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    Structured Review

    PromoCell other primary human cell lines
    Identification of a chemical scaffold with broad anti-myxovirus activity. Chemical structures of the identified scaffold ( A ) and the current lead analog JMN3-003 ( B ). C ) Dose-response curves for JMN3-003 and MeV-Alaska, MuV-South Africa, RSV Long, influenza A/WSN (H1N1), sindbis virus and vaccinia virus. Titers of <t>cell-associated</t> progeny viruses were determined by TCID 50 titration (MeV) or plaque assay (MuV, RSV, sindbis virus, vaccinia virus). For influenza virus, genome copy numbers of released progeny particles were quantified through TaqMan RT-PCR. Titers of released sindbis virus particles were determined by plaque assay. Values reflect averages of at least three experiments ± SD, vaccinia virus titers were determined in duplicate. D and E ) Assessment of metabolic activity of cells after incubation of different established cell <t>lines</t> (D) or <t>primary</t> <t>human</t> cells (E) in the presence of JMN3-003 for 24 hours. Results for human (HeLa, A549, HepG2), primate (Vero-Slam), and canine (MDCK) cell lines and primary human cells (PBMC, smooth muscle, bronchial epithelial) are shown. Values reflect averages of four replicates ± SD.
    Other Primary Human Cell Lines, supplied by PromoCell, used in various techniques. Bioz Stars score: 85/100, based on 375 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases"

    Article Title: Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020069

    Identification of a chemical scaffold with broad anti-myxovirus activity. Chemical structures of the identified scaffold ( A ) and the current lead analog JMN3-003 ( B ). C ) Dose-response curves for JMN3-003 and MeV-Alaska, MuV-South Africa, RSV Long, influenza A/WSN (H1N1), sindbis virus and vaccinia virus. Titers of cell-associated progeny viruses were determined by TCID 50 titration (MeV) or plaque assay (MuV, RSV, sindbis virus, vaccinia virus). For influenza virus, genome copy numbers of released progeny particles were quantified through TaqMan RT-PCR. Titers of released sindbis virus particles were determined by plaque assay. Values reflect averages of at least three experiments ± SD, vaccinia virus titers were determined in duplicate. D and E ) Assessment of metabolic activity of cells after incubation of different established cell lines (D) or primary human cells (E) in the presence of JMN3-003 for 24 hours. Results for human (HeLa, A549, HepG2), primate (Vero-Slam), and canine (MDCK) cell lines and primary human cells (PBMC, smooth muscle, bronchial epithelial) are shown. Values reflect averages of four replicates ± SD.
    Figure Legend Snippet: Identification of a chemical scaffold with broad anti-myxovirus activity. Chemical structures of the identified scaffold ( A ) and the current lead analog JMN3-003 ( B ). C ) Dose-response curves for JMN3-003 and MeV-Alaska, MuV-South Africa, RSV Long, influenza A/WSN (H1N1), sindbis virus and vaccinia virus. Titers of cell-associated progeny viruses were determined by TCID 50 titration (MeV) or plaque assay (MuV, RSV, sindbis virus, vaccinia virus). For influenza virus, genome copy numbers of released progeny particles were quantified through TaqMan RT-PCR. Titers of released sindbis virus particles were determined by plaque assay. Values reflect averages of at least three experiments ± SD, vaccinia virus titers were determined in duplicate. D and E ) Assessment of metabolic activity of cells after incubation of different established cell lines (D) or primary human cells (E) in the presence of JMN3-003 for 24 hours. Results for human (HeLa, A549, HepG2), primate (Vero-Slam), and canine (MDCK) cell lines and primary human cells (PBMC, smooth muscle, bronchial epithelial) are shown. Values reflect averages of four replicates ± SD.

    Techniques Used: Activity Assay, Titration, Plaque Assay, Reverse Transcription Polymerase Chain Reaction, Incubation

    2) Product Images from "miR-146b probably assists miRNA-146a in the suppression of keratinocyte proliferation and inflammatory responses in psoriasis"

    Article Title: miR-146b probably assists miRNA-146a in the suppression of keratinocyte proliferation and inflammatory responses in psoriasis

    Journal: The Journal of investigative dermatology

    doi: 10.1016/j.jid.2017.05.012

    miR-146b inhibits psoriasis-related miR-146a target genes and proliferation of keratinocytes Keratinocytes were transfected either with control (cont) or miR-146a/b mimics or with the control LNA inhibitor (cont) or LNA inhibitor for miR-146a/b (LNA-146). Where indicated, the cells were stimulated with IFN-γ or TNF-α for 48 h or left unstimulated (us). Data represent mean ± SEM. Student’s t-test, * P
    Figure Legend Snippet: miR-146b inhibits psoriasis-related miR-146a target genes and proliferation of keratinocytes Keratinocytes were transfected either with control (cont) or miR-146a/b mimics or with the control LNA inhibitor (cont) or LNA inhibitor for miR-146a/b (LNA-146). Where indicated, the cells were stimulated with IFN-γ or TNF-α for 48 h or left unstimulated (us). Data represent mean ± SEM. Student’s t-test, * P

    Techniques Used: Transfection

    The expression of miR-146a/b in the skin, keratinocytes and fibroblasts ( a–c ) Relative expression of miR-146a/b in lesional (L) and non-lesional (NL) skin from psoriasis (Ps) patients ( a ), human primary keratinocytes (KC), fibroblasts (Fib) or peripheral blood mononuclear cells (PBMCs)( b ) and control skin samples ( c ) was measured by RT-qPCR and is shown compared to the normal skin (cont). ( b ) (n=5). ( d ) ISH images of cont, Ps NL and two Ps L skin biopsies are shown. Blue color shows the expression of miR-146a/b, bar=75 μm. The basement line between the epidermis and dermis is indicated with the dotted line. Stronger signals of miR-146a in the stratum spinosum and miR-146b in the dermis are indicated with black and red arrows, respectively. ( e - g ) Proliferating keratinocytes (KC 2D), reconstituted epidermis (KC 3D) or fibroblasts were stimulated with for 48 h or left unstimulated (us) and subjected to RT-qPCR analysis. Data is shown compared to the mean expression of miR-146a in us cells (=1), (n=4). ( a - c , e - g ) Student’s t-test, * P
    Figure Legend Snippet: The expression of miR-146a/b in the skin, keratinocytes and fibroblasts ( a–c ) Relative expression of miR-146a/b in lesional (L) and non-lesional (NL) skin from psoriasis (Ps) patients ( a ), human primary keratinocytes (KC), fibroblasts (Fib) or peripheral blood mononuclear cells (PBMCs)( b ) and control skin samples ( c ) was measured by RT-qPCR and is shown compared to the normal skin (cont). ( b ) (n=5). ( d ) ISH images of cont, Ps NL and two Ps L skin biopsies are shown. Blue color shows the expression of miR-146a/b, bar=75 μm. The basement line between the epidermis and dermis is indicated with the dotted line. Stronger signals of miR-146a in the stratum spinosum and miR-146b in the dermis are indicated with black and red arrows, respectively. ( e - g ) Proliferating keratinocytes (KC 2D), reconstituted epidermis (KC 3D) or fibroblasts were stimulated with for 48 h or left unstimulated (us) and subjected to RT-qPCR analysis. Data is shown compared to the mean expression of miR-146a in us cells (=1), (n=4). ( a - c , e - g ) Student’s t-test, * P

    Techniques Used: Expressing, Quantitative RT-PCR, In Situ Hybridization

    miR-146a inhibits proliferation and activation-induced apoptosis of human primary keratinocytes Keratinocytes were transfected either with control (cont) or pre-miR-146a (miR-146a) ( a, c ) or with control LNA (cont) or miR-146a inhibitor (LNA-146a) ( b ) for 24 h and then stimulated as indicated for 24 h ( a , b ) or 72 h ( c , d ) or left unstimulated (us). 3 H thymidine was added for another 14h ( a and b ). The percentage of Annexin-V positive cells ( c ) and the viability ( d ), Annexin-V and 7AAD negative cells) of unstimulated (us) or stimulated primary KCs is presented. Data represent the mean ± SEM, Student’s t -test, * P
    Figure Legend Snippet: miR-146a inhibits proliferation and activation-induced apoptosis of human primary keratinocytes Keratinocytes were transfected either with control (cont) or pre-miR-146a (miR-146a) ( a, c ) or with control LNA (cont) or miR-146a inhibitor (LNA-146a) ( b ) for 24 h and then stimulated as indicated for 24 h ( a , b ) or 72 h ( c , d ) or left unstimulated (us). 3 H thymidine was added for another 14h ( a and b ). The percentage of Annexin-V positive cells ( c ) and the viability ( d ), Annexin-V and 7AAD negative cells) of unstimulated (us) or stimulated primary KCs is presented. Data represent the mean ± SEM, Student’s t -test, * P

    Techniques Used: Activation Assay, Transfection

    FERMT1 is a novel miR-146a target ( a ) miR-146a and the mutated binding sites (underlined). Positions indicate the distance from the beginning of FERMT1 3′UTR. ( b ) The relative firefly luciferase (LUC) activity is normalized to the value of control miRNA and empty vector (cont; =1). Data represent the mean ± SEM (n=8). ( c and d ) Keratinocytes were transfected with cont or pre-miR-146a (miR-146a) for 24 h and then stimulated as indicated for 48 h or left unstimulated (us). ( e ) The proliferation was measured with keratinocytes transfected with cont or specific siRNAs for 24 h. ( b , c and e ) Data represent the mean ± SEM. Student’s t-test, * P
    Figure Legend Snippet: FERMT1 is a novel miR-146a target ( a ) miR-146a and the mutated binding sites (underlined). Positions indicate the distance from the beginning of FERMT1 3′UTR. ( b ) The relative firefly luciferase (LUC) activity is normalized to the value of control miRNA and empty vector (cont; =1). Data represent the mean ± SEM (n=8). ( c and d ) Keratinocytes were transfected with cont or pre-miR-146a (miR-146a) for 24 h and then stimulated as indicated for 48 h or left unstimulated (us). ( e ) The proliferation was measured with keratinocytes transfected with cont or specific siRNAs for 24 h. ( b , c and e ) Data represent the mean ± SEM. Student’s t-test, * P

    Techniques Used: Binding Assay, Luciferase, Activity Assay, Plasmid Preparation, Transfection

    3) Product Images from "Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes"

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01387

    Effects of human PGE2-modulated and cytokine-treated CD34 + cells. (A,B) Sustained and robust upregulation of PD-L1 upon culture for 7 days with PGE2 and a cocktail of cytokines (heparin, human SCF, human TPO, human FGF-1, IGFBP2, and Angptl3) on CD34 + cells obtained from T1D patients as compared to those from healthy controls. (C) Effect of PGE2 pulsing on CD34 + cells cultured for 0, 24, 72 h and 6 days on PD-L1 expression on CD34 + cells. (D,E) Bar graphs showing an increase in the number of PD-L1 + CD34 + cells and fold increase in cell number after 7 days of culture with PGE2 supplemented with cytokines. (F,G) Upregulation of PD-L1 expression following culture with PGE2 (shown as percentage) on CD34 + cells was not altered by the freeze/thawing process. (H) PGE2-modulated CD34 + cells abrogate the IFN-γ autoimmune response to insulin-associated 2 (I-A2) autoantigen in vitro , as measured via the quantification of IFN-γ-producing cells in an Elispot assay; Diftavax refers to a vaccine including immunization against tetanus toxoid, difteria, and hemophilus. (I) PGE2-modulated CD34 + cells and PGE2-modulated CD34 + cells cultured for 7 days in STFIA media abrogate the IFN-γ autoimmune response toward insulin-associated 2 (I-A2) autoantigen in vitro , as measured via the quantification of IFN-γ-producing cells in an Elispot assay, even when added at low dose. (J) PGE2-modulated CD34 + cells abrogate the anti-CD3/CD28-stimulated PBMC response in vitro as measured via the quantification of IFN-γ-producing cells in an Elispot assay. Data are expressed as mean ± SEM. Data are representative of at least two independent experiments. * P
    Figure Legend Snippet: Effects of human PGE2-modulated and cytokine-treated CD34 + cells. (A,B) Sustained and robust upregulation of PD-L1 upon culture for 7 days with PGE2 and a cocktail of cytokines (heparin, human SCF, human TPO, human FGF-1, IGFBP2, and Angptl3) on CD34 + cells obtained from T1D patients as compared to those from healthy controls. (C) Effect of PGE2 pulsing on CD34 + cells cultured for 0, 24, 72 h and 6 days on PD-L1 expression on CD34 + cells. (D,E) Bar graphs showing an increase in the number of PD-L1 + CD34 + cells and fold increase in cell number after 7 days of culture with PGE2 supplemented with cytokines. (F,G) Upregulation of PD-L1 expression following culture with PGE2 (shown as percentage) on CD34 + cells was not altered by the freeze/thawing process. (H) PGE2-modulated CD34 + cells abrogate the IFN-γ autoimmune response to insulin-associated 2 (I-A2) autoantigen in vitro , as measured via the quantification of IFN-γ-producing cells in an Elispot assay; Diftavax refers to a vaccine including immunization against tetanus toxoid, difteria, and hemophilus. (I) PGE2-modulated CD34 + cells and PGE2-modulated CD34 + cells cultured for 7 days in STFIA media abrogate the IFN-γ autoimmune response toward insulin-associated 2 (I-A2) autoantigen in vitro , as measured via the quantification of IFN-γ-producing cells in an Elispot assay, even when added at low dose. (J) PGE2-modulated CD34 + cells abrogate the anti-CD3/CD28-stimulated PBMC response in vitro as measured via the quantification of IFN-γ-producing cells in an Elispot assay. Data are expressed as mean ± SEM. Data are representative of at least two independent experiments. * P

    Techniques Used: Cell Culture, Expressing, In Vitro, Enzyme-linked Immunospot

    Prostaglandins (PGs) enhance PD-L1 expression on human CD34 + cells. (A,B) Results of screening of a PG small molecule library tested for the ability to upregulate PD-L1 (MFI) on CD34 + cells obtained from T1D patients. The schematic of the experimental design is shown in (A) . The 3-color coding shown in (B) represents lowest PD-L1 MFI values (blue), median PD-L1 MFI values (pink), and highest PD-L1 MFI values (red). (C,D) Representative flow cytometric analysis and quantitative bar graph of PD-L1 expression on CD34 + cells from T1D patients pre- and post-pharmacologic modulation with PGE2 as compared to CD34 + cells obtained from healthy controls. (E) PD-L1 and CXCR4 expression (mRNA) fold change was quantified for CD34 + cells pre- and post-modulation with PGE2. (F) Migration assay using CD34 + cells pre- and post-modulation with PGE2. (G) Confocal imaging of CD34 + cells pre- and post-modulation with PGE2, showing DAPI (in blue) and PD-L1 (in green) staining. 63× magnification. Scale bar, 50 µm. (H) IDO-1 expression (mRNA) fold change was quantified for CD34 + cells pre- and post-modulation with PGE2. (I,J) Representative flow cytometric analysis (I) and quantitative bar graph (J) of PD-L1 expression on CD34 + cells from T1D patients pre- and post-pharmacologic modulation with four small molecule PGE2 agonists. All data are expressed as mean ± SEM. * P
    Figure Legend Snippet: Prostaglandins (PGs) enhance PD-L1 expression on human CD34 + cells. (A,B) Results of screening of a PG small molecule library tested for the ability to upregulate PD-L1 (MFI) on CD34 + cells obtained from T1D patients. The schematic of the experimental design is shown in (A) . The 3-color coding shown in (B) represents lowest PD-L1 MFI values (blue), median PD-L1 MFI values (pink), and highest PD-L1 MFI values (red). (C,D) Representative flow cytometric analysis and quantitative bar graph of PD-L1 expression on CD34 + cells from T1D patients pre- and post-pharmacologic modulation with PGE2 as compared to CD34 + cells obtained from healthy controls. (E) PD-L1 and CXCR4 expression (mRNA) fold change was quantified for CD34 + cells pre- and post-modulation with PGE2. (F) Migration assay using CD34 + cells pre- and post-modulation with PGE2. (G) Confocal imaging of CD34 + cells pre- and post-modulation with PGE2, showing DAPI (in blue) and PD-L1 (in green) staining. 63× magnification. Scale bar, 50 µm. (H) IDO-1 expression (mRNA) fold change was quantified for CD34 + cells pre- and post-modulation with PGE2. (I,J) Representative flow cytometric analysis (I) and quantitative bar graph (J) of PD-L1 expression on CD34 + cells from T1D patients pre- and post-pharmacologic modulation with four small molecule PGE2 agonists. All data are expressed as mean ± SEM. * P

    Techniques Used: Expressing, Flow Cytometry, Migration, Imaging, Staining

    Profile of murine KL cells. (A,B) Representative flow cytometric analysis and quantitative bar graph of PD-L1 expression on Lineage − c-kit + (KL) cells from NOD mice pre- and post-pharmacologic modulation with PGE2. (C,D) Representative flow cytometric analysis and quantitative bar graph of CXCR4 expression on KL cells from NOD mice pre- and post-pharmacologic modulation with PGE2. (E) Quantitative bar graph of flow cytometric expression of positive and negative costimulatory molecules (CD40, CD80, CD86, PD-L1, PD-L2, and PD-1) and of select pro-inflammatory and anti-inflammatory cytokines (IFN-γ, IL-10, and IL-4) in PGE2-modulated KL cells (PGE2-KL) from NOD mice as compared to those modulated with a selected PGE2 clinical grade agonist (dmPGE2) and compared to unmodulated KL cells isolated from the bone marrow of normoglycemic NOD mice. (F) Flow cytometric expression of selected chemokine receptors (CXCR4, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, and CXCR3) in PGE2-KL from NOD mice as compared to those modulated with a selected PGE2 clinical grade agonist (dmPGE2) and to unmodulated KL cells isolated from the bone marrow of normoglycemic NOD mice. (G) Quantitative bar graph of flow cytometric expression of chemokines receptors (CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR3, and CXCR4) in PGE2-KL from NOD mice as compared to those modulated with a selected PGE2 clinical grade agonist (dmPGE2) and compared to unmodulated KL cells isolated from the bone marrow of normoglycemic NOD mice. Data are expressed as mean ± SEM. Data are representative of at least two independent experiments. * P
    Figure Legend Snippet: Profile of murine KL cells. (A,B) Representative flow cytometric analysis and quantitative bar graph of PD-L1 expression on Lineage − c-kit + (KL) cells from NOD mice pre- and post-pharmacologic modulation with PGE2. (C,D) Representative flow cytometric analysis and quantitative bar graph of CXCR4 expression on KL cells from NOD mice pre- and post-pharmacologic modulation with PGE2. (E) Quantitative bar graph of flow cytometric expression of positive and negative costimulatory molecules (CD40, CD80, CD86, PD-L1, PD-L2, and PD-1) and of select pro-inflammatory and anti-inflammatory cytokines (IFN-γ, IL-10, and IL-4) in PGE2-modulated KL cells (PGE2-KL) from NOD mice as compared to those modulated with a selected PGE2 clinical grade agonist (dmPGE2) and compared to unmodulated KL cells isolated from the bone marrow of normoglycemic NOD mice. (F) Flow cytometric expression of selected chemokine receptors (CXCR4, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, and CXCR3) in PGE2-KL from NOD mice as compared to those modulated with a selected PGE2 clinical grade agonist (dmPGE2) and to unmodulated KL cells isolated from the bone marrow of normoglycemic NOD mice. (G) Quantitative bar graph of flow cytometric expression of chemokines receptors (CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR3, and CXCR4) in PGE2-KL from NOD mice as compared to those modulated with a selected PGE2 clinical grade agonist (dmPGE2) and compared to unmodulated KL cells isolated from the bone marrow of normoglycemic NOD mice. Data are expressed as mean ± SEM. Data are representative of at least two independent experiments. * P

    Techniques Used: Flow Cytometry, Expressing, Mouse Assay, Isolation

    Effects of murine KL cells. (A,B) Representative flow cytometric analysis (A) and quantitative bar graph (B) for IFN-γ + CD4 + T cells isolated from NOD-BDC2.5 TCR Tg mice and stimulated with BDC2.5 peptide in the presence of DCs (Control) or upon coculture with unmodulated KL or with PGE2-KL (at different ratios). (C) Quantitative bar graph of the percentage of CD4 + T cells upon coculture with KL or PGE2-KL. (D) Representative flow cytometric analysis of GFP + PGE2-KL in the PLN of treated NOD mice following 24 h of treatment with GFP + PGE2-KL, demonstrating that they traffic to the PLN following adoptive transfer into NOD mice. (E) Representative flow cytometric analysis of GFP + PGE2-KL in the pancreas of NOD mice 24 h post-infusion with GFP + PGE2-KL, demonstrating the surface phenotype of GFP + cells. Abbreviations: KL, Lineage − c-kit + cells; PGE2-KL, PGE2-modulated KL cells; HSPCs, hematopoietic stem and progenitor cells; GFP, green fluorescent protein; PGE2, prostaglandin E2; DCs, dendritic cells.
    Figure Legend Snippet: Effects of murine KL cells. (A,B) Representative flow cytometric analysis (A) and quantitative bar graph (B) for IFN-γ + CD4 + T cells isolated from NOD-BDC2.5 TCR Tg mice and stimulated with BDC2.5 peptide in the presence of DCs (Control) or upon coculture with unmodulated KL or with PGE2-KL (at different ratios). (C) Quantitative bar graph of the percentage of CD4 + T cells upon coculture with KL or PGE2-KL. (D) Representative flow cytometric analysis of GFP + PGE2-KL in the PLN of treated NOD mice following 24 h of treatment with GFP + PGE2-KL, demonstrating that they traffic to the PLN following adoptive transfer into NOD mice. (E) Representative flow cytometric analysis of GFP + PGE2-KL in the pancreas of NOD mice 24 h post-infusion with GFP + PGE2-KL, demonstrating the surface phenotype of GFP + cells. Abbreviations: KL, Lineage − c-kit + cells; PGE2-KL, PGE2-modulated KL cells; HSPCs, hematopoietic stem and progenitor cells; GFP, green fluorescent protein; PGE2, prostaglandin E2; DCs, dendritic cells.

    Techniques Used: Flow Cytometry, Isolation, Mouse Assay, Adoptive Transfer Assay

    4) Product Images from "Cerebral arteriopathy associated with heterozygous variants in the casitas B-lineage lymphoma gene"

    Article Title: Cerebral arteriopathy associated with heterozygous variants in the casitas B-lineage lymphoma gene

    Journal: Neurology: Genetics

    doi: 10.1212/NXG.0000000000000448

    Enhanced angiogenesis in siRNA CBL-transfected endothelial cells and enhanced proliferation, migration, and PDFGR-induced tyrosine kinase signaling in smooth muscle cells (SMCs) from patients with heterozygous CBL mutation (A) Human umbilical endothelial vein cells (HUVECs) were transfected with siRNA targeting CBL or a scrambled siRNA control and then stimulated with 20 ng/mL VEGF for the indicated periods of time before staining to explore expression of phosphorylated PLC-γ1. Silencing of CBL resulted in significant upregulation of relative phosphorylated-PLC-γ1 expression in siRNA CBL transfected cells compared with control scrambled transfected HUVECs, p = 0.04. (B) Representative images of HUVEC capillary network formation on matrigel for resting endothelial cells, scrambled control siRNA, and CBL siRNA-transfected endothelial cells. Five independent fields were assessed for each well and the mean numbers of tube branches determined. (C) There was enhanced HUVEC capillary network formation in a matrigel assay in siRNA CBL- transfected cells compared with scrambled control cells, p = 0.02. (D and E) Human dermal fibroblast cells (HDFCs) from patients with heterozygous CBL mutations were treated with 5 ng/mL transforming growth factor 1 for 14 days to induce trans-differentiation into SMCs. A standard scratch assay was then performed on cultured monolayer of SMCs derived from HDFCs of healthy controls and patients with CBL mutations. There was an increase in migration of SMC patients with heterozygous CBL mutations compared with control WT/WT cells, p = 0.02. (F) Patient-derived SMC-like cells/myofibroblasts also exhibited enhanced proliferation when compared with control cells, p = 0.045. (G) SMC-like cells/myofibroblasts were stimulated with 20 ng/mL PDGF for the indicated periods of time before staining to explore changes in phosphorylated MAPK expression. There was enhanced phosphorylation of MAPK at 60 minutes induced by PDGF receptor in patient-derived SMCs compared with control cells, p = 0.04. Data were expressed as fold change relative to mean baseline expression for control and plotted as mean of triplicate samples ± SEM. p
    Figure Legend Snippet: Enhanced angiogenesis in siRNA CBL-transfected endothelial cells and enhanced proliferation, migration, and PDFGR-induced tyrosine kinase signaling in smooth muscle cells (SMCs) from patients with heterozygous CBL mutation (A) Human umbilical endothelial vein cells (HUVECs) were transfected with siRNA targeting CBL or a scrambled siRNA control and then stimulated with 20 ng/mL VEGF for the indicated periods of time before staining to explore expression of phosphorylated PLC-γ1. Silencing of CBL resulted in significant upregulation of relative phosphorylated-PLC-γ1 expression in siRNA CBL transfected cells compared with control scrambled transfected HUVECs, p = 0.04. (B) Representative images of HUVEC capillary network formation on matrigel for resting endothelial cells, scrambled control siRNA, and CBL siRNA-transfected endothelial cells. Five independent fields were assessed for each well and the mean numbers of tube branches determined. (C) There was enhanced HUVEC capillary network formation in a matrigel assay in siRNA CBL- transfected cells compared with scrambled control cells, p = 0.02. (D and E) Human dermal fibroblast cells (HDFCs) from patients with heterozygous CBL mutations were treated with 5 ng/mL transforming growth factor 1 for 14 days to induce trans-differentiation into SMCs. A standard scratch assay was then performed on cultured monolayer of SMCs derived from HDFCs of healthy controls and patients with CBL mutations. There was an increase in migration of SMC patients with heterozygous CBL mutations compared with control WT/WT cells, p = 0.02. (F) Patient-derived SMC-like cells/myofibroblasts also exhibited enhanced proliferation when compared with control cells, p = 0.045. (G) SMC-like cells/myofibroblasts were stimulated with 20 ng/mL PDGF for the indicated periods of time before staining to explore changes in phosphorylated MAPK expression. There was enhanced phosphorylation of MAPK at 60 minutes induced by PDGF receptor in patient-derived SMCs compared with control cells, p = 0.04. Data were expressed as fold change relative to mean baseline expression for control and plotted as mean of triplicate samples ± SEM. p

    Techniques Used: Transfection, Migration, Mutagenesis, Staining, Expressing, Planar Chromatography, Matrigel Assay, Wound Healing Assay, Cell Culture, Derivative Assay

    5) Product Images from "Transitional changes in the CRP structure lead to the exposure of proinflammatory binding sites"

    Article Title: Transitional changes in the CRP structure lead to the exposure of proinflammatory binding sites

    Journal: Nature Communications

    doi: 10.1038/ncomms14188

    Model of CRP–microvesicle interaction. pCRP (yellow) binds to the plasma membrane of activated monocytes. It is subsequently released on membrane-derived microvesicles (blue). Microvesicle-associated pCRP is converted to pCRP* (red). pCRP* can activate the complement system by binding C1q (light green) or dissociate into individual mCRP subunits.
    Figure Legend Snippet: Model of CRP–microvesicle interaction. pCRP (yellow) binds to the plasma membrane of activated monocytes. It is subsequently released on membrane-derived microvesicles (blue). Microvesicle-associated pCRP is converted to pCRP* (red). pCRP* can activate the complement system by binding C1q (light green) or dissociate into individual mCRP subunits.

    Techniques Used: Derivative Assay, Binding Assay

    Model of the pCRP conformational rearrangement to pCRP* and interaction of pCRP* with complement C1q. ( a ) The individual subunits of human pCRP (PDB ID: 1B09; ref. 7 ) have been displayed as a molecular surface and coloured to highlight the subunit boundaries. View shown is the membrane binding B face of pCRP, with phosphocholine (cream spheres) and Ca 2+ ions (black spheres) located in the ligand binding site on each subunit. The exposed region of the CHO-binding motif (residues 35–47) is coloured white in each subunit. The cross-sectional size of the pentamer is ∼100 Å (∼10 nm). ( b ) Close-up view of a phosphocholine head group from a microvesicle LPC molecule (light grey, orange, red, blue and white sticks) bound in the ligand binding site of one pCRP subunit (cream cartoon). Some of the residues lining the ligand binding site are labelled (using one letter amino-acid codes) and shown as sticks, the two Ca 2+ ions are green spheres. Salt bridge interactions between the phosphocholine amine group and Glu81 (E81) are indicated by black dashed lines. ( c ) Interaction of pCRP with a model CHO:POPC bilayer (CHO shown as cream coloured sticks; POPC as light grey, orange, red, blue and white sticks). The ligand binding site on each pCRP subunit can bind to a phosphocholine head group of POPC as shown in ( b ) for an LPC molecule. View is from above, looking down onto the pCRP A face and showing the pentameric conformation. The diameter of the pentamer inner annular void is ∼30 Å (∼3 nm). ( d ) Side view showing the interaction between the B face of pCRP and the POPC phosphocholine head groups in the bilayer. The conversion of pCRP to pCRP* is a reversible process. ( e ) pCRP* in a pentameric conformation, same view as in ( c ). As the individual CRP subunits move apart, the neoepitope (residues 199–206, coloured yellow) is exposed and available for anti-mCRP-specific antibody (9C9 or 3H12) binding. The cross-sectional size of the pCRP* pentamer is ∼108 Å (∼10.8 nm). ( f ) The cross-section size of the complement C1q globular head group (PDB ID: 1PK6; ref. 47 ) is ∼50 Å (∼5 nm), this is the C1q domain that interacts with pCRP*. ( g ) and ( h ) The globular head of C1q inserts itself into the inner annular void of pCRP* forcing the subunits further apart. The interacting residues lie on the inner annular surface of the pCRP* pentamer and on the C-terminal surface of the C1q globular head group. The globular head group of C1q is physically unable to bind to pCRP. In ( h ) only one collagen-like fibre is shown (labelled as C1q collagen-like stem). We used part of a free art image for the coiled spring in ( h ) from www.vecteezy.com .
    Figure Legend Snippet: Model of the pCRP conformational rearrangement to pCRP* and interaction of pCRP* with complement C1q. ( a ) The individual subunits of human pCRP (PDB ID: 1B09; ref. 7 ) have been displayed as a molecular surface and coloured to highlight the subunit boundaries. View shown is the membrane binding B face of pCRP, with phosphocholine (cream spheres) and Ca 2+ ions (black spheres) located in the ligand binding site on each subunit. The exposed region of the CHO-binding motif (residues 35–47) is coloured white in each subunit. The cross-sectional size of the pentamer is ∼100 Å (∼10 nm). ( b ) Close-up view of a phosphocholine head group from a microvesicle LPC molecule (light grey, orange, red, blue and white sticks) bound in the ligand binding site of one pCRP subunit (cream cartoon). Some of the residues lining the ligand binding site are labelled (using one letter amino-acid codes) and shown as sticks, the two Ca 2+ ions are green spheres. Salt bridge interactions between the phosphocholine amine group and Glu81 (E81) are indicated by black dashed lines. ( c ) Interaction of pCRP with a model CHO:POPC bilayer (CHO shown as cream coloured sticks; POPC as light grey, orange, red, blue and white sticks). The ligand binding site on each pCRP subunit can bind to a phosphocholine head group of POPC as shown in ( b ) for an LPC molecule. View is from above, looking down onto the pCRP A face and showing the pentameric conformation. The diameter of the pentamer inner annular void is ∼30 Å (∼3 nm). ( d ) Side view showing the interaction between the B face of pCRP and the POPC phosphocholine head groups in the bilayer. The conversion of pCRP to pCRP* is a reversible process. ( e ) pCRP* in a pentameric conformation, same view as in ( c ). As the individual CRP subunits move apart, the neoepitope (residues 199–206, coloured yellow) is exposed and available for anti-mCRP-specific antibody (9C9 or 3H12) binding. The cross-sectional size of the pCRP* pentamer is ∼108 Å (∼10.8 nm). ( f ) The cross-section size of the complement C1q globular head group (PDB ID: 1PK6; ref. 47 ) is ∼50 Å (∼5 nm), this is the C1q domain that interacts with pCRP*. ( g ) and ( h ) The globular head of C1q inserts itself into the inner annular void of pCRP* forcing the subunits further apart. The interacting residues lie on the inner annular surface of the pCRP* pentamer and on the C-terminal surface of the C1q globular head group. The globular head group of C1q is physically unable to bind to pCRP. In ( h ) only one collagen-like fibre is shown (labelled as C1q collagen-like stem). We used part of a free art image for the coiled spring in ( h ) from www.vecteezy.com .

    Techniques Used: Binding Assay, Ligand Binding Assay

    6) Product Images from "Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes"

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01387

    Prostaglandins (PGs) enhance PD-L1 expression on human CD34 + cells. (A,B) Results of screening of a PG small molecule library tested for the ability to upregulate PD-L1 (MFI) on CD34 + cells obtained from T1D patients. The schematic of the experimental design is shown in (A) . The 3-color coding shown in (B) represents lowest PD-L1 MFI values (blue), median PD-L1 MFI values (pink), and highest PD-L1 MFI values (red). (C,D) Representative flow cytometric analysis and quantitative bar graph of PD-L1 expression on CD34 + cells from T1D patients pre- and post-pharmacologic modulation with PGE2 as compared to CD34 + cells obtained from healthy controls. (E) PD-L1 and CXCR4 expression (mRNA) fold change was quantified for CD34 + cells pre- and post-modulation with PGE2. (F) Migration assay using CD34 + cells pre- and post-modulation with PGE2. (G) Confocal imaging of CD34 + cells pre- and post-modulation with PGE2, showing DAPI (in blue) and PD-L1 (in green) staining. 63× magnification. Scale bar, 50 µm. (H) IDO-1 expression (mRNA) fold change was quantified for CD34 + cells pre- and post-modulation with PGE2. (I,J) Representative flow cytometric analysis (I) and quantitative bar graph (J) of PD-L1 expression on CD34 + cells from T1D patients pre- and post-pharmacologic modulation with four small molecule PGE2 agonists. All data are expressed as mean ± SEM. * P
    Figure Legend Snippet: Prostaglandins (PGs) enhance PD-L1 expression on human CD34 + cells. (A,B) Results of screening of a PG small molecule library tested for the ability to upregulate PD-L1 (MFI) on CD34 + cells obtained from T1D patients. The schematic of the experimental design is shown in (A) . The 3-color coding shown in (B) represents lowest PD-L1 MFI values (blue), median PD-L1 MFI values (pink), and highest PD-L1 MFI values (red). (C,D) Representative flow cytometric analysis and quantitative bar graph of PD-L1 expression on CD34 + cells from T1D patients pre- and post-pharmacologic modulation with PGE2 as compared to CD34 + cells obtained from healthy controls. (E) PD-L1 and CXCR4 expression (mRNA) fold change was quantified for CD34 + cells pre- and post-modulation with PGE2. (F) Migration assay using CD34 + cells pre- and post-modulation with PGE2. (G) Confocal imaging of CD34 + cells pre- and post-modulation with PGE2, showing DAPI (in blue) and PD-L1 (in green) staining. 63× magnification. Scale bar, 50 µm. (H) IDO-1 expression (mRNA) fold change was quantified for CD34 + cells pre- and post-modulation with PGE2. (I,J) Representative flow cytometric analysis (I) and quantitative bar graph (J) of PD-L1 expression on CD34 + cells from T1D patients pre- and post-pharmacologic modulation with four small molecule PGE2 agonists. All data are expressed as mean ± SEM. * P

    Techniques Used: Expressing, Flow Cytometry, Migration, Imaging, Staining

    Effects of human PGE2-modulated and cytokine-treated CD34 + cells. (A,B) Sustained and robust upregulation of PD-L1 upon culture for 7 days with PGE2 and a cocktail of cytokines (heparin, human SCF, human TPO, human FGF-1, IGFBP2, and Angptl3) on CD34 + cells obtained from T1D patients as compared to those from healthy controls. (C) Effect of PGE2 pulsing on CD34 + cells cultured for 0, 24, 72 h and 6 days on PD-L1 expression on CD34 + cells. (D,E) Bar graphs showing an increase in the number of PD-L1 + CD34 + cells and fold increase in cell number after 7 days of culture with PGE2 supplemented with cytokines. (F,G) Upregulation of PD-L1 expression following culture with PGE2 (shown as percentage) on CD34 + cells was not altered by the freeze/thawing process. (H) PGE2-modulated CD34 + cells abrogate the IFN-γ autoimmune response to insulin-associated 2 (I-A2) autoantigen in vitro , as measured via the quantification of IFN-γ-producing cells in an Elispot assay; Diftavax refers to a vaccine including immunization against tetanus toxoid, difteria, and hemophilus. (I) PGE2-modulated CD34 + cells and PGE2-modulated CD34 + cells cultured for 7 days in STFIA media abrogate the IFN-γ autoimmune response toward insulin-associated 2 (I-A2) autoantigen in vitro , as measured via the quantification of IFN-γ-producing cells in an Elispot assay, even when added at low dose. (J) PGE2-modulated CD34 + cells abrogate the anti-CD3/CD28-stimulated PBMC response in vitro as measured via the quantification of IFN-γ-producing cells in an Elispot assay. Data are expressed as mean ± SEM. Data are representative of at least two independent experiments. * P
    Figure Legend Snippet: Effects of human PGE2-modulated and cytokine-treated CD34 + cells. (A,B) Sustained and robust upregulation of PD-L1 upon culture for 7 days with PGE2 and a cocktail of cytokines (heparin, human SCF, human TPO, human FGF-1, IGFBP2, and Angptl3) on CD34 + cells obtained from T1D patients as compared to those from healthy controls. (C) Effect of PGE2 pulsing on CD34 + cells cultured for 0, 24, 72 h and 6 days on PD-L1 expression on CD34 + cells. (D,E) Bar graphs showing an increase in the number of PD-L1 + CD34 + cells and fold increase in cell number after 7 days of culture with PGE2 supplemented with cytokines. (F,G) Upregulation of PD-L1 expression following culture with PGE2 (shown as percentage) on CD34 + cells was not altered by the freeze/thawing process. (H) PGE2-modulated CD34 + cells abrogate the IFN-γ autoimmune response to insulin-associated 2 (I-A2) autoantigen in vitro , as measured via the quantification of IFN-γ-producing cells in an Elispot assay; Diftavax refers to a vaccine including immunization against tetanus toxoid, difteria, and hemophilus. (I) PGE2-modulated CD34 + cells and PGE2-modulated CD34 + cells cultured for 7 days in STFIA media abrogate the IFN-γ autoimmune response toward insulin-associated 2 (I-A2) autoantigen in vitro , as measured via the quantification of IFN-γ-producing cells in an Elispot assay, even when added at low dose. (J) PGE2-modulated CD34 + cells abrogate the anti-CD3/CD28-stimulated PBMC response in vitro as measured via the quantification of IFN-γ-producing cells in an Elispot assay. Data are expressed as mean ± SEM. Data are representative of at least two independent experiments. * P

    Techniques Used: Cell Culture, Expressing, In Vitro, Enzyme-linked Immunospot

    Profile of murine KL cells. (A,B) Representative flow cytometric analysis and quantitative bar graph of PD-L1 expression on Lineage − c-kit + (KL) cells from NOD mice pre- and post-pharmacologic modulation with PGE2. (C,D) Representative flow cytometric analysis and quantitative bar graph of CXCR4 expression on KL cells from NOD mice pre- and post-pharmacologic modulation with PGE2. (E) Quantitative bar graph of flow cytometric expression of positive and negative costimulatory molecules (CD40, CD80, CD86, PD-L1, PD-L2, and PD-1) and of select pro-inflammatory and anti-inflammatory cytokines (IFN-γ, IL-10, and IL-4) in PGE2-modulated KL cells (PGE2-KL) from NOD mice as compared to those modulated with a selected PGE2 clinical grade agonist (dmPGE2) and compared to unmodulated KL cells isolated from the bone marrow of normoglycemic NOD mice. (F) Flow cytometric expression of selected chemokine receptors (CXCR4, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, and CXCR3) in PGE2-KL from NOD mice as compared to those modulated with a selected PGE2 clinical grade agonist (dmPGE2) and to unmodulated KL cells isolated from the bone marrow of normoglycemic NOD mice. (G) Quantitative bar graph of flow cytometric expression of chemokines receptors (CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR3, and CXCR4) in PGE2-KL from NOD mice as compared to those modulated with a selected PGE2 clinical grade agonist (dmPGE2) and compared to unmodulated KL cells isolated from the bone marrow of normoglycemic NOD mice. Data are expressed as mean ± SEM. Data are representative of at least two independent experiments. * P
    Figure Legend Snippet: Profile of murine KL cells. (A,B) Representative flow cytometric analysis and quantitative bar graph of PD-L1 expression on Lineage − c-kit + (KL) cells from NOD mice pre- and post-pharmacologic modulation with PGE2. (C,D) Representative flow cytometric analysis and quantitative bar graph of CXCR4 expression on KL cells from NOD mice pre- and post-pharmacologic modulation with PGE2. (E) Quantitative bar graph of flow cytometric expression of positive and negative costimulatory molecules (CD40, CD80, CD86, PD-L1, PD-L2, and PD-1) and of select pro-inflammatory and anti-inflammatory cytokines (IFN-γ, IL-10, and IL-4) in PGE2-modulated KL cells (PGE2-KL) from NOD mice as compared to those modulated with a selected PGE2 clinical grade agonist (dmPGE2) and compared to unmodulated KL cells isolated from the bone marrow of normoglycemic NOD mice. (F) Flow cytometric expression of selected chemokine receptors (CXCR4, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, and CXCR3) in PGE2-KL from NOD mice as compared to those modulated with a selected PGE2 clinical grade agonist (dmPGE2) and to unmodulated KL cells isolated from the bone marrow of normoglycemic NOD mice. (G) Quantitative bar graph of flow cytometric expression of chemokines receptors (CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR3, and CXCR4) in PGE2-KL from NOD mice as compared to those modulated with a selected PGE2 clinical grade agonist (dmPGE2) and compared to unmodulated KL cells isolated from the bone marrow of normoglycemic NOD mice. Data are expressed as mean ± SEM. Data are representative of at least two independent experiments. * P

    Techniques Used: Flow Cytometry, Expressing, Mouse Assay, Isolation

    Effects of murine KL cells. (A,B) Representative flow cytometric analysis (A) and quantitative bar graph (B) for IFN-γ + CD4 + T cells isolated from NOD-BDC2.5 TCR Tg mice and stimulated with BDC2.5 peptide in the presence of DCs (Control) or upon coculture with unmodulated KL or with PGE2-KL (at different ratios). (C) Quantitative bar graph of the percentage of CD4 + T cells upon coculture with KL or PGE2-KL. (D) Representative flow cytometric analysis of GFP + PGE2-KL in the PLN of treated NOD mice following 24 h of treatment with GFP + PGE2-KL, demonstrating that they traffic to the PLN following adoptive transfer into NOD mice. (E) Representative flow cytometric analysis of GFP + PGE2-KL in the pancreas of NOD mice 24 h post-infusion with GFP + PGE2-KL, demonstrating the surface phenotype of GFP + cells. Abbreviations: KL, Lineage − c-kit + cells; PGE2-KL, PGE2-modulated KL cells; HSPCs, hematopoietic stem and progenitor cells; GFP, green fluorescent protein; PGE2, prostaglandin E2; DCs, dendritic cells.
    Figure Legend Snippet: Effects of murine KL cells. (A,B) Representative flow cytometric analysis (A) and quantitative bar graph (B) for IFN-γ + CD4 + T cells isolated from NOD-BDC2.5 TCR Tg mice and stimulated with BDC2.5 peptide in the presence of DCs (Control) or upon coculture with unmodulated KL or with PGE2-KL (at different ratios). (C) Quantitative bar graph of the percentage of CD4 + T cells upon coculture with KL or PGE2-KL. (D) Representative flow cytometric analysis of GFP + PGE2-KL in the PLN of treated NOD mice following 24 h of treatment with GFP + PGE2-KL, demonstrating that they traffic to the PLN following adoptive transfer into NOD mice. (E) Representative flow cytometric analysis of GFP + PGE2-KL in the pancreas of NOD mice 24 h post-infusion with GFP + PGE2-KL, demonstrating the surface phenotype of GFP + cells. Abbreviations: KL, Lineage − c-kit + cells; PGE2-KL, PGE2-modulated KL cells; HSPCs, hematopoietic stem and progenitor cells; GFP, green fluorescent protein; PGE2, prostaglandin E2; DCs, dendritic cells.

    Techniques Used: Flow Cytometry, Isolation, Mouse Assay, Adoptive Transfer Assay

    7) Product Images from "Reduced IL-6 levels and tumor-associated phospho-STAT3 are associated with reduced tumor development in a mouse model of lung cancer chemoprevention with myo-inositol"

    Article Title: Reduced IL-6 levels and tumor-associated phospho-STAT3 are associated with reduced tumor development in a mouse model of lung cancer chemoprevention with myo-inositol

    Journal: International journal of cancer

    doi: 10.1002/ijc.31152

    CC-LR mice show a fall in (A) M1-type antitumoral macrophages and a small rise in (B) protumoral M2-type macrophages. With myo -inositol, there is a recovery of the fall in M1 macrophages with no significant change in M2 macrophages. For M1 macrophages, p=0.002 for LR vs CC-LR on control diet, p=0.03 for LR vs CC-LR on myo -inositol, p=0.008 for CC-LR on control vs myo -inositol diet; for M2 macrophages, p=0.019 for CC-LR vs LR on control diet and p=0.029 for CC-LR vs LR on myo -inositol. C) myo -inositol at 5 mM does not affect healthy PBMC differentiation into an M1-like phenotype (CD68 hi CD86 + CD163 − ) but increases M2-like differentiation (CD68 hi CD86 + CD163 + ). In M2 differentiation media, PBMCs that do not become CD163 + show an M2b-like phenotype. The percentage of these cells decrease with myo -inositol. D–F) myo -inositol decreases IL-6 secretion in M1-like (D, p=0.010), M2-like (E, p=0.001), and M2b-like (F, p=0.022) cells. * indicates p
    Figure Legend Snippet: CC-LR mice show a fall in (A) M1-type antitumoral macrophages and a small rise in (B) protumoral M2-type macrophages. With myo -inositol, there is a recovery of the fall in M1 macrophages with no significant change in M2 macrophages. For M1 macrophages, p=0.002 for LR vs CC-LR on control diet, p=0.03 for LR vs CC-LR on myo -inositol, p=0.008 for CC-LR on control vs myo -inositol diet; for M2 macrophages, p=0.019 for CC-LR vs LR on control diet and p=0.029 for CC-LR vs LR on myo -inositol. C) myo -inositol at 5 mM does not affect healthy PBMC differentiation into an M1-like phenotype (CD68 hi CD86 + CD163 − ) but increases M2-like differentiation (CD68 hi CD86 + CD163 + ). In M2 differentiation media, PBMCs that do not become CD163 + show an M2b-like phenotype. The percentage of these cells decrease with myo -inositol. D–F) myo -inositol decreases IL-6 secretion in M1-like (D, p=0.010), M2-like (E, p=0.001), and M2b-like (F, p=0.022) cells. * indicates p

    Techniques Used: Mouse Assay

    8) Product Images from "Bone marrow stromal cells induce an ALDH+ stem cell-like phenotype and enhance therapy resistance in AML through a TGF-β-p38-ALDH2 pathway"

    Article Title: Bone marrow stromal cells induce an ALDH+ stem cell-like phenotype and enhance therapy resistance in AML through a TGF-β-p38-ALDH2 pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0242809

    Activation of TGF-β1 gene signature in OCI-AML3 cells co-cultured with BM-MSCs. (A, B) OCI-AML3 cells were cultured with or without BM-MSC cells for 3 days. OCI-AML3 cells were FACS sorted to separate them from BM-MSCs and the gene expression analysis was performed by RNA sequencing. Samples were sequenced on the HiSeq Sequencing System. Sequence reads were mapped to human genomics (build hg19) with bowtie2 aligner using RSEM software. R software was used to compare the differential expression between MSC co-culture samples and OCI-AML3 controls. Genes with adjusted p values less than 0.05 and absolute fold changes larger than 2 were considered significant. Analysis of differentially expressed genes using the Ingenuity ® pathway analysis tool revealed activation of a TGF-β1-associated gene signature. (C) Real-time PCR analysis was performed to analyze the expressions of the indicated genes that are differentially regulated by TGF-β1 in OCI-AML3 cells co-cultured with BM-MSCs compared to OCI-AML3 controls. (D) TGF-β1 knockdown MSCs were generated by transfection with plasmid-containing lentiviruses and co-cultured with OCI-AML3 cells for 3 days. ALDH activity was measured by flow cytometry in OCI-AML3 cells cultured with TGF- β1 knockdown MSCs compared to OCI-AML3 cells cultured alone or with control BM-MSCs. Data are plotted as mean values with error bars representing standard error. For C , the Mann-Whitney U test or Student’s t test was used. For D , one-way ANOVA with Tukey's HSD post-hoc test was used.
    Figure Legend Snippet: Activation of TGF-β1 gene signature in OCI-AML3 cells co-cultured with BM-MSCs. (A, B) OCI-AML3 cells were cultured with or without BM-MSC cells for 3 days. OCI-AML3 cells were FACS sorted to separate them from BM-MSCs and the gene expression analysis was performed by RNA sequencing. Samples were sequenced on the HiSeq Sequencing System. Sequence reads were mapped to human genomics (build hg19) with bowtie2 aligner using RSEM software. R software was used to compare the differential expression between MSC co-culture samples and OCI-AML3 controls. Genes with adjusted p values less than 0.05 and absolute fold changes larger than 2 were considered significant. Analysis of differentially expressed genes using the Ingenuity ® pathway analysis tool revealed activation of a TGF-β1-associated gene signature. (C) Real-time PCR analysis was performed to analyze the expressions of the indicated genes that are differentially regulated by TGF-β1 in OCI-AML3 cells co-cultured with BM-MSCs compared to OCI-AML3 controls. (D) TGF-β1 knockdown MSCs were generated by transfection with plasmid-containing lentiviruses and co-cultured with OCI-AML3 cells for 3 days. ALDH activity was measured by flow cytometry in OCI-AML3 cells cultured with TGF- β1 knockdown MSCs compared to OCI-AML3 cells cultured alone or with control BM-MSCs. Data are plotted as mean values with error bars representing standard error. For C , the Mann-Whitney U test or Student’s t test was used. For D , one-way ANOVA with Tukey's HSD post-hoc test was used.

    Techniques Used: Activation Assay, Cell Culture, FACS, Expressing, RNA Sequencing Assay, Sequencing, Software, Co-Culture Assay, Real-time Polymerase Chain Reaction, Generated, Transfection, Plasmid Preparation, Activity Assay, Flow Cytometry, MANN-WHITNEY

    9) Product Images from "Circulating Interleukin-4 Is Associated with a Systemic T Cell Response against Tumor-Associated Antigens in Treatment-Naïve Patients with Resectable Non-Small-Cell Lung Cancer"

    Article Title: Circulating Interleukin-4 Is Associated with a Systemic T Cell Response against Tumor-Associated Antigens in Treatment-Naïve Patients with Resectable Non-Small-Cell Lung Cancer

    Journal: Cancers

    doi: 10.3390/cancers12123496

    Postoperative survival curves predicted using the Cox model. Postoperative RFS after curative-intent surgery for NSCLC was predicted based on the variables serum IL-4 levels and age. Prolonged survival was predicted for younger patients with low serum IL-4 levels than for older patients with high serum IL-4 levels. The favorable effect of younger age on survival was reversed by the combination with higher serum IL-4 levels.
    Figure Legend Snippet: Postoperative survival curves predicted using the Cox model. Postoperative RFS after curative-intent surgery for NSCLC was predicted based on the variables serum IL-4 levels and age. Prolonged survival was predicted for younger patients with low serum IL-4 levels than for older patients with high serum IL-4 levels. The favorable effect of younger age on survival was reversed by the combination with higher serum IL-4 levels.

    Techniques Used:

    Tree-structured dendrogram resulting from the hierarchical clustering analysis based on the correlation matrix of all cytokine and response data. The R-square value is the proportion of variance accounted for by the cluster. For example, the variables IL-4, eotaxin, G-CSF, IL-7 and IL-17A represent a cluster of variables with similar correlations with the response variable (“any_response_PB”; marked with an asterisk). In contrast, the dendrogram shows a marked distance between the variables IL-6 and IL-8 and the response variable, indicating a weak correlation.
    Figure Legend Snippet: Tree-structured dendrogram resulting from the hierarchical clustering analysis based on the correlation matrix of all cytokine and response data. The R-square value is the proportion of variance accounted for by the cluster. For example, the variables IL-4, eotaxin, G-CSF, IL-7 and IL-17A represent a cluster of variables with similar correlations with the response variable (“any_response_PB”; marked with an asterisk). In contrast, the dendrogram shows a marked distance between the variables IL-6 and IL-8 and the response variable, indicating a weak correlation.

    Techniques Used:

    Predicted probability of a TA-specific response based on a multiple logistic model including the variables IL-4, IL-8, and MIP-1b (dichotomized at the optimal cutoff value) and age (continuous variable). The predicted probability of being a responder is visualized based on the three serum cytokines dichotomized at their optimal cutoff values and the variable age. The probabilities of having IL-4 levels above the optimal cutoff value are shown as blue lines, and the probabilities of having IL-4 levels below the cutoff value are shown as red lines. The categorization according to the optimal cutoff value of MIP-1b is shown in the upper and lower rows and the categorization for IL-8 is shown in the left and right columns. The variable age is used as a continuous variable on the x -axis. For example, the probability of being a responder is very high if the serum IL-4 level is below the threshold, the serum IL-8 level is also below the threshold, and the serum MIP-1b level is above the threshold ( d ). In contrast, the likelihood of being a responder in the older age group is very low if the serum IL-8 level is above the threshold and the serum MIP-1b level is also below the threshold ( a ). Likelihood of being a responder if the serum IL-8 level is below the threshold and the serum MIP-1b level is also below the threshold ( b ). Likelihood of being a responder if the serum IL-8 level is above the threshold and the serum MIP-1b level is also above the threshold ( c ).
    Figure Legend Snippet: Predicted probability of a TA-specific response based on a multiple logistic model including the variables IL-4, IL-8, and MIP-1b (dichotomized at the optimal cutoff value) and age (continuous variable). The predicted probability of being a responder is visualized based on the three serum cytokines dichotomized at their optimal cutoff values and the variable age. The probabilities of having IL-4 levels above the optimal cutoff value are shown as blue lines, and the probabilities of having IL-4 levels below the cutoff value are shown as red lines. The categorization according to the optimal cutoff value of MIP-1b is shown in the upper and lower rows and the categorization for IL-8 is shown in the left and right columns. The variable age is used as a continuous variable on the x -axis. For example, the probability of being a responder is very high if the serum IL-4 level is below the threshold, the serum IL-8 level is also below the threshold, and the serum MIP-1b level is above the threshold ( d ). In contrast, the likelihood of being a responder in the older age group is very low if the serum IL-8 level is above the threshold and the serum MIP-1b level is also below the threshold ( a ). Likelihood of being a responder if the serum IL-8 level is below the threshold and the serum MIP-1b level is also below the threshold ( b ). Likelihood of being a responder if the serum IL-8 level is above the threshold and the serum MIP-1b level is also above the threshold ( c ).

    Techniques Used:

    Predicted probability of a response obtained from age-adjusted logistic models. As an example, the age-weighted probability of being a responder based on cytokine levels is shown for the serum cytokines IL-4 ( a ), G-CSF ( b ), IL-17A ( c ), IL-9 ( d ), IL-7 ( e ), and IL-8 ( f ). The blue curves illustrate the probability of having serum cytokine levels below the optimal cutoff, and the red lines represent the probability of having serum cytokine levels above the cutoff.
    Figure Legend Snippet: Predicted probability of a response obtained from age-adjusted logistic models. As an example, the age-weighted probability of being a responder based on cytokine levels is shown for the serum cytokines IL-4 ( a ), G-CSF ( b ), IL-17A ( c ), IL-9 ( d ), IL-7 ( e ), and IL-8 ( f ). The blue curves illustrate the probability of having serum cytokine levels below the optimal cutoff, and the red lines represent the probability of having serum cytokine levels above the cutoff.

    Techniques Used:

    Related Articles

    Isolation:

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: .. We next determined whether freezing/cryopreservation has any effect on PD-L1 expression, by comparing freshly isolated HSPCs with frozen HSPCs, after 7 days of culture using STFIA media pulsed with PGE2 (Figures F,G). .. We observed sustained and conserved PD-L1 expression pre- and post-cryopreservation, suggesting that storage of HSPCs has no detrimental impact on their ex vivo expansion.

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: .. Isolated CD34+ cells from healthy control patients (CTRL) and from T1D patients were cultured in StemSpan SFEMII and pulsed with 10 µM of PGE2 at 24 and 48 h. We first tested the effect of pharmacological modulation with PGE2 by FACS analysis, and our data revealed a slight increase in PD-L1 expression, although not significant, in cultured CD34+ cells from T1D and healthy control patients, with the latter showing a higher percentage of PD-L1 expression as compared to cells from T1D patients (Figures C,D). .. This pattern was further confirmed by confocal imaging, in which PD-L1 surface expression was upregulated in PGE2-treated CD34+ cells as compared to untreated (Figure G).

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: .. Isolated CD34+ cells (HSPCs) obtained from T1D patients and from healthy controls were cultured using StemSpan SFEMII supplemented with the aforementioned human stem cell growth factors (STFIA medium) and pulsed with PGE2 (10 µM) at 24, 96 h and at 7 days at 37°C 5% CO2 . .. PD-L1+ HSPCs were then quantified by FACS analysis at different time points post-culture.

    Cell Culture:

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: .. Isolated CD34+ cells from healthy control patients (CTRL) and from T1D patients were cultured in StemSpan SFEMII and pulsed with 10 µM of PGE2 at 24 and 48 h. We first tested the effect of pharmacological modulation with PGE2 by FACS analysis, and our data revealed a slight increase in PD-L1 expression, although not significant, in cultured CD34+ cells from T1D and healthy control patients, with the latter showing a higher percentage of PD-L1 expression as compared to cells from T1D patients (Figures C,D). .. This pattern was further confirmed by confocal imaging, in which PD-L1 surface expression was upregulated in PGE2-treated CD34+ cells as compared to untreated (Figure G).

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: .. Isolated CD34+ cells (HSPCs) obtained from T1D patients and from healthy controls were cultured using StemSpan SFEMII supplemented with the aforementioned human stem cell growth factors (STFIA medium) and pulsed with PGE2 (10 µM) at 24, 96 h and at 7 days at 37°C 5% CO2 . .. PD-L1+ HSPCs were then quantified by FACS analysis at different time points post-culture.

    other:

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: We next explored the feasibility of pharmacological modulation of PD-L1 with PGE2 in murine HSPCs.

    Expressing:

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: .. We next determined whether freezing/cryopreservation has any effect on PD-L1 expression, by comparing freshly isolated HSPCs with frozen HSPCs, after 7 days of culture using STFIA media pulsed with PGE2 (Figures F,G). .. We observed sustained and conserved PD-L1 expression pre- and post-cryopreservation, suggesting that storage of HSPCs has no detrimental impact on their ex vivo expansion.

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: .. Our screening results performed on ~64 known PGs allowed us to select four PGs, which are analogs to PGE2 and which we show induce relatively high upregulation of PD-L1 expression on human CD34+ cells. .. We therefore sought to test the ability of PGE2-modulated HSPCs to affect the autoimmune response in vitro .

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: .. Isolated CD34+ cells from healthy control patients (CTRL) and from T1D patients were cultured in StemSpan SFEMII and pulsed with 10 µM of PGE2 at 24 and 48 h. We first tested the effect of pharmacological modulation with PGE2 by FACS analysis, and our data revealed a slight increase in PD-L1 expression, although not significant, in cultured CD34+ cells from T1D and healthy control patients, with the latter showing a higher percentage of PD-L1 expression as compared to cells from T1D patients (Figures C,D). .. This pattern was further confirmed by confocal imaging, in which PD-L1 surface expression was upregulated in PGE2-treated CD34+ cells as compared to untreated (Figure G).

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: .. PGE2 Highly Augments PD-L1 Expression in Human HSPCs When Supplemented With Hematopoietic Cytokines In order to improve the strategy used for HSPC expansion and to enhance the function of PGE2-modulated HSPC, we added hematopoietic cytokines (SCF, TPO, FGF-1, IGFBP-2, and Angptl-3 proteins) known as a potent cocktail for HSPC maintenance, into our established culture conditions ( ). .. Isolated CD34+ cells (HSPCs) obtained from T1D patients and from healthy controls were cultured using StemSpan SFEMII supplemented with the aforementioned human stem cell growth factors (STFIA medium) and pulsed with PGE2 (10 µM) at 24, 96 h and at 7 days at 37°C 5% CO2 .

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: .. The expression of another relevant immunoregulatory protein, IDO-1, remained unchanged post-pharmacologic modulation with PGE2 (Figure H). .. We next selected the four PG small molecules [16,16-dimethyl PGE2, 16,16-dimethyl PGE2 4-(4-acetamidobenzamido) phenyl ester, 6-keto PGE1 and 20-ethyl PGE2] that showed the strongest capacity to upregulate PD-L1, based on the results obtained from library screening (Figures I,J).

    Ex Vivo:

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: .. Based on the data herein, ex vivo expa nsion strategies with PGE2 combined with hematopoietic cytokines could generate a novel immunoregulatory HSPC-based approach potentially useful in the treatment of autoimmune T1D, without the detrimental effect of immunosuppressive agent toxicity, which is observed with standard immunotherapy. .. The recent discovery that a pre-established suicide genetic system may control survival and prevent toxicity of HSPCs undergoing ex vivo expansion will implement their use in clinical settings, allowing for easier manipulation of HSPCs and for a cell therapy-based approach in immune-mediated disorders ( ).

    FACS:

    Article Title: Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes
    Article Snippet: .. Isolated CD34+ cells from healthy control patients (CTRL) and from T1D patients were cultured in StemSpan SFEMII and pulsed with 10 µM of PGE2 at 24 and 48 h. We first tested the effect of pharmacological modulation with PGE2 by FACS analysis, and our data revealed a slight increase in PD-L1 expression, although not significant, in cultured CD34+ cells from T1D and healthy control patients, with the latter showing a higher percentage of PD-L1 expression as compared to cells from T1D patients (Figures C,D). .. This pattern was further confirmed by confocal imaging, in which PD-L1 surface expression was upregulated in PGE2-treated CD34+ cells as compared to untreated (Figure G).

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