stable over time in the absence of the PDGF-BB, qRT-PCR revealed an increase in PDGFRβ gene expression at 48 and 72 h post-PDGF-BB treatment in the presence of IFNγ, although this was not statistically significant (Fig. ). αSMA mRNA expression closely followed results from protein analysis. αSMA expression was reduced by both PDGF-BB and IFNγ treatment at all time points (Fig. ).

    Techniques: Western Blot, Software

    Human brain pericytes respond to PDGF-BB signals in vitro . a Pericytes were serum starved for 2 h, then treated with vehicle (lanes 1 and 7) or PDGF-BB (100 ng/mL) (lanes 2–6, and 8) for the indicated times, and analysed by SDS-PAGE. Representative blots from three cases are shown. b Blots from a were analysed and quantified with Image Studio™. Phosphorylated PDGFRβ (Tyr751) (p-PDGFRβ) ( b ), PDGFRβ ( c ), and PDGF-BB ( f ) were normalized to GAPDH; phosphorylated Akt (Ser473) (p-Akt) ( d ) and phosphorylated ERK (Tyr204) (p-ERK) ( e ) were normalized to total Akt and ERK, respectively. **Bottom band in pERK blot is GAPDH

    Journal: Journal of Neuroinflammation

    Article Title: Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

    doi: 10.1186/s12974-016-0722-4

    Figure Lengend Snippet: Human brain pericytes respond to PDGF-BB signals in vitro . a Pericytes were serum starved for 2 h, then treated with vehicle (lanes 1 and 7) or PDGF-BB (100 ng/mL) (lanes 2–6, and 8) for the indicated times, and analysed by SDS-PAGE. Representative blots from three cases are shown. b Blots from a were analysed and quantified with Image Studio™. Phosphorylated PDGFRβ (Tyr751) (p-PDGFRβ) ( b ), PDGFRβ ( c ), and PDGF-BB ( f ) were normalized to GAPDH; phosphorylated Akt (Ser473) (p-Akt) ( d ) and phosphorylated ERK (Tyr204) (p-ERK) ( e ) were normalized to total Akt and ERK, respectively. **Bottom band in pERK blot is GAPDH

    Article Snippet: Although PDGFRβ expression remained relatively stable over time in the absence of the PDGF-BB, qRT-PCR revealed an increase in PDGFRβ gene expression at 48 and 72 h post-PDGF-BB treatment in the presence of IFNγ, although this was not statistically significant (Fig. ). αSMA mRNA expression closely followed results from protein analysis. αSMA expression was reduced by both PDGF-BB and IFNγ treatment at all time points (Fig. ).

    Techniques: In Vitro, SDS Page

    PDGF-BB and VEGF induce greater ERK1/2 activation in VSMC compared with ASMC under hypoxia. VSMC ( A ) and ASMC ( B ) were incubated with PDGF-BB (10 ng/ml) and VEGF-A (10 ng/ml) under hypoxia for 24 h. VEGF-A-neutralizing antibody (α VEGF IgG) and

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-? and VEGFR-2 expression

    doi: 10.1152/ajpheart.00411.2011

    Figure Lengend Snippet: PDGF-BB and VEGF induce greater ERK1/2 activation in VSMC compared with ASMC under hypoxia. VSMC ( A ) and ASMC ( B ) were incubated with PDGF-BB (10 ng/ml) and VEGF-A (10 ng/ml) under hypoxia for 24 h. VEGF-A-neutralizing antibody (α VEGF IgG) and

    Article Snippet: We further established that VSMC proliferation under hypoxia is initiated predominantly by VEGF-A and PDGF-BB via VEGFR-2 and PDGFR-β, respectively.

    Techniques: Activation Assay, Incubation