stat1  (Thermo Fisher)


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    Name:
    STAT1 Monoclonal Antibody C 156 9
    Description:
    STAT1 Monoclonal Antibody for Western Blot IF ICC IHC P
    Catalog Number:
    ma515129
    Price:
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    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher stat1
    STAT3 acts as a downstream mediator for VEGFR2 activation induced by VEGF-A. ( A ) Western blot analysis of T/G HA-VSMC cells transfected with siRNAs for <t>STAT1,</t> STAT3, or STAT5 and treated with 100 ng/mL VEGF-A for 48 hours. ( B ) Western blot analysis of T/G HA-VSMC cells transfected with siRNAs for Myh11 and Smoothlin and quantified the western blot data by Quantity One software. β-actin is a loading control. ** p
    STAT1 Monoclonal Antibody for Western Blot IF ICC IHC P
    https://www.bioz.com/result/stat1/product/Thermo Fisher
    Average 94 stars, based on 46 article reviews
    Price from $9.99 to $1999.99
    stat1 - by Bioz Stars, 2020-11
    94/100 stars

    Images

    1) Product Images from "VEGF-A Stimulates STAT3 Activity via Nitrosylation of Myocardin to Regulate the Expression of Vascular Smooth Muscle Cell Differentiation Markers"

    Article Title: VEGF-A Stimulates STAT3 Activity via Nitrosylation of Myocardin to Regulate the Expression of Vascular Smooth Muscle Cell Differentiation Markers

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02907-6

    STAT3 acts as a downstream mediator for VEGFR2 activation induced by VEGF-A. ( A ) Western blot analysis of T/G HA-VSMC cells transfected with siRNAs for STAT1, STAT3, or STAT5 and treated with 100 ng/mL VEGF-A for 48 hours. ( B ) Western blot analysis of T/G HA-VSMC cells transfected with siRNAs for Myh11 and Smoothlin and quantified the western blot data by Quantity One software. β-actin is a loading control. ** p
    Figure Legend Snippet: STAT3 acts as a downstream mediator for VEGFR2 activation induced by VEGF-A. ( A ) Western blot analysis of T/G HA-VSMC cells transfected with siRNAs for STAT1, STAT3, or STAT5 and treated with 100 ng/mL VEGF-A for 48 hours. ( B ) Western blot analysis of T/G HA-VSMC cells transfected with siRNAs for Myh11 and Smoothlin and quantified the western blot data by Quantity One software. β-actin is a loading control. ** p

    Techniques Used: Activation Assay, Western Blot, Transfection, Software

    2) Product Images from "TRIM14 is a Putative Tumor Suppressor and Regulator of Innate Immune Response in Non-Small Cell Lung Cancer"

    Article Title: TRIM14 is a Putative Tumor Suppressor and Regulator of Innate Immune Response in Non-Small Cell Lung Cancer

    Journal: Scientific Reports

    doi: 10.1038/srep39692

    TRIM14 sensitizes NSCLC cells to anoxic-induced cell death and Type II interferon response. ( a ) Cell cycle progression of isogenic cell lines was assessed by flow cytometry after propidium iodide (PI) staining to determine the percent distribution of G1, S or G2/M-phase cell populations. ( b ) MTS assay was used to measure cell viability of H1650 cells treated with serial dilutions of cisplatin for 48 hours. ( c ) H1650 and H358 cells cultured for 48 hours with a protein kinase inhibitor, Staurosporine (0.5 μM), or under anoxic conditions were fixed and stained for Annexin-V and PI. Flow cytometry was subsequently used to quantitate the percentage of Annexin-V positive cells after treatment. ( d ) Representative flow cytometry analysis of H1650 cells cultured with or without anoxic conditions. ( e ) H1650 and H358 cells were treated with or without 10 U IFNγ for indicated times. Phosphorylation of STAT1 at tyrosine 701, total STAT1 and TRIM14 expression were analyzed by immunoblotting. ( f ) Quantitative RT-PCR using RNA extracted at 4 and 24 hours after IFNγ treatment showed increased transcript levels of ISG56, P21, IFIT1, and OAS1 as compared to untreated cells, but this effect was significantly suppressed in TRIM14-deficient cells. ( g ) Quantitative RT-PCR was used to show that mRNA levels for TRIM14 and IFNB1 were significantly reduced in H1650 xenograft tumors compared to controls (n = 8 tumors with two technical replicates each). Results shown represent more than three biological replicates. (Two-tailed student’s t-test; ****p
    Figure Legend Snippet: TRIM14 sensitizes NSCLC cells to anoxic-induced cell death and Type II interferon response. ( a ) Cell cycle progression of isogenic cell lines was assessed by flow cytometry after propidium iodide (PI) staining to determine the percent distribution of G1, S or G2/M-phase cell populations. ( b ) MTS assay was used to measure cell viability of H1650 cells treated with serial dilutions of cisplatin for 48 hours. ( c ) H1650 and H358 cells cultured for 48 hours with a protein kinase inhibitor, Staurosporine (0.5 μM), or under anoxic conditions were fixed and stained for Annexin-V and PI. Flow cytometry was subsequently used to quantitate the percentage of Annexin-V positive cells after treatment. ( d ) Representative flow cytometry analysis of H1650 cells cultured with or without anoxic conditions. ( e ) H1650 and H358 cells were treated with or without 10 U IFNγ for indicated times. Phosphorylation of STAT1 at tyrosine 701, total STAT1 and TRIM14 expression were analyzed by immunoblotting. ( f ) Quantitative RT-PCR using RNA extracted at 4 and 24 hours after IFNγ treatment showed increased transcript levels of ISG56, P21, IFIT1, and OAS1 as compared to untreated cells, but this effect was significantly suppressed in TRIM14-deficient cells. ( g ) Quantitative RT-PCR was used to show that mRNA levels for TRIM14 and IFNB1 were significantly reduced in H1650 xenograft tumors compared to controls (n = 8 tumors with two technical replicates each). Results shown represent more than three biological replicates. (Two-tailed student’s t-test; ****p

    Techniques Used: Flow Cytometry, Cytometry, Staining, MTS Assay, Cell Culture, Expressing, Quantitative RT-PCR, Two Tailed Test

    3) Product Images from "Activated tyrosine kinases in gastrointestinal stromal tumor with loss of KIT oncoprotein expression"

    Article Title: Activated tyrosine kinases in gastrointestinal stromal tumor with loss of KIT oncoprotein expression

    Journal: Cell Cycle

    doi: 10.1080/15384101.2018.1553335

    (a) Immunoblotting evaluations of KIT-negative GIST cell lines (GIST62 and GIST522) and KIT-positive GIST cell line (GIST882) at 96 hours after infection by lentiviral AXL, EPHA2 , and FAK shRNA constructs. Immunoblots demonstrate the effects of AXL, EPHA2 and FAK knockdown on signaling intermediates (AKT, MAPK p42/44, S6, STAT1, and STAT3), proliferation markers (Cyclin A and PCNA), and cell cycle checkpoint proteins (p27, p21, and p53). Control lanes for each cell line include uninfected cells (untreated lane) and cells infected with empty lentiviral vector. Actin is a loading control. (b) Immunoblotting evaluations of GIST522 and GIST882 cells at 10 days after infection by lentiviral AXL, EPHA2 , and FAK shRNA constructs. AXL shRNA resulted in decreased expression of cyclin A and PCNA, and upregulation of p53, p21, and p27 in GIST522, but not in GIST882. Actin is a loading control. (c) GIST62 and GIST522 cell cultures, evaluated at 6 days after infection by lentiviral AXL shRNA constructs, showing growth inhibition as compared to control cultures infected with empty lentiviral constructs. Scale bars: 100 μm. (d) Cell viability was evaluated in GIST62 (black bars), GIST522 (white bars), and GIST430 (gray bars), at day 8 and day 12 after infection with lentiviral AXL, EPHA2 , or FAK shRNA. Viability was analyzed using the Cell-titer Glo® ATP-based luminescence assay. The data were normalized to empty lentiviral infections, and represent the mean values (± s.d.) from quadruplicate cultures. The experiments were performed in triplicate, and statistically significant differences between vector control and shRNAs are defined as * p
    Figure Legend Snippet: (a) Immunoblotting evaluations of KIT-negative GIST cell lines (GIST62 and GIST522) and KIT-positive GIST cell line (GIST882) at 96 hours after infection by lentiviral AXL, EPHA2 , and FAK shRNA constructs. Immunoblots demonstrate the effects of AXL, EPHA2 and FAK knockdown on signaling intermediates (AKT, MAPK p42/44, S6, STAT1, and STAT3), proliferation markers (Cyclin A and PCNA), and cell cycle checkpoint proteins (p27, p21, and p53). Control lanes for each cell line include uninfected cells (untreated lane) and cells infected with empty lentiviral vector. Actin is a loading control. (b) Immunoblotting evaluations of GIST522 and GIST882 cells at 10 days after infection by lentiviral AXL, EPHA2 , and FAK shRNA constructs. AXL shRNA resulted in decreased expression of cyclin A and PCNA, and upregulation of p53, p21, and p27 in GIST522, but not in GIST882. Actin is a loading control. (c) GIST62 and GIST522 cell cultures, evaluated at 6 days after infection by lentiviral AXL shRNA constructs, showing growth inhibition as compared to control cultures infected with empty lentiviral constructs. Scale bars: 100 μm. (d) Cell viability was evaluated in GIST62 (black bars), GIST522 (white bars), and GIST430 (gray bars), at day 8 and day 12 after infection with lentiviral AXL, EPHA2 , or FAK shRNA. Viability was analyzed using the Cell-titer Glo® ATP-based luminescence assay. The data were normalized to empty lentiviral infections, and represent the mean values (± s.d.) from quadruplicate cultures. The experiments were performed in triplicate, and statistically significant differences between vector control and shRNAs are defined as * p

    Techniques Used: Infection, shRNA, Construct, Western Blot, Plasmid Preparation, Expressing, Inhibition, Luminescence Assay

    4) Product Images from "High-density neutrophils in MGUS and multiple myeloma are dysfunctional and immune-suppressive due to increased STAT3 downstream signaling"

    Article Title: High-density neutrophils in MGUS and multiple myeloma are dysfunctional and immune-suppressive due to increased STAT3 downstream signaling

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-58859-x

    Arginase-1, target of activated STAT3, is increased in MM and MGUS high-density neutrophils. The association between the quantity of ARG1 transcript in MM and MGUS high-density neutrophils with STAT1 ( A ) and STAT3 ( B ) transcripts is shown. Dot-lines represent interval of confidence. ( C ) Arginase expression in healthy, MGUS and MM high-density neutrophils, as detected by qRT-PCR is shown; the differences were evaluated according to ANOVA test. In an independent set of HDNs at steady state, as obtained from peripheral blood of MM, MGUS and healthy subjects, median intensity of fluorescence (MFI) of ARG1 was detected by flow cytometry( D-E ). ( F-H ) ARG1 immunofluorescence staining in HDN isolated by immune-magnetic-based positive selection after density gradient sedimentation from healthy, MGUS and MM subjects. ARG-1 localized in cytosol, in grains larger in MM-HDN than in controls. ( I ) After exposure of healthy HDNs to MM conditioned media obtained from two human myeloma cell lines U266 and OPM2 or 20 ng/mL IL6 or 100 ng/mL LPS for 24 hours, ARG1 was measured by flow cytometry. For more robust statistical evaluation, MFI values were converted to a resolution metric, such as the RD defined as (Median treatment -Median control )/(rSD treatment + rSD control ) to further perform t-test to compare results of different experiments and runs. Stars denote p-value (***p
    Figure Legend Snippet: Arginase-1, target of activated STAT3, is increased in MM and MGUS high-density neutrophils. The association between the quantity of ARG1 transcript in MM and MGUS high-density neutrophils with STAT1 ( A ) and STAT3 ( B ) transcripts is shown. Dot-lines represent interval of confidence. ( C ) Arginase expression in healthy, MGUS and MM high-density neutrophils, as detected by qRT-PCR is shown; the differences were evaluated according to ANOVA test. In an independent set of HDNs at steady state, as obtained from peripheral blood of MM, MGUS and healthy subjects, median intensity of fluorescence (MFI) of ARG1 was detected by flow cytometry( D-E ). ( F-H ) ARG1 immunofluorescence staining in HDN isolated by immune-magnetic-based positive selection after density gradient sedimentation from healthy, MGUS and MM subjects. ARG-1 localized in cytosol, in grains larger in MM-HDN than in controls. ( I ) After exposure of healthy HDNs to MM conditioned media obtained from two human myeloma cell lines U266 and OPM2 or 20 ng/mL IL6 or 100 ng/mL LPS for 24 hours, ARG1 was measured by flow cytometry. For more robust statistical evaluation, MFI values were converted to a resolution metric, such as the RD defined as (Median treatment -Median control )/(rSD treatment + rSD control ) to further perform t-test to compare results of different experiments and runs. Stars denote p-value (***p

    Techniques Used: Expressing, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Immunofluorescence, Staining, Isolation, Selection, Sedimentation

    Pathways transcriptionally dysregulated in MGUS- and MM- HDNs. Our work showed that MM-HDNs had increased expression of IL10RB, IFNGR1/2, TNFR1/2, TLR2, IL17RA/D. While IFN-gamma can activate IFNGR1/2 and IL10 can bind IL10RB in response to unresolved chronic inflammation, to activate STAT1 and STAT3 and promote their nuclear translocation, LPS triggers TLR2, through an adaptor complex which recruits TRAF6 to activate the TAK1 kinase complex can then activate the IKK complex leading to NFkB activation. The increased expression of TNFR1/B and component of their adaptor complex ½ can recruit several transcription factors to amplify the cascade and warrant a robust response. The genes target CD64 and ARG1 are under the transcriptional control of STAT1 and STAT3, as previously reported for other professional phagocytes. The lack of IL17RC excludes that the IL17R complex could be active, but the ligand of the overexpressed IL17RD is still unknown.
    Figure Legend Snippet: Pathways transcriptionally dysregulated in MGUS- and MM- HDNs. Our work showed that MM-HDNs had increased expression of IL10RB, IFNGR1/2, TNFR1/2, TLR2, IL17RA/D. While IFN-gamma can activate IFNGR1/2 and IL10 can bind IL10RB in response to unresolved chronic inflammation, to activate STAT1 and STAT3 and promote their nuclear translocation, LPS triggers TLR2, through an adaptor complex which recruits TRAF6 to activate the TAK1 kinase complex can then activate the IKK complex leading to NFkB activation. The increased expression of TNFR1/B and component of their adaptor complex ½ can recruit several transcription factors to amplify the cascade and warrant a robust response. The genes target CD64 and ARG1 are under the transcriptional control of STAT1 and STAT3, as previously reported for other professional phagocytes. The lack of IL17RC excludes that the IL17R complex could be active, but the ligand of the overexpressed IL17RD is still unknown.

    Techniques Used: Expressing, Translocation Assay, Activation Assay

    5) Product Images from "Diptoindonesin G promotes ERK-mediated nuclear translocation of p-STAT1 (Ser727) and cell differentiation in AML cells"

    Article Title: Diptoindonesin G promotes ERK-mediated nuclear translocation of p-STAT1 (Ser727) and cell differentiation in AML cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.159

    Nuclear translocation of phospho-STAT1 (Ser727) promoted by Dip G. ( a and b ). HL-60 cells were treated with the indicated compounds for 24 h or with Dip G (7.5 μ M) for the indicated times. ( a ) The protein levels of p-STAT1 (S727 and Y701) and STAT1 were analyzed in the whole-cell lysate using western blotting. β -Actin or tubulin was used as a loading control. ( b ) Western blotting analysis for the indicated protein levels in the cytoplasm and nucleus. β -Actin or histone 3 was used as a loading control. ( c ) HL-60 cells were treated with Dip G (7.5 μ M) for 3 or 6 h. The subcellular location of p-STAT1 (S727) and p-STAT1 (Y701) was detected using confocal microscopy. The nuclei are stained with the DNA-binding dye DAPI (4,6-diamidino-2-phenylindole; blue). Scale bar, 10 μ m. The lower panel represents the mean fluorescence intensity (MFI), which is presented in arbitrary units (AU), and the distance from α to ω in the images. ( d and e ). STAT1-WT or STAT1 mutants were overexpressed in HeLa cells. ( d ) Twenty-four hours after transfection, the resulting cells were treated with Dip G (7.5 μ M) for 6 h. Representative photomicrograghic images show the translocation of exogenous STAT1. Scale bar, 10 μ m. ( e ) The resulting cells were treated with Dip G (7.5 μ M) for an additional 6 h. The protein levels of exogenous STAT1 in the cytoplasm and nucleus were analyzed using western blotting. ( f ) STAT1-WT or STAT1 mutants were overexpressed in HL-60 cells using electroporation. The resulting cells were treated with Dip G (7.5 μ M) for 72 h. CD11b expression on gated green-fluorescent protein (GFP)-positive cells was detected using flow cytometry. Data are shown as the mean±S.E.M. of three independent experiments. * P
    Figure Legend Snippet: Nuclear translocation of phospho-STAT1 (Ser727) promoted by Dip G. ( a and b ). HL-60 cells were treated with the indicated compounds for 24 h or with Dip G (7.5 μ M) for the indicated times. ( a ) The protein levels of p-STAT1 (S727 and Y701) and STAT1 were analyzed in the whole-cell lysate using western blotting. β -Actin or tubulin was used as a loading control. ( b ) Western blotting analysis for the indicated protein levels in the cytoplasm and nucleus. β -Actin or histone 3 was used as a loading control. ( c ) HL-60 cells were treated with Dip G (7.5 μ M) for 3 or 6 h. The subcellular location of p-STAT1 (S727) and p-STAT1 (Y701) was detected using confocal microscopy. The nuclei are stained with the DNA-binding dye DAPI (4,6-diamidino-2-phenylindole; blue). Scale bar, 10 μ m. The lower panel represents the mean fluorescence intensity (MFI), which is presented in arbitrary units (AU), and the distance from α to ω in the images. ( d and e ). STAT1-WT or STAT1 mutants were overexpressed in HeLa cells. ( d ) Twenty-four hours after transfection, the resulting cells were treated with Dip G (7.5 μ M) for 6 h. Representative photomicrograghic images show the translocation of exogenous STAT1. Scale bar, 10 μ m. ( e ) The resulting cells were treated with Dip G (7.5 μ M) for an additional 6 h. The protein levels of exogenous STAT1 in the cytoplasm and nucleus were analyzed using western blotting. ( f ) STAT1-WT or STAT1 mutants were overexpressed in HL-60 cells using electroporation. The resulting cells were treated with Dip G (7.5 μ M) for 72 h. CD11b expression on gated green-fluorescent protein (GFP)-positive cells was detected using flow cytometry. Data are shown as the mean±S.E.M. of three independent experiments. * P

    Techniques Used: Translocation Assay, Western Blot, Confocal Microscopy, Staining, Binding Assay, Fluorescence, Transfection, Electroporation, Expressing, Flow Cytometry, Cytometry

    Nuclear translocation of p-STAT1 (Ser727) driven by extracellular signal–regulated kinase (ERK) activation. ( a ) HL-60 cells were treated with the indicated compounds for 24 h or with Dip G (7.5 μ M) for the indicated times. Mitogen-activated protein kinase signaling in the whole-cell lysates were analyzed using western blotting. Tubulin was used as a loading control. ( b ) HL-60 cells were treated with Dip G (7.5 μ M) in the absence or presence of U0126 (1 μ M) or SP610025 (10 μ M) for 72 h. CD11b expression was detected using flow cytometry. Right panel: the percentage of cells expressing CD11b. Data are shown as the mean±S.E.M. of three independent experiments. * P
    Figure Legend Snippet: Nuclear translocation of p-STAT1 (Ser727) driven by extracellular signal–regulated kinase (ERK) activation. ( a ) HL-60 cells were treated with the indicated compounds for 24 h or with Dip G (7.5 μ M) for the indicated times. Mitogen-activated protein kinase signaling in the whole-cell lysates were analyzed using western blotting. Tubulin was used as a loading control. ( b ) HL-60 cells were treated with Dip G (7.5 μ M) in the absence or presence of U0126 (1 μ M) or SP610025 (10 μ M) for 72 h. CD11b expression was detected using flow cytometry. Right panel: the percentage of cells expressing CD11b. Data are shown as the mean±S.E.M. of three independent experiments. * P

    Techniques Used: Translocation Assay, Activation Assay, Western Blot, Expressing, Flow Cytometry, Cytometry

    Induction of STAT1 activation by Dip G in AML cells. ( a ) Heat map of differentiation-related genes modulated by Dip G. HL-60 cells were treated for 24 h in the absence or presence of Dip G (15 μ M). The mRNA expression was detected using an Agilent Human Gene Expression Array. Eighteen upregulated genes and three downregulated genes were used to generate the heat map. Red gene symbols indicate myeloid differentiation markers. ( b ) The results of the microarray were verified using quantitative real-time reverse transcriptase-PCR (RT-PCR) analysis. The samples used for validation were the same samples as those that were used for the microarray. ( c ) HL-60 cells were treated with Dip G (7.5 μ M) for the indicated times and ( d ) primary AML cells were treated with various concentrations of Dip G or ATRA (1 μ M) for 24 h. The mRNA expression of STAT1, IFIT3 and C-X-C motif chemokine ligand 10 (CXCL10) was detected using quantitative real-time RT-PCR analysis. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. Data are shown as the mean±S.E.M. of three independent experiments. * P
    Figure Legend Snippet: Induction of STAT1 activation by Dip G in AML cells. ( a ) Heat map of differentiation-related genes modulated by Dip G. HL-60 cells were treated for 24 h in the absence or presence of Dip G (15 μ M). The mRNA expression was detected using an Agilent Human Gene Expression Array. Eighteen upregulated genes and three downregulated genes were used to generate the heat map. Red gene symbols indicate myeloid differentiation markers. ( b ) The results of the microarray were verified using quantitative real-time reverse transcriptase-PCR (RT-PCR) analysis. The samples used for validation were the same samples as those that were used for the microarray. ( c ) HL-60 cells were treated with Dip G (7.5 μ M) for the indicated times and ( d ) primary AML cells were treated with various concentrations of Dip G or ATRA (1 μ M) for 24 h. The mRNA expression of STAT1, IFIT3 and C-X-C motif chemokine ligand 10 (CXCL10) was detected using quantitative real-time RT-PCR analysis. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. Data are shown as the mean±S.E.M. of three independent experiments. * P

    Techniques Used: Activation Assay, Expressing, Microarray, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    In vivo therapeutic efficacy of Dip G. HL-60 cells were injected subcutaneously into the right flank of NOD/SCID mice. Two weeks later, tumor-bearing mice were distributed into four groups and treated with various doses of Dip G or ATRA intraperitoneally daily for an additional 12 days. ( a ) Tumor volumes were monitored and recorded every 2 days ( n =8–10 mice per group). Left panel: Representative images of the tumors. ( b ) Tumors excised on day 13 were weighed. Tumors excised on day 13 were stained with an antibody specific for ( c ) Ki67 and ( d ) CD11b. Scale bar, 100 μ m. ( e ) The mRNA expression of STAT1, IFIT3 and C-X-C motif chemokine ligand 10 (CXCL10) was detected in tumors excised on day 13 using quantitative real-time reverse transcriptase-CR analysis. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. Data are shown as the mean±S.E.M. of three independent experiments. * P
    Figure Legend Snippet: In vivo therapeutic efficacy of Dip G. HL-60 cells were injected subcutaneously into the right flank of NOD/SCID mice. Two weeks later, tumor-bearing mice were distributed into four groups and treated with various doses of Dip G or ATRA intraperitoneally daily for an additional 12 days. ( a ) Tumor volumes were monitored and recorded every 2 days ( n =8–10 mice per group). Left panel: Representative images of the tumors. ( b ) Tumors excised on day 13 were weighed. Tumors excised on day 13 were stained with an antibody specific for ( c ) Ki67 and ( d ) CD11b. Scale bar, 100 μ m. ( e ) The mRNA expression of STAT1, IFIT3 and C-X-C motif chemokine ligand 10 (CXCL10) was detected in tumors excised on day 13 using quantitative real-time reverse transcriptase-CR analysis. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. Data are shown as the mean±S.E.M. of three independent experiments. * P

    Techniques Used: In Vivo, Injection, Mouse Assay, Staining, Expressing

    6) Product Images from "Autocrine production of IL-11 mediates tumorigenicity in hypoxic cancer cells"

    Article Title: Autocrine production of IL-11 mediates tumorigenicity in hypoxic cancer cells

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI59623

    STAT1 and p38 mediate IL-11–dependent responses under hypoxia.
    Figure Legend Snippet: STAT1 and p38 mediate IL-11–dependent responses under hypoxia.

    Techniques Used:

    7) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    8) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    9) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    10) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    11) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    12) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    13) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    14) Product Images from "Promyelocytic Leukemia Protein (PML) Regulates Endothelial Cell Network Formation and Migration in Response to Tumor Necrosis Factor ? (TNF?) and Interferon ? (IFN?) *"

    Article Title: Promyelocytic Leukemia Protein (PML) Regulates Endothelial Cell Network Formation and Migration in Response to Tumor Necrosis Factor ? (TNF?) and Interferon ? (IFN?) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.340505

    STAT1 participates in the TNFα- and IFNα-induced PML expression and inhibition of EC network formation in HUVECs. A , effects of STAT1 knockdown on PML mRNA levels analyzed by qRT-PCR. B , effects of TNFα and IFNα on endogenous
    Figure Legend Snippet: STAT1 participates in the TNFα- and IFNα-induced PML expression and inhibition of EC network formation in HUVECs. A , effects of STAT1 knockdown on PML mRNA levels analyzed by qRT-PCR. B , effects of TNFα and IFNα on endogenous

    Techniques Used: Expressing, Inhibition, Quantitative RT-PCR

    PML and STAT1 are required for TNFα and IFNα to inhibit integrin β1 (ITGB1) expression. A and B , effects of knockdown of STAT1 ( A ) or PML ( B ) on TNFα- and IFNα-mediated reduction of ITGB1 expression assayed by Western
    Figure Legend Snippet: PML and STAT1 are required for TNFα and IFNα to inhibit integrin β1 (ITGB1) expression. A and B , effects of knockdown of STAT1 ( A ) or PML ( B ) on TNFα- and IFNα-mediated reduction of ITGB1 expression assayed by Western

    Techniques Used: Expressing, Western Blot

    15) Product Images from "TWEAK blockade decreases atherosclerotic lesion size and progression through suppression of STAT1 signaling in diabetic mice"

    Article Title: TWEAK blockade decreases atherosclerotic lesion size and progression through suppression of STAT1 signaling in diabetic mice

    Journal: Scientific Reports

    doi: 10.1038/srep46679

    TWEAK activates STAT1 and induces pro-inflammatory chemokine expression in diabetic mice. ( A ) Representative immunostaining and ( B ) quantification of p-STAT1, p-STAT3 and activated NF-κB in aortic lesions from Tnfsf12 +/+ ApoE −/− and Tnfsf12 −/− ApoE −/− diabetic mice. Values shown are mean ± SD of Tnfsf12 +/+ ApoE −/− (N = 12) and Tnfsf12 −/− ApoE −/− (N = 12) mice. Scale bar, 200 μm. *p
    Figure Legend Snippet: TWEAK activates STAT1 and induces pro-inflammatory chemokine expression in diabetic mice. ( A ) Representative immunostaining and ( B ) quantification of p-STAT1, p-STAT3 and activated NF-κB in aortic lesions from Tnfsf12 +/+ ApoE −/− and Tnfsf12 −/− ApoE −/− diabetic mice. Values shown are mean ± SD of Tnfsf12 +/+ ApoE −/− (N = 12) and Tnfsf12 −/− ApoE −/− (N = 12) mice. Scale bar, 200 μm. *p

    Techniques Used: Expressing, Mouse Assay, Immunostaining

    TWEAK activates STAT1 and induces pro-inflammatory chemokine expression in VSMCs. ( A ) Representative western-blot analysis of pSTAT1 and pSTAT3 in murine VSMCs incubated with low (5.5 mmol/L) or high (35 mmol/L) glucose and rTWEAK (100 ng/mL) for 0–24 hours. Values normalized to tubulin expression are expressed as multiples of control conditions (arbitrarily set to 1). N = 4 independent experiments. Full-length blots are in Supplemental Figure S1 . ( B ) Representative western-blot analysis of pSTAT1 and pSTAT3 in murine VSMCs incubated with low (5.5 mmol/L) or high (35 mmol/L) glucose and rTWEAK (0, 10 or 100 ng/mL) for 24 hours. Values normalized to tubulin expression are expressed as multiples of control conditions (arbitrarily set to 1). N = 4 independent experiments. Full-length blots are in Supplemental Figure S2 . ( C ) Immunodetection of p-STAT1 and Fn14 in murine VSMCs under transfection transfected with either specific siRNA for Fn14 or universal negative control siRNA and stimulated with rTWEAK (100 ng/mL) for 24 hours. N = 4 independent experiments. Full-length blots are in Supplemental Figure S3 . ( D ) Representative western-blot analysis of pSTAT1 in murine VSMCs incubated with low (5.5 mmol/L) or high (35 mmol/L) glucose and rTWEAK (100 ng/mL) for 24 hours in presence or absence of JAK inhibitor (250 nM). N = 4 independent experiments. Full-length blots are in Supplemental Figure S4 . ( E ) Quantitative real time PCR analysis of CCL5, CXCL10, ICAM-1, VCAM-1 and TNF-α mRNA expression in murine VSMCs stimulated with TWEAK, in the presence of low or high glucose concentrations. Values shown are mean ± SD. N = 6 experiments *p
    Figure Legend Snippet: TWEAK activates STAT1 and induces pro-inflammatory chemokine expression in VSMCs. ( A ) Representative western-blot analysis of pSTAT1 and pSTAT3 in murine VSMCs incubated with low (5.5 mmol/L) or high (35 mmol/L) glucose and rTWEAK (100 ng/mL) for 0–24 hours. Values normalized to tubulin expression are expressed as multiples of control conditions (arbitrarily set to 1). N = 4 independent experiments. Full-length blots are in Supplemental Figure S1 . ( B ) Representative western-blot analysis of pSTAT1 and pSTAT3 in murine VSMCs incubated with low (5.5 mmol/L) or high (35 mmol/L) glucose and rTWEAK (0, 10 or 100 ng/mL) for 24 hours. Values normalized to tubulin expression are expressed as multiples of control conditions (arbitrarily set to 1). N = 4 independent experiments. Full-length blots are in Supplemental Figure S2 . ( C ) Immunodetection of p-STAT1 and Fn14 in murine VSMCs under transfection transfected with either specific siRNA for Fn14 or universal negative control siRNA and stimulated with rTWEAK (100 ng/mL) for 24 hours. N = 4 independent experiments. Full-length blots are in Supplemental Figure S3 . ( D ) Representative western-blot analysis of pSTAT1 in murine VSMCs incubated with low (5.5 mmol/L) or high (35 mmol/L) glucose and rTWEAK (100 ng/mL) for 24 hours in presence or absence of JAK inhibitor (250 nM). N = 4 independent experiments. Full-length blots are in Supplemental Figure S4 . ( E ) Quantitative real time PCR analysis of CCL5, CXCL10, ICAM-1, VCAM-1 and TNF-α mRNA expression in murine VSMCs stimulated with TWEAK, in the presence of low or high glucose concentrations. Values shown are mean ± SD. N = 6 experiments *p

    Techniques Used: Expressing, Western Blot, Incubation, Immunodetection, Transfection, Negative Control, Real-time Polymerase Chain Reaction

    Anti-TWEAK mAb treatment diminished plaque progression and STAT1 activation in diabetic mice. ( A ) Representative images (bright field and polarized light) of Sirius Red staining and ( B ) quantification of Sirius red and Oil-Red-O in the aortic root of diabetic mice treated with anti-TWEAK or IgG treated (N = 13 in each group) mice. Values shown are mean ± SD. *p
    Figure Legend Snippet: Anti-TWEAK mAb treatment diminished plaque progression and STAT1 activation in diabetic mice. ( A ) Representative images (bright field and polarized light) of Sirius Red staining and ( B ) quantification of Sirius red and Oil-Red-O in the aortic root of diabetic mice treated with anti-TWEAK or IgG treated (N = 13 in each group) mice. Values shown are mean ± SD. *p

    Techniques Used: Activation Assay, Mouse Assay, Staining

    16) Product Images from "Activated tyrosine kinases in gastrointestinal stromal tumor with loss of KIT oncoprotein expression"

    Article Title: Activated tyrosine kinases in gastrointestinal stromal tumor with loss of KIT oncoprotein expression

    Journal: Cell Cycle

    doi: 10.1080/15384101.2018.1553335

    (a) Immunoblotting evaluations of KIT-negative GIST cell lines (GIST62 and GIST522) and KIT-positive GIST cell line (GIST882) at 96 hours after infection by lentiviral AXL, EPHA2 , and FAK shRNA constructs. Immunoblots demonstrate the effects of AXL, EPHA2 and FAK knockdown on signaling intermediates (AKT, MAPK p42/44, S6, STAT1, and STAT3), proliferation markers (Cyclin A and PCNA), and cell cycle checkpoint proteins (p27, p21, and p53). Control lanes for each cell line include uninfected cells (untreated lane) and cells infected with empty lentiviral vector. Actin is a loading control. (b) Immunoblotting evaluations of GIST522 and GIST882 cells at 10 days after infection by lentiviral AXL, EPHA2 , and FAK shRNA constructs. AXL shRNA resulted in decreased expression of cyclin A and PCNA, and upregulation of p53, p21, and p27 in GIST522, but not in GIST882. Actin is a loading control. (c) GIST62 and GIST522 cell cultures, evaluated at 6 days after infection by lentiviral AXL shRNA constructs, showing growth inhibition as compared to control cultures infected with empty lentiviral constructs. Scale bars: 100 μm. (d) Cell viability was evaluated in GIST62 (black bars), GIST522 (white bars), and GIST430 (gray bars), at day 8 and day 12 after infection with lentiviral AXL, EPHA2 , or FAK shRNA. Viability was analyzed using the Cell-titer Glo® ATP-based luminescence assay. The data were normalized to empty lentiviral infections, and represent the mean values (± s.d.) from quadruplicate cultures. The experiments were performed in triplicate, and statistically significant differences between vector control and shRNAs are defined as * p
    Figure Legend Snippet: (a) Immunoblotting evaluations of KIT-negative GIST cell lines (GIST62 and GIST522) and KIT-positive GIST cell line (GIST882) at 96 hours after infection by lentiviral AXL, EPHA2 , and FAK shRNA constructs. Immunoblots demonstrate the effects of AXL, EPHA2 and FAK knockdown on signaling intermediates (AKT, MAPK p42/44, S6, STAT1, and STAT3), proliferation markers (Cyclin A and PCNA), and cell cycle checkpoint proteins (p27, p21, and p53). Control lanes for each cell line include uninfected cells (untreated lane) and cells infected with empty lentiviral vector. Actin is a loading control. (b) Immunoblotting evaluations of GIST522 and GIST882 cells at 10 days after infection by lentiviral AXL, EPHA2 , and FAK shRNA constructs. AXL shRNA resulted in decreased expression of cyclin A and PCNA, and upregulation of p53, p21, and p27 in GIST522, but not in GIST882. Actin is a loading control. (c) GIST62 and GIST522 cell cultures, evaluated at 6 days after infection by lentiviral AXL shRNA constructs, showing growth inhibition as compared to control cultures infected with empty lentiviral constructs. Scale bars: 100 μm. (d) Cell viability was evaluated in GIST62 (black bars), GIST522 (white bars), and GIST430 (gray bars), at day 8 and day 12 after infection with lentiviral AXL, EPHA2 , or FAK shRNA. Viability was analyzed using the Cell-titer Glo® ATP-based luminescence assay. The data were normalized to empty lentiviral infections, and represent the mean values (± s.d.) from quadruplicate cultures. The experiments were performed in triplicate, and statistically significant differences between vector control and shRNAs are defined as * p

    Techniques Used: Infection, shRNA, Construct, Western Blot, Plasmid Preparation, Expressing, Inhibition, Luminescence Assay

    17) Product Images from "Autoimmune Regulator Deficiency Results in a Decrease in STAT1 Levels in Human Monocytes"

    Article Title: Autoimmune Regulator Deficiency Results in a Decrease in STAT1 Levels in Human Monocytes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00820

    The decrease in STAT1 protein and pSTAT1 levels seen in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patients correlates with lower monocyte interferon (IFN)-γR2 receptor expression and the presence of type I IFN autoantibodies. (A) Sera from 8 APECED patients and 100 healthy donors were evaluated for the presence of autoantibodies against IFN-γ, IFN-α, and IFN-ω. Shown is autoantibody immunoreactivity against the indicated cytokines expressed as fluorescence intensity using a particle-based multiplex assay. (B) IFN-γ receptor 1 and 2 levels were measured in CD14 + monocytes of healthy donors ( n = 8) and APECED patients ( n = 8). Summary data on mean fluorescence intensity and representative histogram FACS plots are shown. ** p
    Figure Legend Snippet: The decrease in STAT1 protein and pSTAT1 levels seen in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patients correlates with lower monocyte interferon (IFN)-γR2 receptor expression and the presence of type I IFN autoantibodies. (A) Sera from 8 APECED patients and 100 healthy donors were evaluated for the presence of autoantibodies against IFN-γ, IFN-α, and IFN-ω. Shown is autoantibody immunoreactivity against the indicated cytokines expressed as fluorescence intensity using a particle-based multiplex assay. (B) IFN-γ receptor 1 and 2 levels were measured in CD14 + monocytes of healthy donors ( n = 8) and APECED patients ( n = 8). Summary data on mean fluorescence intensity and representative histogram FACS plots are shown. ** p

    Techniques Used: Expressing, Fluorescence, Multiplex Assay, FACS

    Autoimmune regulator deficiency results in a decrease in STAT1 protein levels in human monocytes. (A) Representative depiction of pSTAT1 level at rest and up to 30 min after interferon (IFN)-γ stimulation in CD14 + cells of a STAT1 gain-of-function (GOF) patient (orange), an autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patient (red), a patient with the autosomal dominant form of IFN-γR1 deficiency (black) and a healthy donor (blue) (B) Representative depiction of STAT1 protein level at rest and up to 30 min after IFN-γ stimulation in CD14 + cells of a STAT1 GOF patient (orange), an APECED patient (red), and a healthy donor (blue). Protein and phosphorylation levels are expressed in geometric mean of fluorescence (Geo. Mean), as measured by flow cytometry. STAT1 total protein (C) and pSTAT1 (D) levels in CD14 + cells of APECED patients ( n = 8; red dots) and healthy donors ( n = 13; blue dots) at rest (time 0) and up to 60 min after IFN-γ stimulation. Total protein and phosphorylation levels are expressed in % of the same-day control average values, for each time point—0, 15, 30, and 60 min, separately. (E) Area under the curve of CD14 + cells STAT1 phosphorylation vs. time in APECED patients ( n = 8; red dots) and healthy donors ( n = 13; blue dots). (F) STAT1 mRNA level, relative to glyceraldehyde-3-phosphate dehydrogenase, in peripheral blood mononuclear cells of healthy donors ( n = 10) and APECED patients ( n = 8) at rest. ns, not significant. * p
    Figure Legend Snippet: Autoimmune regulator deficiency results in a decrease in STAT1 protein levels in human monocytes. (A) Representative depiction of pSTAT1 level at rest and up to 30 min after interferon (IFN)-γ stimulation in CD14 + cells of a STAT1 gain-of-function (GOF) patient (orange), an autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patient (red), a patient with the autosomal dominant form of IFN-γR1 deficiency (black) and a healthy donor (blue) (B) Representative depiction of STAT1 protein level at rest and up to 30 min after IFN-γ stimulation in CD14 + cells of a STAT1 GOF patient (orange), an APECED patient (red), and a healthy donor (blue). Protein and phosphorylation levels are expressed in geometric mean of fluorescence (Geo. Mean), as measured by flow cytometry. STAT1 total protein (C) and pSTAT1 (D) levels in CD14 + cells of APECED patients ( n = 8; red dots) and healthy donors ( n = 13; blue dots) at rest (time 0) and up to 60 min after IFN-γ stimulation. Total protein and phosphorylation levels are expressed in % of the same-day control average values, for each time point—0, 15, 30, and 60 min, separately. (E) Area under the curve of CD14 + cells STAT1 phosphorylation vs. time in APECED patients ( n = 8; red dots) and healthy donors ( n = 13; blue dots). (F) STAT1 mRNA level, relative to glyceraldehyde-3-phosphate dehydrogenase, in peripheral blood mononuclear cells of healthy donors ( n = 10) and APECED patients ( n = 8) at rest. ns, not significant. * p

    Techniques Used: Fluorescence, Flow Cytometry, Cytometry

    18) Product Images from "Selective blockade of the inhibitory Fc? receptor (Fc?RIIB) in human dendritic cells and monocytes induces a type I interferon response program"

    Article Title: Selective blockade of the inhibitory Fc? receptor (Fc?RIIB) in human dendritic cells and monocytes induces a type I interferon response program

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20062545

    Role of activating FcγRs in the FcR-mediated DC maturation and induction of P-STAT1. (A) Expression of FcγRI, RIIA, RIIB, and RIIIA on DCs generated from CD14+ monocytes by flow cytometry. Day 5 monocyte-derived IDCs were examined for the expression of FcγR1, FcγRIIA, FcγRIIB, and FcγRIII by flow cytometry. The area in gray represents staining with isotype control antibodies. Figure represents one of six similar experiments. (B) Down-regulation of anti-FcγRIIB antibody induced DC maturation by blocking antibodies against activating FcγR. IDCs ( n = 4) were treated with isotype control antibody or anti-FcγRIIB antibody either with blocking antibodies against CD32A and CD16 antibody (αCD32A + αCD16; clone IV.3 and 3G8, both at 10 μg/ml) or their isotype control antibodies (Isotype; mouse IgG2a and IgG1, respectively). 24 h later, the expression of CD80 and CD83 was monitored by flow cytometry, and the double-positive cells were used to assess DC maturation. Change in maturation between isotype-treated and anti-FcγRIIB–treated DCs was considered as 100%. The figure shows the percentage of decrease in maturation of DCs treated with blocking antibodies against the activating FcγR (CD32A and CD16). Data are a summary of four similar experiments. *, P
    Figure Legend Snippet: Role of activating FcγRs in the FcR-mediated DC maturation and induction of P-STAT1. (A) Expression of FcγRI, RIIA, RIIB, and RIIIA on DCs generated from CD14+ monocytes by flow cytometry. Day 5 monocyte-derived IDCs were examined for the expression of FcγR1, FcγRIIA, FcγRIIB, and FcγRIII by flow cytometry. The area in gray represents staining with isotype control antibodies. Figure represents one of six similar experiments. (B) Down-regulation of anti-FcγRIIB antibody induced DC maturation by blocking antibodies against activating FcγR. IDCs ( n = 4) were treated with isotype control antibody or anti-FcγRIIB antibody either with blocking antibodies against CD32A and CD16 antibody (αCD32A + αCD16; clone IV.3 and 3G8, both at 10 μg/ml) or their isotype control antibodies (Isotype; mouse IgG2a and IgG1, respectively). 24 h later, the expression of CD80 and CD83 was monitored by flow cytometry, and the double-positive cells were used to assess DC maturation. Change in maturation between isotype-treated and anti-FcγRIIB–treated DCs was considered as 100%. The figure shows the percentage of decrease in maturation of DCs treated with blocking antibodies against the activating FcγR (CD32A and CD16). Data are a summary of four similar experiments. *, P

    Techniques Used: Expressing, Generated, Flow Cytometry, Cytometry, Derivative Assay, Staining, Blocking Assay

    Mechanism of FcγR-mediated induction of IFN response. (A) Up-regulation of P-STAT1 in DCs or monocytes treated with anti-FcγRIIB mAb versus isotype control. Immature Mo-DCs ( n = 8) or freshly isolated monocytes ( n = 7) were treated with anti-FcγRIIB antibody (RIIB) or isotype control antibody (Iso). 24 h later, P-STAT1 expression was examined by flow cytometry. The histogram shows fold change in expression of P-STAT1 in anti-FcγRIIB antibody–treated versus isotype control antibody–treated cells. *, P
    Figure Legend Snippet: Mechanism of FcγR-mediated induction of IFN response. (A) Up-regulation of P-STAT1 in DCs or monocytes treated with anti-FcγRIIB mAb versus isotype control. Immature Mo-DCs ( n = 8) or freshly isolated monocytes ( n = 7) were treated with anti-FcγRIIB antibody (RIIB) or isotype control antibody (Iso). 24 h later, P-STAT1 expression was examined by flow cytometry. The histogram shows fold change in expression of P-STAT1 in anti-FcγRIIB antibody–treated versus isotype control antibody–treated cells. *, P

    Techniques Used: Isolation, Expressing, Flow Cytometry, Cytometry

    Induction of IFN response by anti-FcγRIIB antibody is not inhibited by blocking antibodies against IFN-α and IFN-γ. (A) Up-regulation of P-STAT1 by exogenous IFN-α is blocked by antibodies against IFN-α. Monocyte-derived IDCs were either left untreated (DC−) or treated with IFN-α2b (1,000 U/ml intron A). The DCs were either treated with a combination of blocking antibodies against IFN-α, as well as IFNAR (αIFNAR; both at 10 μg/ml) or with their isotype control antibodies (Isotype; mouse IgG1 and mouse IgG2a, respectively; both at 10 μg/ml) for 45 min before treatment with IFN-α. The DCs were analyzed for their expression of P-STAT1 by flow cytometry. Gray area of the histogram shows staining of the DCs with isotype control (Mouse IgG2a) for P-STAT1 antibody. The graph represents one of two similar experiments. (B) Effect of blocking antibodies against IFN-α on anti-FcγRIIB–mediated induction of P-STAT1. Monocyte-derived IDCs were treated with anti-FcγRIIB antibody (5 μg/ml RIIB) or mouse IgG1 isotype control antibody (Iso). DCs treated with anti-FcγRIIB antibody were either treated with blocking antibodies against IFN-α and IFNAR (10 μg/ml RIIB + αIFNa + αIFNAR) or isotype control antibody (RIIB + Isotype; 10 μg/ml mouse IgG2a and 10 μg/ml IgG1, respectively) for 45 min before the addition of anti-FcγRIIB antibody. 24 h later, flow cytometry was performed to examine the expression of P-STAT1. Some DCs were also stained with mouse IgG2a, which is isotype control for the P-STAT1 antibody. One of two similar experiments. (C) Effect of anti-IFNAR blocking antibody on anti-FcγRIIB–mediated induction of P-STAT1. Freshly isolated PBMCs were treated with IFN-α2b (1,000 U/ml intron A) or anti-FcγRIIB antibody and an isotype control antibody either in the presence of 20 μg/ml IFNAR antibody (IFNAR Ab) or isotype control antibody (mouse IgG2a). 1 h later, flow cytometry was performed to examine the expression of P-STAT1 on CD14+ monocytes. Data shown are the summary of three similar experiments. *, P
    Figure Legend Snippet: Induction of IFN response by anti-FcγRIIB antibody is not inhibited by blocking antibodies against IFN-α and IFN-γ. (A) Up-regulation of P-STAT1 by exogenous IFN-α is blocked by antibodies against IFN-α. Monocyte-derived IDCs were either left untreated (DC−) or treated with IFN-α2b (1,000 U/ml intron A). The DCs were either treated with a combination of blocking antibodies against IFN-α, as well as IFNAR (αIFNAR; both at 10 μg/ml) or with their isotype control antibodies (Isotype; mouse IgG1 and mouse IgG2a, respectively; both at 10 μg/ml) for 45 min before treatment with IFN-α. The DCs were analyzed for their expression of P-STAT1 by flow cytometry. Gray area of the histogram shows staining of the DCs with isotype control (Mouse IgG2a) for P-STAT1 antibody. The graph represents one of two similar experiments. (B) Effect of blocking antibodies against IFN-α on anti-FcγRIIB–mediated induction of P-STAT1. Monocyte-derived IDCs were treated with anti-FcγRIIB antibody (5 μg/ml RIIB) or mouse IgG1 isotype control antibody (Iso). DCs treated with anti-FcγRIIB antibody were either treated with blocking antibodies against IFN-α and IFNAR (10 μg/ml RIIB + αIFNa + αIFNAR) or isotype control antibody (RIIB + Isotype; 10 μg/ml mouse IgG2a and 10 μg/ml IgG1, respectively) for 45 min before the addition of anti-FcγRIIB antibody. 24 h later, flow cytometry was performed to examine the expression of P-STAT1. Some DCs were also stained with mouse IgG2a, which is isotype control for the P-STAT1 antibody. One of two similar experiments. (C) Effect of anti-IFNAR blocking antibody on anti-FcγRIIB–mediated induction of P-STAT1. Freshly isolated PBMCs were treated with IFN-α2b (1,000 U/ml intron A) or anti-FcγRIIB antibody and an isotype control antibody either in the presence of 20 μg/ml IFNAR antibody (IFNAR Ab) or isotype control antibody (mouse IgG2a). 1 h later, flow cytometry was performed to examine the expression of P-STAT1 on CD14+ monocytes. Data shown are the summary of three similar experiments. *, P

    Techniques Used: Blocking Assay, Derivative Assay, Expressing, Flow Cytometry, Cytometry, Staining, Isolation

    FcγR-mediated DC maturation can be inhibited by STAT1 knockdown. (A) Day 4 IDCs were electroporated with 10 μg of STAT1 siRNA (STAT1) or nontargeting siRNA (Control). Some DCs were cultured without electroporation (No electr). The DCs were harvested 48 and 72 h after electroporation, and a Western blot was performed to detect total STAT1 protein. Data are representative of two separate experiments. (B and C) DCs were harvested 72 h after electroporation with either STAT1 siRNA or nontargeting control siRNA. Some nonelectroporated DCs were also harvested. The DCs were treated with anti-FcγRIIB antibody or isotype control antibody. Some of the DCs electroporated with STAT1 siRNA were also treated with the inflammatory cytokine cocktail. 24 h later, DC maturation was determined by examining the expression of CD80, CD83, and CD11c by flow cytometry. (B) Summary of data in four independent experiments (mean ± the SD; *, P
    Figure Legend Snippet: FcγR-mediated DC maturation can be inhibited by STAT1 knockdown. (A) Day 4 IDCs were electroporated with 10 μg of STAT1 siRNA (STAT1) or nontargeting siRNA (Control). Some DCs were cultured without electroporation (No electr). The DCs were harvested 48 and 72 h after electroporation, and a Western blot was performed to detect total STAT1 protein. Data are representative of two separate experiments. (B and C) DCs were harvested 72 h after electroporation with either STAT1 siRNA or nontargeting control siRNA. Some nonelectroporated DCs were also harvested. The DCs were treated with anti-FcγRIIB antibody or isotype control antibody. Some of the DCs electroporated with STAT1 siRNA were also treated with the inflammatory cytokine cocktail. 24 h later, DC maturation was determined by examining the expression of CD80, CD83, and CD11c by flow cytometry. (B) Summary of data in four independent experiments (mean ± the SD; *, P

    Techniques Used: Cell Culture, Electroporation, Western Blot, Expressing, Flow Cytometry, Cytometry

    19) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    20) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    21) Product Images from "Critical role of the endogenous interferon ligand–receptors in type I and type II interferons response"

    Article Title: Critical role of the endogenous interferon ligand–receptors in type I and type II interferons response

    Journal: Immunology

    doi: 10.1111/imm.12273

    Modulation of interferon- α (IFN- α ) and IFN- β responses in haematopoietic cells. (a) EMSA analysis of signal transducer and activator of transcription 1 (STAT1) activation in splenocytes (SPs) isolated from wild-type, IFNG −/−
    Figure Legend Snippet: Modulation of interferon- α (IFN- α ) and IFN- β responses in haematopoietic cells. (a) EMSA analysis of signal transducer and activator of transcription 1 (STAT1) activation in splenocytes (SPs) isolated from wild-type, IFNG −/−

    Techniques Used: Activation Assay, Isolation

    22) Product Images from "IL-27 inhibits epithelial-mesenchymal transition and angiogenic factor production in a STAT1-dominant pathway in human non-small cell lung cancer"

    Article Title: IL-27 inhibits epithelial-mesenchymal transition and angiogenic factor production in a STAT1-dominant pathway in human non-small cell lung cancer

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-32-97

    Inhibition of in vitro cell migration dependent upon STAT1 activation. (A) A549 cells were treated with IL-27 (50 ng/mL) at 60 ~ 70% confluency for 24 hours and a scratch was created in the cell monolayer. The same fields were observed for cell migration using phase contrast microscopy after 24 hours of IL-27 treatment. (B) The scratch technique was utilized to measure cell migration for A549 cells that were transfected with STAT1 siRNA (40 nM) for 24 hours prior to with or without IL-27 (50 ng/mL) exposure. (C) The motility assay was employed to measure cell migration after Stattic (7.5 nM) pre-treatment for 1 hour prior to IL-27 exposure (50 ng/mL), and changes in cell migration were observed for 60 hours. Scale bar, 200 μm. (D and E) Cell migration evaluated using transwell chambers. A549 sells transfected with STAT siRNAs for 24 hrs, control siRNA-transfected or untreated cells (D) followed by 1 hour of Stattic treatment (E) were plated 24 h after treatment with IL-27 on 96-well transwell plates. After 48 hours, cells that migrated through the pores to the under surface of the membrane and bottom wells were labeled with Calcein-AM. Migration rate was calculated using fluorescence as described in Materials and Methods.
    Figure Legend Snippet: Inhibition of in vitro cell migration dependent upon STAT1 activation. (A) A549 cells were treated with IL-27 (50 ng/mL) at 60 ~ 70% confluency for 24 hours and a scratch was created in the cell monolayer. The same fields were observed for cell migration using phase contrast microscopy after 24 hours of IL-27 treatment. (B) The scratch technique was utilized to measure cell migration for A549 cells that were transfected with STAT1 siRNA (40 nM) for 24 hours prior to with or without IL-27 (50 ng/mL) exposure. (C) The motility assay was employed to measure cell migration after Stattic (7.5 nM) pre-treatment for 1 hour prior to IL-27 exposure (50 ng/mL), and changes in cell migration were observed for 60 hours. Scale bar, 200 μm. (D and E) Cell migration evaluated using transwell chambers. A549 sells transfected with STAT siRNAs for 24 hrs, control siRNA-transfected or untreated cells (D) followed by 1 hour of Stattic treatment (E) were plated 24 h after treatment with IL-27 on 96-well transwell plates. After 48 hours, cells that migrated through the pores to the under surface of the membrane and bottom wells were labeled with Calcein-AM. Migration rate was calculated using fluorescence as described in Materials and Methods.

    Techniques Used: Inhibition, In Vitro, Migration, Activation Assay, Microscopy, Transfection, Motility Assay, Labeling, Fluorescence

    JAK-dependent activation of STAT1 and STAT3 by IL-27 treatment. A549 cells were cultured in the presence of JAK inhibitor I (1-100 nM) for 1 hour prior to IL-27 (50 ng/mL) exposure for 24 hours. The activated and total amounts of STAT1 and STAT3 proteins were detected by Western blot. The densitometric measurements of total amounts of STAT1 and STAT3 were taken using Image J1.45o. The values above the figures represent relative density of the bands compared to control DMSO that was set to 1 after normalized to GAPDH.
    Figure Legend Snippet: JAK-dependent activation of STAT1 and STAT3 by IL-27 treatment. A549 cells were cultured in the presence of JAK inhibitor I (1-100 nM) for 1 hour prior to IL-27 (50 ng/mL) exposure for 24 hours. The activated and total amounts of STAT1 and STAT3 proteins were detected by Western blot. The densitometric measurements of total amounts of STAT1 and STAT3 were taken using Image J1.45o. The values above the figures represent relative density of the bands compared to control DMSO that was set to 1 after normalized to GAPDH.

    Techniques Used: Activation Assay, Cell Culture, Western Blot

    Acquisition of a more epithelial phenotype by inhibition of STAT1 expression in IL-27 treated cells. (A) A549 cells were transfected with a non-targeting control or STAT1 siRNAs (40 nM) for 6 hours prior to IL-27 (50 ng/mL) exposure for 15 or 30 minutes. Activated and total amounts of STAT1 and STAT3 proteins were detected by Western blot. GAPDH was used as a loading control. (B) Stattic (7.5 nM) or its diluent (DMSO) was added to A549 cells for 1 hour prior to IL-27 (50 ng/mL) exposure for 15 or 30 minutes. Activated and total amounts of STAT1 and STAT3 proteins were detected by Western blot. (C) After transfection with STAT1 siRNA (40 nM) for 6 hours or Stattic (7.5 nM) pre-treatment for 1 hour, A549 cells were exposed to IL-27 (50 ng/mL) for 24 hours. Morphologic changes were documented and photographed by phase contrast microscopy (50 × magnification). Scale bar, 100 μm.
    Figure Legend Snippet: Acquisition of a more epithelial phenotype by inhibition of STAT1 expression in IL-27 treated cells. (A) A549 cells were transfected with a non-targeting control or STAT1 siRNAs (40 nM) for 6 hours prior to IL-27 (50 ng/mL) exposure for 15 or 30 minutes. Activated and total amounts of STAT1 and STAT3 proteins were detected by Western blot. GAPDH was used as a loading control. (B) Stattic (7.5 nM) or its diluent (DMSO) was added to A549 cells for 1 hour prior to IL-27 (50 ng/mL) exposure for 15 or 30 minutes. Activated and total amounts of STAT1 and STAT3 proteins were detected by Western blot. (C) After transfection with STAT1 siRNA (40 nM) for 6 hours or Stattic (7.5 nM) pre-treatment for 1 hour, A549 cells were exposed to IL-27 (50 ng/mL) for 24 hours. Morphologic changes were documented and photographed by phase contrast microscopy (50 × magnification). Scale bar, 100 μm.

    Techniques Used: Inhibition, Expressing, Transfection, Western Blot, Microscopy

    Increased expression of epithelial and decreased expression of mesenchymal markers by a dominant STAT1 pathway. After transfection with STAT1 siRNA (40 nM) for 6 hours or Stattic (7.5 nM) pre-treatment for 1 hour, A549 cells were exposed to IL-27 (50 ng/mL) for 24 hours. Proteins responsible for the epithelial phenotype (E-cadherin and γ-catenin) and the mesenchymal phenotype (N-cadherin and vimentin) were detected by Western blot. Changes in Snail levels were also demonstrated by Western blot. Activated and total amounts of STAT1 and STAT3 were also detected, and GAPDH was used as a loading control. Densitometric measurements of the bands were taken using Image J1.45o. The values above the figures represent relative density of the bands normalized to GAPDH.
    Figure Legend Snippet: Increased expression of epithelial and decreased expression of mesenchymal markers by a dominant STAT1 pathway. After transfection with STAT1 siRNA (40 nM) for 6 hours or Stattic (7.5 nM) pre-treatment for 1 hour, A549 cells were exposed to IL-27 (50 ng/mL) for 24 hours. Proteins responsible for the epithelial phenotype (E-cadherin and γ-catenin) and the mesenchymal phenotype (N-cadherin and vimentin) were detected by Western blot. Changes in Snail levels were also demonstrated by Western blot. Activated and total amounts of STAT1 and STAT3 were also detected, and GAPDH was used as a loading control. Densitometric measurements of the bands were taken using Image J1.45o. The values above the figures represent relative density of the bands normalized to GAPDH.

    Techniques Used: Expressing, Transfection, Western Blot

    Down-regulation of angiogenic factors and up-regulation of angiostatic factors by STAT1-dependent pathway. (A-F) Protein concentrations of VEGF (A, B) , IL-8/CXCL8 (C, D) , CXCL5 (E, F) secreted by A549 cells were measured by ELISA. A549 cells were either transfected with STAT1 siRNAs (40 nM) or control siRNA for 24 hours and further treated with or without Stattic (7.5 nM) for 1 hour followed by IL-27 (50 ng/mL) treatment for 24 hours. The cell culture supernatants were used for ELISA. * p vs. no treatment, ** p vs. IL-27 by student t- test.
    Figure Legend Snippet: Down-regulation of angiogenic factors and up-regulation of angiostatic factors by STAT1-dependent pathway. (A-F) Protein concentrations of VEGF (A, B) , IL-8/CXCL8 (C, D) , CXCL5 (E, F) secreted by A549 cells were measured by ELISA. A549 cells were either transfected with STAT1 siRNAs (40 nM) or control siRNA for 24 hours and further treated with or without Stattic (7.5 nM) for 1 hour followed by IL-27 (50 ng/mL) treatment for 24 hours. The cell culture supernatants were used for ELISA. * p vs. no treatment, ** p vs. IL-27 by student t- test.

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

    IL-27-mediated activation of STAT1 and STAT3. (A) A549 cells were treated with IL-27 (50 ng/mL) for up to 72 hours. The tyrosine phosphorylated, or activated, forms of STAT1 and STAT3 ( P -STAT1 and P -STAT3) as well as the total amounts of the transcriptional factors (T-STAT1 and T-STAT3) were detected by Western blot. (B) Seven human NSCLC cell lines (H1703, H292, H157, H1437, H460, H1650, and H358) were cultured with the diluent of IL-27 (0.1% PBS/BSA) or IL-27 (50 ng/mL) for 24 hours and the activated and total amounts of STAT1 and STAT3 proteins were measured by Western blot. The densitometric measurements of total amounts of STAT1 and STAT3 were taken using Image J1.45o. The values above the figures represent relative density of the bands normalized to GAPDH. (C-D) A549 cells were treated with IL-27 (50 ng/mL) for 15 minutes, and stained with anti-tyrosine phosphorylated STAT1 (C) (green) and STAT3 (D) (green) antibodies for immunofluorescence microscopy (50 × magnification). The cells were counterstained with DAPI (blue). The white arrows indicate cells with nuclear activation of STAT1 or STAT3 by IL-27 treatment. Scale bar, 100 μm. (E) Expression of IL-27 receptor (TCCR) on cultured A549 cells. (F) Expression of IL-27 receptor (TCCR) on A549 cells after treatment with or without IL-27 (50 ng/mL) for 24 hours.
    Figure Legend Snippet: IL-27-mediated activation of STAT1 and STAT3. (A) A549 cells were treated with IL-27 (50 ng/mL) for up to 72 hours. The tyrosine phosphorylated, or activated, forms of STAT1 and STAT3 ( P -STAT1 and P -STAT3) as well as the total amounts of the transcriptional factors (T-STAT1 and T-STAT3) were detected by Western blot. (B) Seven human NSCLC cell lines (H1703, H292, H157, H1437, H460, H1650, and H358) were cultured with the diluent of IL-27 (0.1% PBS/BSA) or IL-27 (50 ng/mL) for 24 hours and the activated and total amounts of STAT1 and STAT3 proteins were measured by Western blot. The densitometric measurements of total amounts of STAT1 and STAT3 were taken using Image J1.45o. The values above the figures represent relative density of the bands normalized to GAPDH. (C-D) A549 cells were treated with IL-27 (50 ng/mL) for 15 minutes, and stained with anti-tyrosine phosphorylated STAT1 (C) (green) and STAT3 (D) (green) antibodies for immunofluorescence microscopy (50 × magnification). The cells were counterstained with DAPI (blue). The white arrows indicate cells with nuclear activation of STAT1 or STAT3 by IL-27 treatment. Scale bar, 100 μm. (E) Expression of IL-27 receptor (TCCR) on cultured A549 cells. (F) Expression of IL-27 receptor (TCCR) on A549 cells after treatment with or without IL-27 (50 ng/mL) for 24 hours.

    Techniques Used: Activation Assay, Western Blot, Cell Culture, Staining, Immunofluorescence, Microscopy, Expressing

    23) Product Images from "STAT1 is required for IL-6 mediated Bcl6 induction for early Tfh differentiation"

    Article Title: STAT1 is required for IL-6 mediated Bcl6 induction for early Tfh differentiation

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1203032

    Both STAT1 and STAT3 are required for early Tfh differentiation of CD4 T cells (A) Ctrl- or STAT1 KD SM (CD45.1 + ) cells were stimulated with IL-6 for pSTAT stainings. Overlaid histograms of pSTAT1 and pSTAT3. (B) Ctrl- or STAT1 KD SM cells were transferred into B6 mice that were infected with LCMV. Gates indicate Bcl6 hi CXCR5 hi at day 2 after infection. % of Bcl6 + CXCR5 + Tfh cells among total SM cells. (C–E) Ctrl-, STAT1 KD , STAT3 −/− , or STAT3 −/− STAT1 KD SM cells were transferred into B6 mice. SM analyzed at day 3 after infection. (C) Gates indicate Bcl6 + CXCR5 + Tfh cells. (D) % of Tfh cells calculated. (E) CXCR5 MFIs were normalized with cell size of donor cells (CXCR5 MFI /FSC MFI ). Data are representative of two (C–E) and three (A–B) independent experiments (n = 4–5 mice per group). ** P
    Figure Legend Snippet: Both STAT1 and STAT3 are required for early Tfh differentiation of CD4 T cells (A) Ctrl- or STAT1 KD SM (CD45.1 + ) cells were stimulated with IL-6 for pSTAT stainings. Overlaid histograms of pSTAT1 and pSTAT3. (B) Ctrl- or STAT1 KD SM cells were transferred into B6 mice that were infected with LCMV. Gates indicate Bcl6 hi CXCR5 hi at day 2 after infection. % of Bcl6 + CXCR5 + Tfh cells among total SM cells. (C–E) Ctrl-, STAT1 KD , STAT3 −/− , or STAT3 −/− STAT1 KD SM cells were transferred into B6 mice. SM analyzed at day 3 after infection. (C) Gates indicate Bcl6 + CXCR5 + Tfh cells. (D) % of Tfh cells calculated. (E) CXCR5 MFIs were normalized with cell size of donor cells (CXCR5 MFI /FSC MFI ). Data are representative of two (C–E) and three (A–B) independent experiments (n = 4–5 mice per group). ** P

    Techniques Used: Mouse Assay, Infection

    STAT1 is necessary for early Tfh differentiation after VACV infection Ctrl- or STAT1 KD SM (CD45.1 + ) cells were transferred into B6 mice that were infected with VACV-gpc. Bcl6 + CXCR5 + Tfh cells gated and calculated as % of total SM cells at day 2 (A) and 3 (B) after infection. Data are representative of two independent experiments (n = 4–5 mice per group). ** P
    Figure Legend Snippet: STAT1 is necessary for early Tfh differentiation after VACV infection Ctrl- or STAT1 KD SM (CD45.1 + ) cells were transferred into B6 mice that were infected with VACV-gpc. Bcl6 + CXCR5 + Tfh cells gated and calculated as % of total SM cells at day 2 (A) and 3 (B) after infection. Data are representative of two independent experiments (n = 4–5 mice per group). ** P

    Techniques Used: Infection, Mouse Assay, Gel Permeation Chromatography

    24) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    25) Product Images from "Melanoma response to anti-PD-L1 immunotherapy requires JAK1 signaling, but not JAK2"

    Article Title: Melanoma response to anti-PD-L1 immunotherapy requires JAK1 signaling, but not JAK2

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2018.1438106

    JAK1 shows a greater contribution to IFN-γ stimulated expression of p-STAT1, p-STAT3, and p-STAT5 expression than JAK2. (A) Western blots demonstrating the effect of RUX or BSK pretreatment on the IFN-γ stimulated expression levels of p-STAT1, p-STAT3, and p-STAT5 in BRAF-mutant and BRAF-wildtype melanoma cell lines. Actin was used as a loading control. (B) Western blots demonstrating IFN-γ-induced p-STAT1, p-STAT3, and p-STAT5 expression levels in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of non-targeting control siRNA (NTC), specific JAK1 siRNA (siJAK1), or JAK2 siRNA (siJAK2) transfection. (C) Western blots demonstrating IFN-γ-induced p-STAT1, p-STAT3, and p-STAT5 expression levels in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of non -targeting control siRNA (NTC), specific JAK1 siRNA (siJAK1), and JAK2 siRNA (siJAK2) transfection.
    Figure Legend Snippet: JAK1 shows a greater contribution to IFN-γ stimulated expression of p-STAT1, p-STAT3, and p-STAT5 expression than JAK2. (A) Western blots demonstrating the effect of RUX or BSK pretreatment on the IFN-γ stimulated expression levels of p-STAT1, p-STAT3, and p-STAT5 in BRAF-mutant and BRAF-wildtype melanoma cell lines. Actin was used as a loading control. (B) Western blots demonstrating IFN-γ-induced p-STAT1, p-STAT3, and p-STAT5 expression levels in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of non-targeting control siRNA (NTC), specific JAK1 siRNA (siJAK1), or JAK2 siRNA (siJAK2) transfection. (C) Western blots demonstrating IFN-γ-induced p-STAT1, p-STAT3, and p-STAT5 expression levels in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of non -targeting control siRNA (NTC), specific JAK1 siRNA (siJAK1), and JAK2 siRNA (siJAK2) transfection.

    Techniques Used: Expressing, Western Blot, Mutagenesis, Transfection

    Knockdown of PTPN2 promotes IFN-γ-induced expression of p-STAT1, p-STAT3, and p-STAT5 along with MHC-I, MHC-II, and PD-L1 and potentiates anti-PD-L1 response. (A) Western blots show IFN-γ-induced p-STAT1, p-STAT3, and p-STAT5 expression in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of nontargeting control siRNA (NTC) or specific PTPN2 siRNA (siPTPN2) transfection. Knockdown efficiency was confirmed by examining PTPN2 expression and actin was used as a loading control. (B) Western blots show IFN-γ-induced MHC-I, MHC-II, and PD-L1 expression in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of nontargeting control siRNA (NTC) or specific PTPN2 siRNA (siPTPN2) transfection. (C) Schema for the in vivo experiment with results shown in panels D and E. (D) Tumor growth curves of YUMM1.1 with 5 mice in each group (mean ± SEM) with anti-PD-L1, or isotype control treatment under shPTPN2 conditions. (E) Waterfall plot of YUMM1.1 tumors with anti-PD-L1, or isotype control treatment under shPTPN2 conditions. The data are expressed as the percentage of tumor volume change from baseline.
    Figure Legend Snippet: Knockdown of PTPN2 promotes IFN-γ-induced expression of p-STAT1, p-STAT3, and p-STAT5 along with MHC-I, MHC-II, and PD-L1 and potentiates anti-PD-L1 response. (A) Western blots show IFN-γ-induced p-STAT1, p-STAT3, and p-STAT5 expression in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of nontargeting control siRNA (NTC) or specific PTPN2 siRNA (siPTPN2) transfection. Knockdown efficiency was confirmed by examining PTPN2 expression and actin was used as a loading control. (B) Western blots show IFN-γ-induced MHC-I, MHC-II, and PD-L1 expression in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of nontargeting control siRNA (NTC) or specific PTPN2 siRNA (siPTPN2) transfection. (C) Schema for the in vivo experiment with results shown in panels D and E. (D) Tumor growth curves of YUMM1.1 with 5 mice in each group (mean ± SEM) with anti-PD-L1, or isotype control treatment under shPTPN2 conditions. (E) Waterfall plot of YUMM1.1 tumors with anti-PD-L1, or isotype control treatment under shPTPN2 conditions. The data are expressed as the percentage of tumor volume change from baseline.

    Techniques Used: Expressing, Western Blot, Mutagenesis, Transfection, In Vivo, Mouse Assay

    Knockdown of JAK1 partially recovers IFN-γ-induced inhibition of cell proliferation. (A) Graphs show IFN-γ-induced inhibition of cell proliferation in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of nontargeting control (NTC) or specific siRNA (siJAK1, siJAK2, siSTAT1, siSTAT3, siSTAT5A, and siSTAT5B) transfection during a 96 hr period. The box graphs denote an absorbance ratio of IFN-γ treatment to control group under each specific siRNA transfection conditions. The absorbance ratio of control group under NTC transfection condition at 96 hr was used as the baseline. (B) Western blots demonstrating knockdown efficiency of siRNA transfection by examining JAK1, JAK2, STAT1, STAT3, STAT5A or STAT5B expression.
    Figure Legend Snippet: Knockdown of JAK1 partially recovers IFN-γ-induced inhibition of cell proliferation. (A) Graphs show IFN-γ-induced inhibition of cell proliferation in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of nontargeting control (NTC) or specific siRNA (siJAK1, siJAK2, siSTAT1, siSTAT3, siSTAT5A, and siSTAT5B) transfection during a 96 hr period. The box graphs denote an absorbance ratio of IFN-γ treatment to control group under each specific siRNA transfection conditions. The absorbance ratio of control group under NTC transfection condition at 96 hr was used as the baseline. (B) Western blots demonstrating knockdown efficiency of siRNA transfection by examining JAK1, JAK2, STAT1, STAT3, STAT5A or STAT5B expression.

    Techniques Used: Inhibition, Mutagenesis, Transfection, Western Blot, Expressing

    STAT1 is the primary molecule downstream of JAKs involved in IFN-γ-induced expression of MHC-I, MHC-II, and PD-L1. (A) Western blots show IFN-γ-induced MHC-I, MHC-II, and PD-L1 expression in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of non-targeting control siRNA (NTC), specific siRNA for STAT1 (siSTAT1), STAT3 (siSTAT3), STAT5A (siSTAT5A), and STAT5B (siSTAT5B) transfection. Knockdown efficiency was confirmed by examining STAT1, STAT3, STAT5A, or STAT5B expression and actin was used as a loading control. (B) Western blots show IFN-γ-induced PD-L1 expression in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of non-targeting control siRNA (NTC), specific siRNA for STAT5A (siSTAT5A) and STAT5B (siSTAT5B) transfection.
    Figure Legend Snippet: STAT1 is the primary molecule downstream of JAKs involved in IFN-γ-induced expression of MHC-I, MHC-II, and PD-L1. (A) Western blots show IFN-γ-induced MHC-I, MHC-II, and PD-L1 expression in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of non-targeting control siRNA (NTC), specific siRNA for STAT1 (siSTAT1), STAT3 (siSTAT3), STAT5A (siSTAT5A), and STAT5B (siSTAT5B) transfection. Knockdown efficiency was confirmed by examining STAT1, STAT3, STAT5A, or STAT5B expression and actin was used as a loading control. (B) Western blots show IFN-γ-induced PD-L1 expression in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of non-targeting control siRNA (NTC), specific siRNA for STAT5A (siSTAT5A) and STAT5B (siSTAT5B) transfection.

    Techniques Used: Expressing, Western Blot, Mutagenesis, Transfection

    Co-administration of ruxolitinib with anti-PD-L1 antibody partially blocks the immunotherapy efficacy. (A) Schema for the in vivo experiment with results shown in panels B, C and D. (B) Tumor growth curves of YUMM2.1 with at least 5 mice in each group (mean ± SEM) after NVP-BSK805 or ruxolitinib along with anti-PD-L1, or isotype control treatment. (C) The scatter plots illustrate the final tumor volume of YUMM2.1 with at least 5 mice in each group (mean ± SEM) after NVP-BSK805 or ruxolitinib along with anti-PD-L1, or isotype control treatment. (D) Western blots from tumor lysates harvested 1 hr after the last of three consecutive doses demonstrating the effect of NVP-BSK805 or ruxolitinib treatment on STAT1, STAT3 and STAT5 phosphorylation of YUMM2.1 in vivo model tumor samples. Actin was used as a loading control.
    Figure Legend Snippet: Co-administration of ruxolitinib with anti-PD-L1 antibody partially blocks the immunotherapy efficacy. (A) Schema for the in vivo experiment with results shown in panels B, C and D. (B) Tumor growth curves of YUMM2.1 with at least 5 mice in each group (mean ± SEM) after NVP-BSK805 or ruxolitinib along with anti-PD-L1, or isotype control treatment. (C) The scatter plots illustrate the final tumor volume of YUMM2.1 with at least 5 mice in each group (mean ± SEM) after NVP-BSK805 or ruxolitinib along with anti-PD-L1, or isotype control treatment. (D) Western blots from tumor lysates harvested 1 hr after the last of three consecutive doses demonstrating the effect of NVP-BSK805 or ruxolitinib treatment on STAT1, STAT3 and STAT5 phosphorylation of YUMM2.1 in vivo model tumor samples. Actin was used as a loading control.

    Techniques Used: In Vivo, Mouse Assay, Western Blot

    26) Product Images from "A Human STAT1 Gain-of-Function Mutation Impairs CD8+ T Cell Responses against Gammaherpesvirus 68"

    Article Title: A Human STAT1 Gain-of-Function Mutation Impairs CD8+ T Cell Responses against Gammaherpesvirus 68

    Journal: Journal of Virology

    doi: 10.1128/JVI.00307-19

    Hyperphosphorylation of p-STAT1 in IFN-γ-treated STAT1 R274W T cells. Splenocytes from 6- to 8-week-old STAT1 R274W and WT littermate control mice were treated with vehicle (medium) or 100 ng/ml IFN-γ for 15 min followed by flow cytometric analysis of p-STAT1 expression levels in CD8 + and CD4 + T cells, B cells, and NK cells. (A) Representative flow cytometry histograms of p-STAT1 in CD8 + T cells, CD4 + T cells, B cells, and NK cells with and without treatment with IFN-γ. (B through E) Percent p-STAT1 + , number of p-STAT1 + , and MFI of p-STAT1 in CD8 + T cells (B), CD4 + T cells (C), B220 + B cells (D), and NK1.1 + NK cells (E). All data represent the mean of n = 5 to 9 samples per genotype from three independent experiments and were analyzed by unpaired t test (****, P
    Figure Legend Snippet: Hyperphosphorylation of p-STAT1 in IFN-γ-treated STAT1 R274W T cells. Splenocytes from 6- to 8-week-old STAT1 R274W and WT littermate control mice were treated with vehicle (medium) or 100 ng/ml IFN-γ for 15 min followed by flow cytometric analysis of p-STAT1 expression levels in CD8 + and CD4 + T cells, B cells, and NK cells. (A) Representative flow cytometry histograms of p-STAT1 in CD8 + T cells, CD4 + T cells, B cells, and NK cells with and without treatment with IFN-γ. (B through E) Percent p-STAT1 + , number of p-STAT1 + , and MFI of p-STAT1 in CD8 + T cells (B), CD4 + T cells (C), B220 + B cells (D), and NK1.1 + NK cells (E). All data represent the mean of n = 5 to 9 samples per genotype from three independent experiments and were analyzed by unpaired t test (****, P

    Techniques Used: Mouse Assay, Expressing, Flow Cytometry

    Heterozygous STAT1 R274W mice are more susceptible to γHV68 and have diminished expression of ISGs and IFN-γ during infection. WT and STAT1 R274W mice were intraperitoneally inoculated with 10 6 PFU of γHV68 or PBS control (mock, day 0). Mice were euthanized and the spleen harvested at days 0, 4, 8, and 14 after infection. (A) Viral genome copies in the splenocytes as measured by qPCR on days 4, 8, and 14 after infection. (B) Expression of open reading frame 50 (ORF50), ORF73, and MK3 in the spleen as measured by qPCR on day 14 after infection. (C) Levels of infectious virus in splenocytes as measured by plaque-forming assay on day 14 after infection. (D to F) Expression levels of CXCL10 (D), IRF1 (E), and IFN-γ (F) in the spleen as measured by qRT-PCR on days 0, 4, 8, and 14 after infection. All data represent the mean of n = 8 to 14 samples per genotype at each time point from at least two independent experiments and were analyzed by unpaired t test (****, P
    Figure Legend Snippet: Heterozygous STAT1 R274W mice are more susceptible to γHV68 and have diminished expression of ISGs and IFN-γ during infection. WT and STAT1 R274W mice were intraperitoneally inoculated with 10 6 PFU of γHV68 or PBS control (mock, day 0). Mice were euthanized and the spleen harvested at days 0, 4, 8, and 14 after infection. (A) Viral genome copies in the splenocytes as measured by qPCR on days 4, 8, and 14 after infection. (B) Expression of open reading frame 50 (ORF50), ORF73, and MK3 in the spleen as measured by qPCR on day 14 after infection. (C) Levels of infectious virus in splenocytes as measured by plaque-forming assay on day 14 after infection. (D to F) Expression levels of CXCL10 (D), IRF1 (E), and IFN-γ (F) in the spleen as measured by qRT-PCR on days 0, 4, 8, and 14 after infection. All data represent the mean of n = 8 to 14 samples per genotype at each time point from at least two independent experiments and were analyzed by unpaired t test (****, P

    Techniques Used: Mouse Assay, Expressing, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    The STAT1 R274W mutation impairs antigen-specific CD8 + T cell responses during γHV68 infection. WT and STAT1 R274W mice were inoculated intraperitoneally at age 5 to 6 weeks with 10 6 PFU of γHV68 or PBS control (mock, day 0). Infected mice were euthanized and splenocytes harvested at days 0, 4, 8, and 14 for flow cytometric analysis. (A) Representative dot plots of antigen-specific CD8 + T cells quantitated by flow cytometric analysis following ORF6 tetramer staining. (B and C) Quantitation of the percentage (B) and number (C) of ORF6 tetramer + CD8 + T cells by flow cytometry on days 0, 4, 8, and 14 after infection. (D and E) Representative dot plots of TNF-α- (D) and IFN-γ-expressing (E) CD8 + T cells at day 14 were quantitated by flow cytometric analysis after restimulation with or without (NS) the immunodominant ORF6 peptide. (F-G) Percentage (F) and number (G) of TNF-α-expressing CD8 + T cells. (H-I) Percentage (H) and number (I) of IFN-γ-expressing CD8 + T cells. Data represent the mean of n = 4 to 8 mice per genotype pooled from two independent experiments. Results were analyzed by unpaired t test. ****, P
    Figure Legend Snippet: The STAT1 R274W mutation impairs antigen-specific CD8 + T cell responses during γHV68 infection. WT and STAT1 R274W mice were inoculated intraperitoneally at age 5 to 6 weeks with 10 6 PFU of γHV68 or PBS control (mock, day 0). Infected mice were euthanized and splenocytes harvested at days 0, 4, 8, and 14 for flow cytometric analysis. (A) Representative dot plots of antigen-specific CD8 + T cells quantitated by flow cytometric analysis following ORF6 tetramer staining. (B and C) Quantitation of the percentage (B) and number (C) of ORF6 tetramer + CD8 + T cells by flow cytometry on days 0, 4, 8, and 14 after infection. (D and E) Representative dot plots of TNF-α- (D) and IFN-γ-expressing (E) CD8 + T cells at day 14 were quantitated by flow cytometric analysis after restimulation with or without (NS) the immunodominant ORF6 peptide. (F-G) Percentage (F) and number (G) of TNF-α-expressing CD8 + T cells. (H-I) Percentage (H) and number (I) of IFN-γ-expressing CD8 + T cells. Data represent the mean of n = 4 to 8 mice per genotype pooled from two independent experiments. Results were analyzed by unpaired t test. ****, P

    Techniques Used: Mutagenesis, Infection, Mouse Assay, Staining, Quantitation Assay, Flow Cytometry, Expressing

    Flow cytometric analysis of CD4 + and CD8 + T cell subsets in the spleens of naive and γHV68-infected mice. At age 5 to 6 weeks, WT and STAT1 R274W mice were inoculated intraperitoneally with 10 6 PFU of γHV68 or PBS control (mock). Infected mice were euthanized and splenocytes harvested at day 14 for flow cytometric analysis. Splenocytes were cultured for 6 h with brefeldin A in the presence or absence (NS) of PMA-ionomycin (PMA/IOM). (A to C) Percentage (A), number (B), and IL-17A MFI (C) of IL-17A-producing CD4 + T cells from the spleens of mock- and γHV68-infected mice. (D to F) Percentage (D), number (E), and IL-17A MFI (F) of IL-17A-producing CD8 + T cells from the spleens of mock- and γHV68-infected mice. (G to I) Percentage (G), number (H), and IFN-γ MFI (I) of IFN-γ-producing CD4 + T cells from the spleens of mock- and γHV68-infected mice. (J to L) Percentage (J), number (K), and IFN-γ MFI (L) of IFN-γ-producing CD8 + T cells from the spleens of mock- and γHV68-infected mice. Data represent the mean of n = 14 to 16 samples from two independent experiments. Results were analyzed by unpaired t test. ****, P
    Figure Legend Snippet: Flow cytometric analysis of CD4 + and CD8 + T cell subsets in the spleens of naive and γHV68-infected mice. At age 5 to 6 weeks, WT and STAT1 R274W mice were inoculated intraperitoneally with 10 6 PFU of γHV68 or PBS control (mock). Infected mice were euthanized and splenocytes harvested at day 14 for flow cytometric analysis. Splenocytes were cultured for 6 h with brefeldin A in the presence or absence (NS) of PMA-ionomycin (PMA/IOM). (A to C) Percentage (A), number (B), and IL-17A MFI (C) of IL-17A-producing CD4 + T cells from the spleens of mock- and γHV68-infected mice. (D to F) Percentage (D), number (E), and IL-17A MFI (F) of IL-17A-producing CD8 + T cells from the spleens of mock- and γHV68-infected mice. (G to I) Percentage (G), number (H), and IFN-γ MFI (I) of IFN-γ-producing CD4 + T cells from the spleens of mock- and γHV68-infected mice. (J to L) Percentage (J), number (K), and IFN-γ MFI (L) of IFN-γ-producing CD8 + T cells from the spleens of mock- and γHV68-infected mice. Data represent the mean of n = 14 to 16 samples from two independent experiments. Results were analyzed by unpaired t test. ****, P

    Techniques Used: Infection, Mouse Assay, Cell Culture

    Multistep γHV68 growth curve analysis and gene expression in primary BMDMs and MEFs generated from WT and heterozygous STAT1 R274W mice. (A and B) BMDMs and MEFs were infected with γHV68 at a multiplicity of infection (MOI) of 0.05. The medium was harvested for quantitation of infectious virus by plaque assay at the indicated time points. (C) BMDMs were pretreated with medium or IFN-γ at 100 ng/ml for 12 h before being infected with γHV68 at an MOI of 0.05. At the indicated time point after infection, the virus titer was determined by plaque assay. (D) Relative gene expression levels in BMDMs infected with γHV68 at an MOI of 10 for 0, 6, 12, and 24 h, followed by harvesting of cells for RNA isolation and qRT-PCR analysis. (E) Relative gene expression in MEFs infected with γHV68 at an MOI of 10 for 12 h, followed by harvesting of cells for RNA isolation and qRT-PCR analysis. Growth curve data in panels A through C represent the mean plus or minus standard error of the mean (SEM) of two independent experiments with 3 technical replicates for each growth curve experiment. Data in panel D represent the mean ±SEM of 4 or 5 biological replicates pooled from two independent experiments. Data in panel E represent the mean ±SEM of 4 biological replicates pooled from two independent experiments. Data in panels A, B, and C were analyzed by 2-way analysis of variance (ANOVA). Data in panels D and E were analyzed by unpaired t test. *, P
    Figure Legend Snippet: Multistep γHV68 growth curve analysis and gene expression in primary BMDMs and MEFs generated from WT and heterozygous STAT1 R274W mice. (A and B) BMDMs and MEFs were infected with γHV68 at a multiplicity of infection (MOI) of 0.05. The medium was harvested for quantitation of infectious virus by plaque assay at the indicated time points. (C) BMDMs were pretreated with medium or IFN-γ at 100 ng/ml for 12 h before being infected with γHV68 at an MOI of 0.05. At the indicated time point after infection, the virus titer was determined by plaque assay. (D) Relative gene expression levels in BMDMs infected with γHV68 at an MOI of 10 for 0, 6, 12, and 24 h, followed by harvesting of cells for RNA isolation and qRT-PCR analysis. (E) Relative gene expression in MEFs infected with γHV68 at an MOI of 10 for 12 h, followed by harvesting of cells for RNA isolation and qRT-PCR analysis. Growth curve data in panels A through C represent the mean plus or minus standard error of the mean (SEM) of two independent experiments with 3 technical replicates for each growth curve experiment. Data in panel D represent the mean ±SEM of 4 or 5 biological replicates pooled from two independent experiments. Data in panel E represent the mean ±SEM of 4 biological replicates pooled from two independent experiments. Data in panels A, B, and C were analyzed by 2-way analysis of variance (ANOVA). Data in panels D and E were analyzed by unpaired t test. *, P

    Techniques Used: Expressing, Generated, Mouse Assay, Infection, Quantitation Assay, Plaque Assay, Isolation, Quantitative RT-PCR

    Generation and initial characterization of heterozygous STAT1 R274W knock-in mice. (A) Schematic functional domain map of STAT1 with corresponding alignment of the highly conserved coiled-coil domain amino acid sequence, including arginine 274 (R274). (B) Electropherogram of WT and STAT1 R274W mutant alleles within exon 10. Three nucleotides and one amino acid (R274W) were altered in STAT1 exon 10. (C) Expression of indicated ISGs in the spleens of 5- to 7-week-old STAT1 R274W mice and WT littermates. Gene expression was measured by reverse transcription-quantitative PCR (qRT-PCR). Relative gene expression (or fold change) was calculated using the threshold cycle (ΔΔ C T ) method after normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data represent the mean of 10 to 12 spleens per group pooled from two independent experiments. (D and E) Expression levels of CXCL10, IRF1, and IFIT1 in WT and STAT1 R274W BMDMs (D) and MEFs (E) 6 h after stimulation with 100 ng/ml of recombinant murine IFN-γ. Data represent the mean of three independent experiments from separate WT and STAT1 R274W primary cell lines. BMDMs (F and G) and MEFs (H and I) were stimulated with 100 ng/ml IFN-γ for 30 min, and the phosphorylation of STAT1 (p-STAT1) was assessed by Western blotting with antibody to p-STAT1 (Tyr701), using STAT1 and GAPDH as loading controls. Relative p-STAT1 density was quantified using ImageJ software and was normalized to GAPDH ( n = 3). (J) Representative flow cytometry histograms of p-STAT1 levels in BMDMs with and without 100 ng/ml IFN-γ treatment for 15 or 30 min. (K) Mean fluorescence intensity (MFI) of p-STAT1 in WT and STAT1 R274W BMDMs with or without 100 ng/ml IFN-γ treatment for 15 or 30 min. (L) Time course of ISG expression in WT or STAT1 R274W BMDMs at 0, 3, and 6 h after IFN-γ stimulation. Data in panels J through L represent the mean of n = 4 to 5 mice per group, pooled from two independent experiments. All data were analyzed by unpaired t test ( P > 0.1 for all comparisons).
    Figure Legend Snippet: Generation and initial characterization of heterozygous STAT1 R274W knock-in mice. (A) Schematic functional domain map of STAT1 with corresponding alignment of the highly conserved coiled-coil domain amino acid sequence, including arginine 274 (R274). (B) Electropherogram of WT and STAT1 R274W mutant alleles within exon 10. Three nucleotides and one amino acid (R274W) were altered in STAT1 exon 10. (C) Expression of indicated ISGs in the spleens of 5- to 7-week-old STAT1 R274W mice and WT littermates. Gene expression was measured by reverse transcription-quantitative PCR (qRT-PCR). Relative gene expression (or fold change) was calculated using the threshold cycle (ΔΔ C T ) method after normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data represent the mean of 10 to 12 spleens per group pooled from two independent experiments. (D and E) Expression levels of CXCL10, IRF1, and IFIT1 in WT and STAT1 R274W BMDMs (D) and MEFs (E) 6 h after stimulation with 100 ng/ml of recombinant murine IFN-γ. Data represent the mean of three independent experiments from separate WT and STAT1 R274W primary cell lines. BMDMs (F and G) and MEFs (H and I) were stimulated with 100 ng/ml IFN-γ for 30 min, and the phosphorylation of STAT1 (p-STAT1) was assessed by Western blotting with antibody to p-STAT1 (Tyr701), using STAT1 and GAPDH as loading controls. Relative p-STAT1 density was quantified using ImageJ software and was normalized to GAPDH ( n = 3). (J) Representative flow cytometry histograms of p-STAT1 levels in BMDMs with and without 100 ng/ml IFN-γ treatment for 15 or 30 min. (K) Mean fluorescence intensity (MFI) of p-STAT1 in WT and STAT1 R274W BMDMs with or without 100 ng/ml IFN-γ treatment for 15 or 30 min. (L) Time course of ISG expression in WT or STAT1 R274W BMDMs at 0, 3, and 6 h after IFN-γ stimulation. Data in panels J through L represent the mean of n = 4 to 5 mice per group, pooled from two independent experiments. All data were analyzed by unpaired t test ( P > 0.1 for all comparisons).

    Techniques Used: Knock-In, Mouse Assay, Functional Assay, Sequencing, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Recombinant, Western Blot, Software, Flow Cytometry, Fluorescence

    Flow cytometric quantitation of naive (T N ), effector memory (T EM ), and central memory (T CM ) CD8 + and CD4 + T cell subsets in the spleens of naive and γHV68-infected mice. WT and STAT1 R274W mice were inoculated intraperitoneally at age 5 to 6 weeks with 10 6 PFU of γHV68 or PBS control (mock, day 0). Infected mice were euthanized and splenocytes harvested at days 0, 4, 8, and 14 for flow cytometric analysis of CD8 + and CD4 + naive (CD44 lo CD62L hi ) T cells, effector memory (CD44 hi CD62L lo ) T cells, and central memory (CD44 hi CD62L hi ) T cells. (A and B) Percentage and numbers of naive (T N ), effector memory (T EM ), and central memory (T CM ) CD8 + (A) and CD4 + (B) T cell subsets at the indicated time points. Data represent the mean ±SEM of n = 4 to 8 mice per genotype at each time point. Data were analyzed by 2-way ANOVA. ****, P
    Figure Legend Snippet: Flow cytometric quantitation of naive (T N ), effector memory (T EM ), and central memory (T CM ) CD8 + and CD4 + T cell subsets in the spleens of naive and γHV68-infected mice. WT and STAT1 R274W mice were inoculated intraperitoneally at age 5 to 6 weeks with 10 6 PFU of γHV68 or PBS control (mock, day 0). Infected mice were euthanized and splenocytes harvested at days 0, 4, 8, and 14 for flow cytometric analysis of CD8 + and CD4 + naive (CD44 lo CD62L hi ) T cells, effector memory (CD44 hi CD62L lo ) T cells, and central memory (CD44 hi CD62L hi ) T cells. (A and B) Percentage and numbers of naive (T N ), effector memory (T EM ), and central memory (T CM ) CD8 + (A) and CD4 + (B) T cell subsets at the indicated time points. Data represent the mean ±SEM of n = 4 to 8 mice per genotype at each time point. Data were analyzed by 2-way ANOVA. ****, P

    Techniques Used: Quantitation Assay, Infection, Mouse Assay

    Virus-specific antibody responses and flow cytometric quantitation of splenic lymphocyte subsets in γHV68-infected WT and STAT1 R274W mice. At ages of 5 to 6 weeks, WT and STAT1 R274W mice were inoculated intraperitoneally with 10 6 PFU of γHV68 or PBS control (mock, day 0). (A and B) Serum was harvested on day 14 after infection. Virus-specific IgM (A) and IgG (B) levels were measured by ELISA. Data represent the mean of n = 16 biological replicates pooled from two independent experiments. P > 0.1 by unpaired t test. (C) Spleen weight prior to isolation of leukocytes. (D to G) Mice were euthanized and the spleens harvested at days 0, 4, 8, and 14 after infection for flow cytometric analysis. Numbers of CD45 + leukocytes (D), CD19 + B cells (E), CD8 + T cells (F), and CD4 + T cells (G). Data represent the mean of n = 4 to 8 samples from two independent experiments. Data were analyzed by unpaired t test. ****, P
    Figure Legend Snippet: Virus-specific antibody responses and flow cytometric quantitation of splenic lymphocyte subsets in γHV68-infected WT and STAT1 R274W mice. At ages of 5 to 6 weeks, WT and STAT1 R274W mice were inoculated intraperitoneally with 10 6 PFU of γHV68 or PBS control (mock, day 0). (A and B) Serum was harvested on day 14 after infection. Virus-specific IgM (A) and IgG (B) levels were measured by ELISA. Data represent the mean of n = 16 biological replicates pooled from two independent experiments. P > 0.1 by unpaired t test. (C) Spleen weight prior to isolation of leukocytes. (D to G) Mice were euthanized and the spleens harvested at days 0, 4, 8, and 14 after infection for flow cytometric analysis. Numbers of CD45 + leukocytes (D), CD19 + B cells (E), CD8 + T cells (F), and CD4 + T cells (G). Data represent the mean of n = 4 to 8 samples from two independent experiments. Data were analyzed by unpaired t test. ****, P

    Techniques Used: Quantitation Assay, Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Isolation

    Impaired STAT1 R274W antigen-specific CD8 + T cell responses after γHV68 infection of WT and STAT1 R274W mixed bone marrow chimeric mice. (A to K) STAT1 R274W and WT littermate animals were lethally irradiated followed by intravenous injection of a 1:1 mixture of CD45.1 WT bone marrow cells and either CD45.2 WT or STAT1 R274W bone marrow cells. This resulted in generation of WT/WT chimeras and WT/STAT1 R274W chimeras. Six weeks later, after reconstitution of similar numbers of circulating leukocytes, mice were intraperitoneally inoculated with 10 6 PFU of γHV68 and then euthanized 14 days after infection for virological and immunological analysis (A). (B) Viral DNA levels in the spleen as measured by qPCR. (C) Infectious virus levels in the spleens of mixed bone marrow chimeric mice as measured by plaque assay. The dashed line indicates the limit of detection of the assay at 10 PFU/g of tissue. (D and E) Flow cytometric analysis of the percentage (D) and number (E) of CD45.1 + and CD45.2 + leukocytes in the spleens of mixed bone marrow chimeric mice. (F to H) Numbers of CD19 + B cells, CD4 + T cells, and CD8 + T cells in the spleen after infection as measured by flow cytometry. (I) Representative dot plots of ORF6 tetramer + CD8 + T cells in mixed bone marrow chimeric mice. (J and K) The percentage (J) and number (K) of ORF6 tetramer + CD8 + T cells in mixed bone marrow chimeric mice. Data represent the mean of n = 8 to 9 chimeric mice per group pooled from two independent experiments. Results were analyzed by unpaired t test. ****, P
    Figure Legend Snippet: Impaired STAT1 R274W antigen-specific CD8 + T cell responses after γHV68 infection of WT and STAT1 R274W mixed bone marrow chimeric mice. (A to K) STAT1 R274W and WT littermate animals were lethally irradiated followed by intravenous injection of a 1:1 mixture of CD45.1 WT bone marrow cells and either CD45.2 WT or STAT1 R274W bone marrow cells. This resulted in generation of WT/WT chimeras and WT/STAT1 R274W chimeras. Six weeks later, after reconstitution of similar numbers of circulating leukocytes, mice were intraperitoneally inoculated with 10 6 PFU of γHV68 and then euthanized 14 days after infection for virological and immunological analysis (A). (B) Viral DNA levels in the spleen as measured by qPCR. (C) Infectious virus levels in the spleens of mixed bone marrow chimeric mice as measured by plaque assay. The dashed line indicates the limit of detection of the assay at 10 PFU/g of tissue. (D and E) Flow cytometric analysis of the percentage (D) and number (E) of CD45.1 + and CD45.2 + leukocytes in the spleens of mixed bone marrow chimeric mice. (F to H) Numbers of CD19 + B cells, CD4 + T cells, and CD8 + T cells in the spleen after infection as measured by flow cytometry. (I) Representative dot plots of ORF6 tetramer + CD8 + T cells in mixed bone marrow chimeric mice. (J and K) The percentage (J) and number (K) of ORF6 tetramer + CD8 + T cells in mixed bone marrow chimeric mice. Data represent the mean of n = 8 to 9 chimeric mice per group pooled from two independent experiments. Results were analyzed by unpaired t test. ****, P

    Techniques Used: Infection, Mouse Assay, Irradiation, Injection, Real-time Polymerase Chain Reaction, Plaque Assay, Flow Cytometry

    27) Product Images from "PLA2G1B is involved in CD4 anergy and CD4 lymphopenia in HIV-infected patients"

    Article Title: PLA2G1B is involved in CD4 anergy and CD4 lymphopenia in HIV-infected patients

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI131842

    Effect of PLA2G1B on CD4 + T cell subpopulations, specificity, and reversion. ( A ) Dose effect of PLA2G1B (IL-7, n = 4; IL-2, n = 3; IL-4, n = 5) and ( B ) of 1% HD plasma (IL-7, n = 4; IL-2 and IL-4, n = 3) and VP plasma ( n = 5) on IL-2–, IL-4–, and IL-7–induced p-STAT NT in CD4 + T cells. ( C ) Effects of PLA2G1B (IL-7, n = 4; IFN-α, n = 5) and ( D ) plasma (HD [ n = 4] or VP [ n = 5], 1%) on IL-7–induced p-STAT5 NT and IFN-α–induced p-STAT1 NT in CD4 + T cells ( n = 5 donors). ( E ) The effect of PLA2G1B (30 minutes) on IL-7–induced p-STAT5 NT was analyzed in total (HD T CD4 + :IL-7), naive (HD T CD4 + CD45RA + :IL-7), and memory (HD T CD4 + CD45RA – :IL-7) CD4 + T cells from the same donor in response to IL-7 ( n = 3 donors). ( F ) Percentage of CD127 + cells among and ( G ) CD127 expression (Δ anti-CD127 MFI minus isotype control MFI) on CD45RA + and CD45RA – CD4 + = 3 donors). ( H ) Effect of PLA2G1B (250 nM) on aMMD induction in CD4 + T cells ( n = 5) and CD8 + T cells ( n = 8) and ( I ) on IL-7–induced p-STAT5 NT in CD8 + T cells (dose effect, n = 3). In A – I , results are shown as the mean ± SD. * P
    Figure Legend Snippet: Effect of PLA2G1B on CD4 + T cell subpopulations, specificity, and reversion. ( A ) Dose effect of PLA2G1B (IL-7, n = 4; IL-2, n = 3; IL-4, n = 5) and ( B ) of 1% HD plasma (IL-7, n = 4; IL-2 and IL-4, n = 3) and VP plasma ( n = 5) on IL-2–, IL-4–, and IL-7–induced p-STAT NT in CD4 + T cells. ( C ) Effects of PLA2G1B (IL-7, n = 4; IFN-α, n = 5) and ( D ) plasma (HD [ n = 4] or VP [ n = 5], 1%) on IL-7–induced p-STAT5 NT and IFN-α–induced p-STAT1 NT in CD4 + T cells ( n = 5 donors). ( E ) The effect of PLA2G1B (30 minutes) on IL-7–induced p-STAT5 NT was analyzed in total (HD T CD4 + :IL-7), naive (HD T CD4 + CD45RA + :IL-7), and memory (HD T CD4 + CD45RA – :IL-7) CD4 + T cells from the same donor in response to IL-7 ( n = 3 donors). ( F ) Percentage of CD127 + cells among and ( G ) CD127 expression (Δ anti-CD127 MFI minus isotype control MFI) on CD45RA + and CD45RA – CD4 + = 3 donors). ( H ) Effect of PLA2G1B (250 nM) on aMMD induction in CD4 + T cells ( n = 5) and CD8 + T cells ( n = 8) and ( I ) on IL-7–induced p-STAT5 NT in CD8 + T cells (dose effect, n = 3). In A – I , results are shown as the mean ± SD. * P

    Techniques Used: Expressing

    28) Product Images from "Differential roles of STAT1 and STAT2 in the sensitivity of JAK2V617F- vs. BCR-ABL-positive cells to interferon alpha"

    Article Title: Differential roles of STAT1 and STAT2 in the sensitivity of JAK2V617F- vs. BCR-ABL-positive cells to interferon alpha

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-019-0722-9

    Histone modifications in the promoter region of interferon target genes differ between 32D-BCR-ABL- and 32D-JAK2V617F-positive cells. a Enrichment analysis of transcription factor binding sites (TFBS) in acetylation (H3K9ac) peaks based on ChIP-seq data. Differentially regulated genes were analyzed for the presence of TFBS in the acetylation peaks, and it was tested if the number of TFBS is significantly different. b 32D-BCR-ABL-transduced (blue) and 32D-JAK2V617F-transduced (red) cells were analyzed for changes of H3K9 acetylation in the promoter region of Stat1 , Stat2 , Irf1 , and Irf9 after 4 h of 1 μM TKI and/or 100 U/ml IFNa treatment by ChIP-PCR and were tested for differences in comparison to the corresponding DMSO-treated cell line. * p
    Figure Legend Snippet: Histone modifications in the promoter region of interferon target genes differ between 32D-BCR-ABL- and 32D-JAK2V617F-positive cells. a Enrichment analysis of transcription factor binding sites (TFBS) in acetylation (H3K9ac) peaks based on ChIP-seq data. Differentially regulated genes were analyzed for the presence of TFBS in the acetylation peaks, and it was tested if the number of TFBS is significantly different. b 32D-BCR-ABL-transduced (blue) and 32D-JAK2V617F-transduced (red) cells were analyzed for changes of H3K9 acetylation in the promoter region of Stat1 , Stat2 , Irf1 , and Irf9 after 4 h of 1 μM TKI and/or 100 U/ml IFNa treatment by ChIP-PCR and were tested for differences in comparison to the corresponding DMSO-treated cell line. * p

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    Simplified overview of ISG regulation by STAT1 and STAT2 in BCR-ABL- and JAK2V617F-positive cells. In BCR-ABL-expressing cells, STAT2 is partially phosphorylated leading to ISG repression [ 43 ] and is not capable to induce STAT1 expression. In addition, chromatin marks negatively regulating gene expression are present (i.e., H3K27me3). STAT2, although not phosphorylated in JAK2V617F-positive cells, can induce STAT1 expression in the presence of histone marks representing active promoters (i.e., H3K9ac and H3K27ac). Upon stimulation with IFNa, STAT2 is essential for STAT1 phosphorylation at its tyrosine residue Y701 in BCR-ABL-positive cells (indicated by red arrow). The ISGF3 complex is formed and ISG expression is induced. IFNa stimulation of JAK2V617F-positive cells leads to ISGF3 complex formation and ISG as well as STAT1 promoter access. The equilibrium of STAT1/STAT1 and STAT1/STAT3 dimers shifts in dependence of the amount of active STAT2. STAT1/3 illustrates different dimer options: STAT1/STAT1, STAT1/STAT3, and STAT3/STAT3. In BCR-ABL-expressing cells, STAT3 is phosphorylated after IFNa binding to its receptor. P, tyrosine phosphorylation
    Figure Legend Snippet: Simplified overview of ISG regulation by STAT1 and STAT2 in BCR-ABL- and JAK2V617F-positive cells. In BCR-ABL-expressing cells, STAT2 is partially phosphorylated leading to ISG repression [ 43 ] and is not capable to induce STAT1 expression. In addition, chromatin marks negatively regulating gene expression are present (i.e., H3K27me3). STAT2, although not phosphorylated in JAK2V617F-positive cells, can induce STAT1 expression in the presence of histone marks representing active promoters (i.e., H3K9ac and H3K27ac). Upon stimulation with IFNa, STAT2 is essential for STAT1 phosphorylation at its tyrosine residue Y701 in BCR-ABL-positive cells (indicated by red arrow). The ISGF3 complex is formed and ISG expression is induced. IFNa stimulation of JAK2V617F-positive cells leads to ISGF3 complex formation and ISG as well as STAT1 promoter access. The equilibrium of STAT1/STAT1 and STAT1/STAT3 dimers shifts in dependence of the amount of active STAT2. STAT1/3 illustrates different dimer options: STAT1/STAT1, STAT1/STAT3, and STAT3/STAT3. In BCR-ABL-expressing cells, STAT3 is phosphorylated after IFNa binding to its receptor. P, tyrosine phosphorylation

    Techniques Used: Expressing, Binding Assay

    Reconstitution of 32D-BCR-ABL and 32D-JAK2V617F STAT1ko and STAT2ko cells. 32D-BCR-ABL and 32D-JAK2V617F cells (depicted as WT, respectively), which passed through the CRISPR STAT KO process but showed no knockout, were used as control cell lines. 32D-BCR-ABL ( a ) and 32D-JAK2V617F ( b ) STAT1ko or STAT2ko cells reconstituted with wt-STAT1, wt-STAT2, STAT1Y701F (Y/F), or STAT2Y689F (Y/F) were applied in a MTT assay and treated with the indicated concentrations of IFNa (0–10 4 U/ml) for 72 h. MTT assays have been performed four times in independent experiments, and untreated controls were analyzed with one-way ANOVA and Dunn’s multiple comparison test. Further statistical analysis can be found in Additional file 9 : Figure S7A, B. c Western blot analysis of the 32D cell lines depicted in a and b treated for 4 h with 100 U/ml IFNa or left untreated. Phosphorylation of STAT1, STAT2, and STAT3 was analyzed. GAPDH served as the loading control. d mRNA expression of interferon-stimulated genes in the indicated cell lines. Stat2 qPCR primer detected the ectopically expressed Stat2 mRNA, explaining the strong upregulation, and endogenous Stat2 can thus not be evaluated in the reconstituted experiments. Gene expression was calculated as a percentage of Gapdh , and the mean values ± SD are depicted. * p
    Figure Legend Snippet: Reconstitution of 32D-BCR-ABL and 32D-JAK2V617F STAT1ko and STAT2ko cells. 32D-BCR-ABL and 32D-JAK2V617F cells (depicted as WT, respectively), which passed through the CRISPR STAT KO process but showed no knockout, were used as control cell lines. 32D-BCR-ABL ( a ) and 32D-JAK2V617F ( b ) STAT1ko or STAT2ko cells reconstituted with wt-STAT1, wt-STAT2, STAT1Y701F (Y/F), or STAT2Y689F (Y/F) were applied in a MTT assay and treated with the indicated concentrations of IFNa (0–10 4 U/ml) for 72 h. MTT assays have been performed four times in independent experiments, and untreated controls were analyzed with one-way ANOVA and Dunn’s multiple comparison test. Further statistical analysis can be found in Additional file 9 : Figure S7A, B. c Western blot analysis of the 32D cell lines depicted in a and b treated for 4 h with 100 U/ml IFNa or left untreated. Phosphorylation of STAT1, STAT2, and STAT3 was analyzed. GAPDH served as the loading control. d mRNA expression of interferon-stimulated genes in the indicated cell lines. Stat2 qPCR primer detected the ectopically expressed Stat2 mRNA, explaining the strong upregulation, and endogenous Stat2 can thus not be evaluated in the reconstituted experiments. Gene expression was calculated as a percentage of Gapdh , and the mean values ± SD are depicted. * p

    Techniques Used: CRISPR, Knock-Out, MTT Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction

    STAT1 or STAT2 knockout alters IFNa responsiveness only in JAK2V617F-positive cells. a 32D-BCR-ABL-WT, 32D-BCR-ABL-STAT1ko, and 32D-BCR-ABL-STAT2ko or b 32D-JAK2V617F-WT, 32D-JAK2V617F-STAT1ko, and 32D-JAK2V617F-STAT2ko cells were treated for 4 h with TKI (1 μM imatinib and ruxolitinib, respectively) or IFNa (100 U/ml) or a combination of both. SDS-Page and Western blotting were performed, and the indicated immunostainings were carried out. GAPDH served as the loading control. c 32D-BCR-ABL-WT, 32D-BCR-ABL-STAT1ko, and 32D-BCR-ABL-STAT2ko or d 32D-JAK2V617F-WT, 32D-JAK2V617F-STAT1ko, and 32D-JAK2V617F-STAT2ko cells were treated with increasing concentrations of IFNa, and cell viability was measured by MTT assay. e Indicated 32D cells were treated with TKI (0.5 μM imatinib and 0.1 μM ruxolitinib, respectively) or IFNa (100 U/ml) or a combination of both for 48 h, due to the rapid growth of untreated BCR-ABL-positive cells. PI staining was performed to discriminate between living and dead cells. Mean values ± SD are depicted. f Measurement of Stat1 , Stat2 , Irf7 , and Irf9 mRNA expression in the indicated cell lines. Expression was calculated as a percentage of Gapdh , and the mean values ± SD are depicted. * p
    Figure Legend Snippet: STAT1 or STAT2 knockout alters IFNa responsiveness only in JAK2V617F-positive cells. a 32D-BCR-ABL-WT, 32D-BCR-ABL-STAT1ko, and 32D-BCR-ABL-STAT2ko or b 32D-JAK2V617F-WT, 32D-JAK2V617F-STAT1ko, and 32D-JAK2V617F-STAT2ko cells were treated for 4 h with TKI (1 μM imatinib and ruxolitinib, respectively) or IFNa (100 U/ml) or a combination of both. SDS-Page and Western blotting were performed, and the indicated immunostainings were carried out. GAPDH served as the loading control. c 32D-BCR-ABL-WT, 32D-BCR-ABL-STAT1ko, and 32D-BCR-ABL-STAT2ko or d 32D-JAK2V617F-WT, 32D-JAK2V617F-STAT1ko, and 32D-JAK2V617F-STAT2ko cells were treated with increasing concentrations of IFNa, and cell viability was measured by MTT assay. e Indicated 32D cells were treated with TKI (0.5 μM imatinib and 0.1 μM ruxolitinib, respectively) or IFNa (100 U/ml) or a combination of both for 48 h, due to the rapid growth of untreated BCR-ABL-positive cells. PI staining was performed to discriminate between living and dead cells. Mean values ± SD are depicted. f Measurement of Stat1 , Stat2 , Irf7 , and Irf9 mRNA expression in the indicated cell lines. Expression was calculated as a percentage of Gapdh , and the mean values ± SD are depicted. * p

    Techniques Used: Knock-Out, SDS Page, Western Blot, MTT Assay, Staining, Expressing

    29) Product Images from "Human papillomavirus 16 E2 regulates keratinocyte gene expression relevant to cancer and the viral life cycle"

    Article Title: Human papillomavirus 16 E2 regulates keratinocyte gene expression relevant to cancer and the viral life cycle

    Journal: bioRxiv

    doi: 10.1101/461715

    E2 represses U-IGF3 gene expression in multiple NOKs+E2 clonal cell lines. A) Western blots using antibodies against the indicated cellular proteins were carried out on extracts from the E2 clones. β-actin is shown as a loading control. B) Expression levels of a sub-set of U-ISGF3 genes in NOKs and NOKs clones expressing E2. Results are expressed as fold change from that observed in parental NOKs and represent the average of three independent experiments. C ) Expression levels of STAT1, IRF9 and IFNκ genes in NOKs and NOKs clones expressing E2. Results are expressed as fold change from that observed in parental NOKs and represent the average of three independent experiments. Bars marked with * in B and C are significantly different from NOKs (p-value
    Figure Legend Snippet: E2 represses U-IGF3 gene expression in multiple NOKs+E2 clonal cell lines. A) Western blots using antibodies against the indicated cellular proteins were carried out on extracts from the E2 clones. β-actin is shown as a loading control. B) Expression levels of a sub-set of U-ISGF3 genes in NOKs and NOKs clones expressing E2. Results are expressed as fold change from that observed in parental NOKs and represent the average of three independent experiments. C ) Expression levels of STAT1, IRF9 and IFNκ genes in NOKs and NOKs clones expressing E2. Results are expressed as fold change from that observed in parental NOKs and represent the average of three independent experiments. Bars marked with * in B and C are significantly different from NOKs (p-value

    Techniques Used: Expressing, Western Blot, Clone Assay, Significance Assay

    HPV16 E2, E6 and E7 can repress innate immune gene expression in NOKs. A) Western blot of NOKs cells transduced and selected following infection with retroviruses expressing HA-tagged E6 (lane 1), E7 (lane 2) and E2 (lane 3) with an HA antibody. GAPDH is shown as a loading control. B) Western blot of NOKs cells transduced and selected following infection with retroviruses expressing HA-tagged E6 (lane 1), E7 (lane 2) and E2 (lane 3) with a p53 antibody. GAPDH is shown as a loading control. There were lanes between the NOKs (lane 1) and the viral protein expressing NOKs lanes (2-4) that have been removed for clarity, but the NOKs sample image is taken from the same membrane and exposure. C) Western blotting of NOKs cells and NOKs expressing the indicated viral proteins with MX1 and IFIT1 antibodies. β-actin is shown as a loading control. D) Expression levels of a sub-set of U-ISGF3 genes in NOKs and NOKs expressing the indicated viral proteins. Results are expressed as fold change from that observed in parental NOKs and represent the average of three independent experiments. Standard error bars are shown. E) Expression of STAT1 and IFNκ genes in NOKs and NOKs expressing the indicated viral proteins. F) Transduction with retroviral vectors does not induce repression of U-ISGF3 proteins. Results are expressed as fold change from that observed in parental NOKs and represent the average of three independent experiments. Standard error bars are shown. Bars marked with * in D and E are significantly different from NOKs (p-value
    Figure Legend Snippet: HPV16 E2, E6 and E7 can repress innate immune gene expression in NOKs. A) Western blot of NOKs cells transduced and selected following infection with retroviruses expressing HA-tagged E6 (lane 1), E7 (lane 2) and E2 (lane 3) with an HA antibody. GAPDH is shown as a loading control. B) Western blot of NOKs cells transduced and selected following infection with retroviruses expressing HA-tagged E6 (lane 1), E7 (lane 2) and E2 (lane 3) with a p53 antibody. GAPDH is shown as a loading control. There were lanes between the NOKs (lane 1) and the viral protein expressing NOKs lanes (2-4) that have been removed for clarity, but the NOKs sample image is taken from the same membrane and exposure. C) Western blotting of NOKs cells and NOKs expressing the indicated viral proteins with MX1 and IFIT1 antibodies. β-actin is shown as a loading control. D) Expression levels of a sub-set of U-ISGF3 genes in NOKs and NOKs expressing the indicated viral proteins. Results are expressed as fold change from that observed in parental NOKs and represent the average of three independent experiments. Standard error bars are shown. E) Expression of STAT1 and IFNκ genes in NOKs and NOKs expressing the indicated viral proteins. F) Transduction with retroviral vectors does not induce repression of U-ISGF3 proteins. Results are expressed as fold change from that observed in parental NOKs and represent the average of three independent experiments. Standard error bars are shown. Bars marked with * in D and E are significantly different from NOKs (p-value

    Techniques Used: Expressing, Western Blot, Infection, Transduction, Significance Assay

    E2 also targets innate immune gene repression in NTERT, a TERT immortalized foreskin cell line. A) Expression of HA tagged E2 in the NTERT detected by western blotting of protein extracts with an E2 antibody. β-actin is shown as a loading control. Western blots for IFIT1 and MX1 on protein extracts from the E2 positive and negative cells. GAPDH is shown as a loading control. B) Expression of a sub-set of the U-ISGF3 gene set in the NTERT and NTERT+E2 cells. Results are expressed relative to NTERT levels equaling 1 and are representative of three independent experiments. C) Expression levels of STAT1 and IFNk genes in NTERT and NTERT+E2. Results are expressed relative to NTERT levels equaling 1 and are representative of three independent experiments. Bars marked with * in B and C are significantly different from NOKs (p-value
    Figure Legend Snippet: E2 also targets innate immune gene repression in NTERT, a TERT immortalized foreskin cell line. A) Expression of HA tagged E2 in the NTERT detected by western blotting of protein extracts with an E2 antibody. β-actin is shown as a loading control. Western blots for IFIT1 and MX1 on protein extracts from the E2 positive and negative cells. GAPDH is shown as a loading control. B) Expression of a sub-set of the U-ISGF3 gene set in the NTERT and NTERT+E2 cells. Results are expressed relative to NTERT levels equaling 1 and are representative of three independent experiments. C) Expression levels of STAT1 and IFNk genes in NTERT and NTERT+E2. Results are expressed relative to NTERT levels equaling 1 and are representative of three independent experiments. Bars marked with * in B and C are significantly different from NOKs (p-value

    Techniques Used: Expressing, Western Blot, Significance Assay

    30) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    31) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    32) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    33) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    34) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    35) Product Images from "Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *"

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179572

    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Figure Legend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Techniques Used: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
    Figure Legend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    36) Product Images from "Dengue Virus Inhibits Alpha Interferon Signaling by Reducing STAT2 Expression"

    Article Title: Dengue Virus Inhibits Alpha Interferon Signaling by Reducing STAT2 Expression

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.9.5414-5420.2005

    Dengue virus RNA replication inhibits STAT1 and STAT2 phosphorylation in response to IFN-α and reduces steady-state levels of STAT2. (A) K562, K562.ΔCprME-PAC2A, and cured K562 cells; (B) THP-1 and THP-1.ΔCprME-PAC2A cells. Cells
    Figure Legend Snippet: Dengue virus RNA replication inhibits STAT1 and STAT2 phosphorylation in response to IFN-α and reduces steady-state levels of STAT2. (A) K562, K562.ΔCprME-PAC2A, and cured K562 cells; (B) THP-1 and THP-1.ΔCprME-PAC2A cells. Cells

    Techniques Used:

    37) Product Images from "STAT1 is required for IL-6 mediated Bcl6 induction for early Tfh differentiation"

    Article Title: STAT1 is required for IL-6 mediated Bcl6 induction for early Tfh differentiation

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1203032

    Both STAT1 and STAT3 are required for early Tfh differentiation of CD4 T cells (A) Ctrl- or STAT1 KD SM (CD45.1 + ) cells were stimulated with IL-6 for pSTAT stainings. Overlaid histograms of pSTAT1 and pSTAT3. (B) Ctrl- or STAT1 KD SM cells were transferred into B6 mice that were infected with LCMV. Gates indicate Bcl6 hi CXCR5 hi at day 2 after infection. % of Bcl6 + CXCR5 + Tfh cells among total SM cells. (C–E) Ctrl-, STAT1 KD , STAT3 −/− , or STAT3 −/− STAT1 KD SM cells were transferred into B6 mice. SM analyzed at day 3 after infection. (C) Gates indicate Bcl6 + CXCR5 + Tfh cells. (D) % of Tfh cells calculated. (E) CXCR5 MFIs were normalized with cell size of donor cells (CXCR5 MFI /FSC MFI ). Data are representative of two (C–E) and three (A–B) independent experiments (n = 4–5 mice per group). ** P
    Figure Legend Snippet: Both STAT1 and STAT3 are required for early Tfh differentiation of CD4 T cells (A) Ctrl- or STAT1 KD SM (CD45.1 + ) cells were stimulated with IL-6 for pSTAT stainings. Overlaid histograms of pSTAT1 and pSTAT3. (B) Ctrl- or STAT1 KD SM cells were transferred into B6 mice that were infected with LCMV. Gates indicate Bcl6 hi CXCR5 hi at day 2 after infection. % of Bcl6 + CXCR5 + Tfh cells among total SM cells. (C–E) Ctrl-, STAT1 KD , STAT3 −/− , or STAT3 −/− STAT1 KD SM cells were transferred into B6 mice. SM analyzed at day 3 after infection. (C) Gates indicate Bcl6 + CXCR5 + Tfh cells. (D) % of Tfh cells calculated. (E) CXCR5 MFIs were normalized with cell size of donor cells (CXCR5 MFI /FSC MFI ). Data are representative of two (C–E) and three (A–B) independent experiments (n = 4–5 mice per group). ** P

    Techniques Used: Mouse Assay, Infection

    STAT1 is necessary for early Tfh differentiation after VACV infection Ctrl- or STAT1 KD SM (CD45.1 + ) cells were transferred into B6 mice that were infected with VACV-gpc. Bcl6 + CXCR5 + Tfh cells gated and calculated as % of total SM cells at day 2 (A) and 3 (B) after infection. Data are representative of two independent experiments (n = 4–5 mice per group). ** P
    Figure Legend Snippet: STAT1 is necessary for early Tfh differentiation after VACV infection Ctrl- or STAT1 KD SM (CD45.1 + ) cells were transferred into B6 mice that were infected with VACV-gpc. Bcl6 + CXCR5 + Tfh cells gated and calculated as % of total SM cells at day 2 (A) and 3 (B) after infection. Data are representative of two independent experiments (n = 4–5 mice per group). ** P

    Techniques Used: Infection, Mouse Assay, Gel Permeation Chromatography

    38) Product Images from "STAT1 is required for IL-6 mediated Bcl6 induction for early Tfh differentiation"

    Article Title: STAT1 is required for IL-6 mediated Bcl6 induction for early Tfh differentiation

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1203032

    Both STAT1 and STAT3 are required for early Tfh differentiation of CD4 T cells (A) Ctrl- or STAT1 KD SM (CD45.1 + ) cells were stimulated with IL-6 for pSTAT stainings. Overlaid histograms of pSTAT1 and pSTAT3. (B) Ctrl- or STAT1 KD SM cells were transferred into B6 mice that were infected with LCMV. Gates indicate Bcl6 hi CXCR5 hi at day 2 after infection. % of Bcl6 + CXCR5 + Tfh cells among total SM cells. (C–E) Ctrl-, STAT1 KD , STAT3 −/− , or STAT3 −/− STAT1 KD SM cells were transferred into B6 mice. SM analyzed at day 3 after infection. (C) Gates indicate Bcl6 + CXCR5 + Tfh cells. (D) % of Tfh cells calculated. (E) CXCR5 MFIs were normalized with cell size of donor cells (CXCR5 MFI /FSC MFI ). Data are representative of two (C–E) and three (A–B) independent experiments (n = 4–5 mice per group). ** P
    Figure Legend Snippet: Both STAT1 and STAT3 are required for early Tfh differentiation of CD4 T cells (A) Ctrl- or STAT1 KD SM (CD45.1 + ) cells were stimulated with IL-6 for pSTAT stainings. Overlaid histograms of pSTAT1 and pSTAT3. (B) Ctrl- or STAT1 KD SM cells were transferred into B6 mice that were infected with LCMV. Gates indicate Bcl6 hi CXCR5 hi at day 2 after infection. % of Bcl6 + CXCR5 + Tfh cells among total SM cells. (C–E) Ctrl-, STAT1 KD , STAT3 −/− , or STAT3 −/− STAT1 KD SM cells were transferred into B6 mice. SM analyzed at day 3 after infection. (C) Gates indicate Bcl6 + CXCR5 + Tfh cells. (D) % of Tfh cells calculated. (E) CXCR5 MFIs were normalized with cell size of donor cells (CXCR5 MFI /FSC MFI ). Data are representative of two (C–E) and three (A–B) independent experiments (n = 4–5 mice per group). ** P

    Techniques Used: Mouse Assay, Infection

    STAT1 is necessary for early Tfh differentiation after VACV infection Ctrl- or STAT1 KD SM (CD45.1 + ) cells were transferred into B6 mice that were infected with VACV-gpc. Bcl6 + CXCR5 + Tfh cells gated and calculated as % of total SM cells at day 2 (A) and 3 (B) after infection. Data are representative of two independent experiments (n = 4–5 mice per group). ** P
    Figure Legend Snippet: STAT1 is necessary for early Tfh differentiation after VACV infection Ctrl- or STAT1 KD SM (CD45.1 + ) cells were transferred into B6 mice that were infected with VACV-gpc. Bcl6 + CXCR5 + Tfh cells gated and calculated as % of total SM cells at day 2 (A) and 3 (B) after infection. Data are representative of two independent experiments (n = 4–5 mice per group). ** P

    Techniques Used: Infection, Mouse Assay, Gel Permeation Chromatography

    39) Product Images from "STAT1 is required for IL-6 mediated Bcl6 induction for early Tfh differentiation"

    Article Title: STAT1 is required for IL-6 mediated Bcl6 induction for early Tfh differentiation

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1203032

    Both STAT1 and STAT3 are required for early Tfh differentiation of CD4 T cells (A) Ctrl- or STAT1 KD SM (CD45.1 + ) cells were stimulated with IL-6 for pSTAT stainings. Overlaid histograms of pSTAT1 and pSTAT3. (B) Ctrl- or STAT1 KD SM cells were transferred into B6 mice that were infected with LCMV. Gates indicate Bcl6 hi CXCR5 hi at day 2 after infection. % of Bcl6 + CXCR5 + Tfh cells among total SM cells. (C–E) Ctrl-, STAT1 KD , STAT3 −/− , or STAT3 −/− STAT1 KD SM cells were transferred into B6 mice. SM analyzed at day 3 after infection. (C) Gates indicate Bcl6 + CXCR5 + Tfh cells. (D) % of Tfh cells calculated. (E) CXCR5 MFIs were normalized with cell size of donor cells (CXCR5 MFI /FSC MFI ). Data are representative of two (C–E) and three (A–B) independent experiments (n = 4–5 mice per group). ** P
    Figure Legend Snippet: Both STAT1 and STAT3 are required for early Tfh differentiation of CD4 T cells (A) Ctrl- or STAT1 KD SM (CD45.1 + ) cells were stimulated with IL-6 for pSTAT stainings. Overlaid histograms of pSTAT1 and pSTAT3. (B) Ctrl- or STAT1 KD SM cells were transferred into B6 mice that were infected with LCMV. Gates indicate Bcl6 hi CXCR5 hi at day 2 after infection. % of Bcl6 + CXCR5 + Tfh cells among total SM cells. (C–E) Ctrl-, STAT1 KD , STAT3 −/− , or STAT3 −/− STAT1 KD SM cells were transferred into B6 mice. SM analyzed at day 3 after infection. (C) Gates indicate Bcl6 + CXCR5 + Tfh cells. (D) % of Tfh cells calculated. (E) CXCR5 MFIs were normalized with cell size of donor cells (CXCR5 MFI /FSC MFI ). Data are representative of two (C–E) and three (A–B) independent experiments (n = 4–5 mice per group). ** P

    Techniques Used: Mouse Assay, Infection

    STAT1 is necessary for early Tfh differentiation after VACV infection Ctrl- or STAT1 KD SM (CD45.1 + ) cells were transferred into B6 mice that were infected with VACV-gpc. Bcl6 + CXCR5 + Tfh cells gated and calculated as % of total SM cells at day 2 (A) and 3 (B) after infection. Data are representative of two independent experiments (n = 4–5 mice per group). ** P
    Figure Legend Snippet: STAT1 is necessary for early Tfh differentiation after VACV infection Ctrl- or STAT1 KD SM (CD45.1 + ) cells were transferred into B6 mice that were infected with VACV-gpc. Bcl6 + CXCR5 + Tfh cells gated and calculated as % of total SM cells at day 2 (A) and 3 (B) after infection. Data are representative of two independent experiments (n = 4–5 mice per group). ** P

    Techniques Used: Infection, Mouse Assay, Gel Permeation Chromatography

    40) Product Images from "Tumor STAT1 Transcription Factor Activity Enhances Breast Tumor Growth and Immune Suppression Mediated by Myeloid-derived Suppressor Cells *"

    Article Title: Tumor STAT1 Transcription Factor Activity Enhances Breast Tumor Growth and Immune Suppression Mediated by Myeloid-derived Suppressor Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.441402

    Tumor STAT1 expression and MDSC recruitment in human breast tumors correlates with disease progression. A and B , immunohistochemical staining of phospho-STAT1 in human DCIS and invasive tumor tissue sections. A , representative images of DCIS and invasive carcinoma. Nuclear staining with hematoxylin is indicated in purple , and cells staining positive for phospho-STAT1 (Tyr 701 ; Cell Signaling) using 3,3′-diaminobenzidine are in brown (×20 magnification). The arrows indicate phospho-STAT1-positive cells. B , histogram quantifying the total number of phospho-STAT1 + cells/total hematoxylin + tumor cells. Data are mean ± S.E., 5 slides/tumor group, compiled from two independent experiments (a total of 10 samples for each group). **, p ≤ 0.01, Student's t test. C and D , immunofluorescence analysis of the expression of CD33 + MDSCs in human DCIS and invasive tumor tissue sections. Black scale bar , 50 μm. C , representative images of DCIS and invasive carcinoma. Nuclear staining with DAPI is indicated in blue , and cells staining positive for anti-CD33 + (BD Biosciences) are in red . The arrows indicate CD33 + cells. D , histogram quantifying CD33 + staining in tumors. Total numbers of defined red CD33 + cells were counted per field and averaged from five randomly selected fields at the same magnification (×10) for each slide. White scale bar , 50 μm. Data are mean ± S.E. ( error bars ), 10 tumor slides/group, compiled from two independent experiments. ***, p ≤ 0.001, Student's t test.
    Figure Legend Snippet: Tumor STAT1 expression and MDSC recruitment in human breast tumors correlates with disease progression. A and B , immunohistochemical staining of phospho-STAT1 in human DCIS and invasive tumor tissue sections. A , representative images of DCIS and invasive carcinoma. Nuclear staining with hematoxylin is indicated in purple , and cells staining positive for phospho-STAT1 (Tyr 701 ; Cell Signaling) using 3,3′-diaminobenzidine are in brown (×20 magnification). The arrows indicate phospho-STAT1-positive cells. B , histogram quantifying the total number of phospho-STAT1 + cells/total hematoxylin + tumor cells. Data are mean ± S.E., 5 slides/tumor group, compiled from two independent experiments (a total of 10 samples for each group). **, p ≤ 0.01, Student's t test. C and D , immunofluorescence analysis of the expression of CD33 + MDSCs in human DCIS and invasive tumor tissue sections. Black scale bar , 50 μm. C , representative images of DCIS and invasive carcinoma. Nuclear staining with DAPI is indicated in blue , and cells staining positive for anti-CD33 + (BD Biosciences) are in red . The arrows indicate CD33 + cells. D , histogram quantifying CD33 + staining in tumors. Total numbers of defined red CD33 + cells were counted per field and averaged from five randomly selected fields at the same magnification (×10) for each slide. White scale bar , 50 μm. Data are mean ± S.E. ( error bars ), 10 tumor slides/group, compiled from two independent experiments. ***, p ≤ 0.001, Student's t test.

    Techniques Used: Expressing, Immunohistochemistry, Staining, Immunofluorescence

    Recruitment of Gr1 + CD11b + MDSCs and suppressive function MDSCs via arginase 1. A , Gr1 + CD11b + MDSCs recruited to STAT1-overexpressing tumors are suppressive MDSCs. Arginase activity was assessed in TM40D-MB tumor extracts and purified Gr1 + cell lysates from TM40D-MB tumors and compared with control CD11b + cells from naive bone marrow. Data are mean ± S.D. ( error bars ) and are representative of two independent experiments. **, p ≤ 0.01, Student's t test. U / L , units/liter. B , tumor CD11b + cells suppress T cell proliferation. T cells co-cultured with Cd11b + cells from TM40D-MB primary tumor exhibited suppressed T cell proliferation. Data are mean ± S.D. C and D , STAT1 tumor-derived factors promote direct migration of CD11b + myeloid cells in vitro . Splenocytes were harvested from naive mice, and CD11b + cells were purified using MACS columns (Miltenyi Biotec). Filtered cell culture supernatants from each tumor cell group were harvested after 48 h in culture and plated in the bottom wells of a 5-μm 96-well chemotaxis plate (Neuroprobe), with serum-free medium as a control. CD11b + cells ( C ) or CD11b − MNCs ( D ) were resuspended at 1 × 10 6 /ml in serum-free RPMI medium and plated onto the membrane and incubated for 4 h at 37 °C. Migrated cells were counted using inverted microscopy and ImageJ software. Experiments were performed in triplicate, and data are representative of two independent experiments. Data are mean ± S.D. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; Student's t test.
    Figure Legend Snippet: Recruitment of Gr1 + CD11b + MDSCs and suppressive function MDSCs via arginase 1. A , Gr1 + CD11b + MDSCs recruited to STAT1-overexpressing tumors are suppressive MDSCs. Arginase activity was assessed in TM40D-MB tumor extracts and purified Gr1 + cell lysates from TM40D-MB tumors and compared with control CD11b + cells from naive bone marrow. Data are mean ± S.D. ( error bars ) and are representative of two independent experiments. **, p ≤ 0.01, Student's t test. U / L , units/liter. B , tumor CD11b + cells suppress T cell proliferation. T cells co-cultured with Cd11b + cells from TM40D-MB primary tumor exhibited suppressed T cell proliferation. Data are mean ± S.D. C and D , STAT1 tumor-derived factors promote direct migration of CD11b + myeloid cells in vitro . Splenocytes were harvested from naive mice, and CD11b + cells were purified using MACS columns (Miltenyi Biotec). Filtered cell culture supernatants from each tumor cell group were harvested after 48 h in culture and plated in the bottom wells of a 5-μm 96-well chemotaxis plate (Neuroprobe), with serum-free medium as a control. CD11b + cells ( C ) or CD11b − MNCs ( D ) were resuspended at 1 × 10 6 /ml in serum-free RPMI medium and plated onto the membrane and incubated for 4 h at 37 °C. Migrated cells were counted using inverted microscopy and ImageJ software. Experiments were performed in triplicate, and data are representative of two independent experiments. Data are mean ± S.D. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; Student's t test.

    Techniques Used: Activity Assay, Purification, Cell Culture, Derivative Assay, Migration, In Vitro, Mouse Assay, Magnetic Cell Separation, Chemotaxis Assay, Incubation, Inverted Microscopy, Software

    STAT1 modulates tumor cytokine production in tumor cells in vitro . A , analysis of tumor cytokine expression by real-time RT-PCR. RNA was harvested from TM40D-MB-pSM2 and TM40D-MB-shSTAT1 tumor cells grown in culture for 48 h, with Dox (1 μg/ml) added to the TM40D-TetOn and TM40D-STAT1C tumor cells. Reverse transcribed cDNA was assessed by real-time RT-PCR using SYBR Green and the appropriate primer pairs, with GAPDH as the endogenous control. Representative gels for each cytokine assessed by real-time RT-PCR are shown to the right ( B ). C , TNFα is a driver of tumor-derived CD11b + cell migration. TM40D-MB primary tumor CD11b + cell migration is attenuated when TNFα in TM40D-STAT1 conditioned medium is inhibited. Experiments were performed in triplicate and are representative of two independent experiments. Data are mean ± S.E. ( error bars ). **, p ≤ 0.01; Student's t test.
    Figure Legend Snippet: STAT1 modulates tumor cytokine production in tumor cells in vitro . A , analysis of tumor cytokine expression by real-time RT-PCR. RNA was harvested from TM40D-MB-pSM2 and TM40D-MB-shSTAT1 tumor cells grown in culture for 48 h, with Dox (1 μg/ml) added to the TM40D-TetOn and TM40D-STAT1C tumor cells. Reverse transcribed cDNA was assessed by real-time RT-PCR using SYBR Green and the appropriate primer pairs, with GAPDH as the endogenous control. Representative gels for each cytokine assessed by real-time RT-PCR are shown to the right ( B ). C , TNFα is a driver of tumor-derived CD11b + cell migration. TM40D-MB primary tumor CD11b + cell migration is attenuated when TNFα in TM40D-STAT1 conditioned medium is inhibited. Experiments were performed in triplicate and are representative of two independent experiments. Data are mean ± S.E. ( error bars ). **, p ≤ 0.01; Student's t test.

    Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, SYBR Green Assay, Derivative Assay, Migration

    STAT1 expression in tumor cells correlates with tumor growth rates in vivo . A and B , low metastatic TM40D tumor cells were transduced with a Dox-inducible retroviral vector (TetOn, Clontech) containing a constitutive STAT1 cDNA construct (TM40D-STAT1C) or vector control (TM40D-TetOn). Cells were cultured with or without Dox (1 μg/ml) as indicated for 48 h. Highly metastatic TM40D-MB cells were transduced with an shRNA against STAT1 (TM40D-MB-shSTAT1) or vector control (TM40D-MB-pSM2). Cells were additionally treated with or without IFN-γ (5 ng/ml) for 24 h as a positive control for STAT1 induction. A , cells were lysed, and total protein was assessed by immunoblot for STAT1α and phospho-Tyr 701 -STAT1 (Cell Signaling) expression, with α-actin as a loading control. B , histogram depicting -fold protein expression differences (compared with TM40D) of STAT1α in all cells tested. Data are representative of at least three independent experiments. C , orthotopic injection into the bilateral mammary fat pads of BALB/c mice with either 1 × 10 6 low STAT1-expressing tumor cells transduced with vector (TM40D-TetOn) or constitutive STAT1-expressing cells (TM40D-STAT1C). Mice were administered Dox (2 mg/ml) in drinking water and monitored for tumor growth by caliper measurement, and mice were euthanized at maximum tumor size (2 cm). For day 8 (the earliest significant difference detected), asterisks indicate p = 0.011, Student's t test. Results represent a total of 10 animals in each tumor group, compiled from two independent experiments. D , mice were implanted with either high STAT1-expressing cells transduced with vector (TM40D-MB-pSM2) or TM40D-MB shSTAT1 knockdown cells (TM40D-MB-shSTAT1). Mice were monitored for tumor growth, and maximum tumor size was scored as end point for survival. *, p = 0.02, log rank test. Results represent a total of 10 animals in each tumor group, compiled from two independent experiments.
    Figure Legend Snippet: STAT1 expression in tumor cells correlates with tumor growth rates in vivo . A and B , low metastatic TM40D tumor cells were transduced with a Dox-inducible retroviral vector (TetOn, Clontech) containing a constitutive STAT1 cDNA construct (TM40D-STAT1C) or vector control (TM40D-TetOn). Cells were cultured with or without Dox (1 μg/ml) as indicated for 48 h. Highly metastatic TM40D-MB cells were transduced with an shRNA against STAT1 (TM40D-MB-shSTAT1) or vector control (TM40D-MB-pSM2). Cells were additionally treated with or without IFN-γ (5 ng/ml) for 24 h as a positive control for STAT1 induction. A , cells were lysed, and total protein was assessed by immunoblot for STAT1α and phospho-Tyr 701 -STAT1 (Cell Signaling) expression, with α-actin as a loading control. B , histogram depicting -fold protein expression differences (compared with TM40D) of STAT1α in all cells tested. Data are representative of at least three independent experiments. C , orthotopic injection into the bilateral mammary fat pads of BALB/c mice with either 1 × 10 6 low STAT1-expressing tumor cells transduced with vector (TM40D-TetOn) or constitutive STAT1-expressing cells (TM40D-STAT1C). Mice were administered Dox (2 mg/ml) in drinking water and monitored for tumor growth by caliper measurement, and mice were euthanized at maximum tumor size (2 cm). For day 8 (the earliest significant difference detected), asterisks indicate p = 0.011, Student's t test. Results represent a total of 10 animals in each tumor group, compiled from two independent experiments. D , mice were implanted with either high STAT1-expressing cells transduced with vector (TM40D-MB-pSM2) or TM40D-MB shSTAT1 knockdown cells (TM40D-MB-shSTAT1). Mice were monitored for tumor growth, and maximum tumor size was scored as end point for survival. *, p = 0.02, log rank test. Results represent a total of 10 animals in each tumor group, compiled from two independent experiments.

    Techniques Used: Expressing, In Vivo, Transduction, Plasmid Preparation, Construct, Cell Culture, shRNA, Positive Control, Injection, Mouse Assay

    STAT1 modulates T cell recruitment in the tumor microenvironment. A–C , tumors were harvested from mice at maximum tumor size, and live CD45 + cells were assessed by FACS for populations of cells staining for CD4-PE and CD8α-PerCP-Cy5.5. Shown are representative pictures ( A ) and a histogram quantifying the total tumor frequencies of CD4 + ( B ) and CD8 + ( C ) T cells. Data are mean ± S.D. ( error bars ), 5 mice/TM40D-TetOn and TM40D-MB-pSM2 tumor group, and 9 mice/TM40D-STAT1C and TM40D-MB-shSTAT1 tumor group. Data are representative of two independent experiments. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001, Mann-Whitney test.
    Figure Legend Snippet: STAT1 modulates T cell recruitment in the tumor microenvironment. A–C , tumors were harvested from mice at maximum tumor size, and live CD45 + cells were assessed by FACS for populations of cells staining for CD4-PE and CD8α-PerCP-Cy5.5. Shown are representative pictures ( A ) and a histogram quantifying the total tumor frequencies of CD4 + ( B ) and CD8 + ( C ) T cells. Data are mean ± S.D. ( error bars ), 5 mice/TM40D-TetOn and TM40D-MB-pSM2 tumor group, and 9 mice/TM40D-STAT1C and TM40D-MB-shSTAT1 tumor group. Data are representative of two independent experiments. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001, Mann-Whitney test.

    Techniques Used: Mouse Assay, FACS, Staining, MANN-WHITNEY

    STAT1 promotes systemic Gr1 + CD11b + MDSC expansion and primary tumor recruitment. Spleens and hind leg bones were harvested from mice at maximum tumor size. Shown are representative pictures ( A ) and a histogram ( B ) quantifying the frequency of live splenocytes analyzed by flow cytometry (BD FACS Canto II) positive for expression of MDSC surface markers Gr1-PerCP-Cy5.5 and CD11b-PE. Data are mean ± S.D. ( error bars ), 5 mice/tumor group. C , histogram quantifying the frequency of live Gr1 + CD11b + bone marrow cells analyzed by FACS. Data are mean ± S.D., 5 mice/tumor group. Data are representative of two independent experiments. D and E , formalin-fixed sections of tumors from three mice in each tumor group were prepared as described. Slides were stained with biotinylated anti-mouse Gr1 (BD Pharmingen) and counterstained with hematoxylin (eBioscience). For each tumor group, 15 slides/tumor were counted and quantified using ImageJ. D , representative pictures of Gr1 + cells (in red ) for each tumor group. Black scale bar , 50 μm. E , histogram quantifying the total number of Gr1 + cell counts for each tumor group. Data are mean ± S.E., 3 mice/tumor group. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001, Student's t test.
    Figure Legend Snippet: STAT1 promotes systemic Gr1 + CD11b + MDSC expansion and primary tumor recruitment. Spleens and hind leg bones were harvested from mice at maximum tumor size. Shown are representative pictures ( A ) and a histogram ( B ) quantifying the frequency of live splenocytes analyzed by flow cytometry (BD FACS Canto II) positive for expression of MDSC surface markers Gr1-PerCP-Cy5.5 and CD11b-PE. Data are mean ± S.D. ( error bars ), 5 mice/tumor group. C , histogram quantifying the frequency of live Gr1 + CD11b + bone marrow cells analyzed by FACS. Data are mean ± S.D., 5 mice/tumor group. Data are representative of two independent experiments. D and E , formalin-fixed sections of tumors from three mice in each tumor group were prepared as described. Slides were stained with biotinylated anti-mouse Gr1 (BD Pharmingen) and counterstained with hematoxylin (eBioscience). For each tumor group, 15 slides/tumor were counted and quantified using ImageJ. D , representative pictures of Gr1 + cells (in red ) for each tumor group. Black scale bar , 50 μm. E , histogram quantifying the total number of Gr1 + cell counts for each tumor group. Data are mean ± S.E., 3 mice/tumor group. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001, Student's t test.

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, FACS, Expressing, Staining

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    Article Snippet: .. Western Blotting The protein levels of STAT1, P-STAT1, caspase3, caspase9, C-caspase3, C-caspase9, cytochrome c, Bax, GADPH, and Bcl-2 were detected via Western blotting. .. Total cellular proteins were isolated using a radio immunoprecipitation assay (RIPA) lysis buffer [with 1% phenylmethane sulfonyl fluoride (PMSF)] and the protein concentration was subsequently quantified using the bicinchoninic acid assay (BCA) protein assay kit (Beyotime).

    other:

    Article Title: Secretome of Hypoxic Endothelial Cells Stimulates Bone Marrow-Derived Mesenchymal Stem Cells to Enhance Alternative Activation of Macrophages
    Article Snippet: The immunoblotting of p-STAT1 and NF-κB were used to assess the intracellular signaling induced by IFN-γ and TNF-α, respectively.

    Staining:

    Article Title: The Protective Effect of Luteolin in Glucocorticoid-Induced Osteonecrosis of the Femoral Head
    Article Snippet: .. The cells were then blocked with 5% goat serum for 1 h at 37°C, rinsed three times with PBS, stained with antibodies against P-STAT1 (1:200) and C- caspase3 (1:200) at 4°C overnight, and then stained with Alexa Fluor® 594 or Fluor® 488-conjugated secondary antibody (1:400) for 1 h. Following this, the cells were stained by DAPI (Invitrogen) for 1 min. .. Finally, the cells were imaged via fluorescence microscopy (Olympus Inc, Tokyo, Japan).

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  • 95
    Thermo Fisher p stat1
    Hypoxic endothelial cells activate the MSCs intracellular signaling pathways of IFN-γ and TNF-α. The culture media obtained from endothelial cells subjected to hypoxia for 4 h were used for culturing HCELL-positive MSCs in the presence of the inhibitors of signaling pathways of IFN-γ ( a ) and TNF-α (b), whereas those from intact endothelial cells were used for assessing recombinant IFN-γ ( a ) and TNF-α ( b ) stimulation. After 24 hours’ culture in the depicted experimental settings, MSCs pellets were subjected to Western blotting to measure <t>p-STAT1</t> and NF-κB for assessing IFN-γ and TNF-α pathway activation respectively, while the supernatant was used to measure IL-13 level with ELISA. The representative Western blottings are shown. The quantitative results of Western blotting were adjusted according to β-actin level. n = 8 in each group; a, p
    P Stat1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher stat1 knockdown sequence
    Effect of FGF23 on the phosphorylation of <t>STAT1</t> induced by erythropoietin. Notes: mIMCD3 cells were stimulated with the following: 5 U/mL erythropoietin alone; 10 ng/mL FGF23 alone or 5 U/mL erythropoietin plus 10, 20 or 50 ng/mL FGF23. Phosphorylated STAT1 was detected by Western blotting. Abbreviations: PC, positive control for phosphorylated STAT1 (control cell extract); none, unstimulated cells; Epo, erythropoietin; p-STAT1, phosphorylated STAT1; GAPDH, control for total protein loading.
    Stat1 Knockdown Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp stat1 hs01014003 m1
    miR-146a-5p and miR-155-5p mechanism of action under normal conditions and early stages of inflammation ( 2 , 9 , 34 ). During the early stages of inflammation in GVHD, the TLR and Janus Kinase <t>(JAK)-STAT1</t> pathway are activated by LPS and IFN-γ, respectively. Under such conditions, miR-146a-5p and miR-155-5p have reduced expression levels. This results in minor alterations of the mRNA target levels and may therefore represent subclinical early inflammatory GVHD.
    Gene Exp Stat1 Hs01014003 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hypoxic endothelial cells activate the MSCs intracellular signaling pathways of IFN-γ and TNF-α. The culture media obtained from endothelial cells subjected to hypoxia for 4 h were used for culturing HCELL-positive MSCs in the presence of the inhibitors of signaling pathways of IFN-γ ( a ) and TNF-α (b), whereas those from intact endothelial cells were used for assessing recombinant IFN-γ ( a ) and TNF-α ( b ) stimulation. After 24 hours’ culture in the depicted experimental settings, MSCs pellets were subjected to Western blotting to measure p-STAT1 and NF-κB for assessing IFN-γ and TNF-α pathway activation respectively, while the supernatant was used to measure IL-13 level with ELISA. The representative Western blottings are shown. The quantitative results of Western blotting were adjusted according to β-actin level. n = 8 in each group; a, p

    Journal: International Journal of Molecular Sciences

    Article Title: Secretome of Hypoxic Endothelial Cells Stimulates Bone Marrow-Derived Mesenchymal Stem Cells to Enhance Alternative Activation of Macrophages

    doi: 10.3390/ijms21124409

    Figure Lengend Snippet: Hypoxic endothelial cells activate the MSCs intracellular signaling pathways of IFN-γ and TNF-α. The culture media obtained from endothelial cells subjected to hypoxia for 4 h were used for culturing HCELL-positive MSCs in the presence of the inhibitors of signaling pathways of IFN-γ ( a ) and TNF-α (b), whereas those from intact endothelial cells were used for assessing recombinant IFN-γ ( a ) and TNF-α ( b ) stimulation. After 24 hours’ culture in the depicted experimental settings, MSCs pellets were subjected to Western blotting to measure p-STAT1 and NF-κB for assessing IFN-γ and TNF-α pathway activation respectively, while the supernatant was used to measure IL-13 level with ELISA. The representative Western blottings are shown. The quantitative results of Western blotting were adjusted according to β-actin level. n = 8 in each group; a, p

    Article Snippet: The immunoblotting of p-STAT1 and NF-κB were used to assess the intracellular signaling induced by IFN-γ and TNF-α, respectively.

    Techniques: Recombinant, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay

    Interferon response-like signalling is upregulated in amoeboid cells. ( a ) Ten most significant GO Biological Process terms enriched in the genes affected concordantly by both iRhoA and dasatinib (DAS) treatment. Only transcripts with adjusted p -value ≤ 0.25 and fold change > 1.5 in either direction were considered differentially expressed. Network plot was generated using ShinyGO v0.61 online tool [ 30 ]. ( b ) Gene expression (log2 fold change) of STAT1, STAT2, STAT3 and IRF9 in cells after mesenchymal–amoeboid transition (MAT) in 3D collagen (48 h) determined by RT-qPCR, normalized to control cells without MAT induction. ( c ) Immunoblotting detection of Stat transcription factors Stat1, Stat2 and Stat3 in HT1080 cells before and after MAT. p -values: ** p

    Journal: Cancers

    Article Title: Sustained Inflammatory Signalling through Stat1/Stat2/IRF9 Is Associated with Amoeboid Phenotype of Melanoma Cells

    doi: 10.3390/cancers12092450

    Figure Lengend Snippet: Interferon response-like signalling is upregulated in amoeboid cells. ( a ) Ten most significant GO Biological Process terms enriched in the genes affected concordantly by both iRhoA and dasatinib (DAS) treatment. Only transcripts with adjusted p -value ≤ 0.25 and fold change > 1.5 in either direction were considered differentially expressed. Network plot was generated using ShinyGO v0.61 online tool [ 30 ]. ( b ) Gene expression (log2 fold change) of STAT1, STAT2, STAT3 and IRF9 in cells after mesenchymal–amoeboid transition (MAT) in 3D collagen (48 h) determined by RT-qPCR, normalized to control cells without MAT induction. ( c ) Immunoblotting detection of Stat transcription factors Stat1, Stat2 and Stat3 in HT1080 cells before and after MAT. p -values: ** p

    Article Snippet: The following primary antibodies were used: P-Stat1 (Thermo Fisher Scientific, Waltham, MA, USA; #MA5-15071), P-Stat2 (CST, Danvers, MA, USA; #88410), P-Stat3 (CST; #9145), Stat1 (Thermo Fisher Scientific; #MA5-15129), Stat2 (CST; #72604), Stat3 (CST; #12640), IRF9 (CST; #76684), Jak1 (CST; #3344), MX1 (CST; #37849) and GAPDH (Thermo Fisher Scientific; #MA5-15738).

    Techniques: Generated, Expressing, Quantitative RT-PCR

    Role of IFN signalling in melanoma invasion plasticity. ( a ) Inhibition of Jak1/2 by Ruxolitinib promotes the elongated, mesenchymal phenotype of melanoma cells cultured in 3D collagen (48 h). ( b ) Representative image of WM3629 cells after 48 h in 3D collagen treated with DMSO or Ruxolitinib. ( c ) Quantification of morphology of WM3629 cells treated with IFNβ alone or IFNβ plus Ruxolitinib after 48 h in collagen. ( d ) Quantification of morphology of melanoma cells cultured in 3D collagen for 48 h after treatment with IFNs (overall exposure to IFNs took 96 h). ( e ) Representative images of WM3629 cells after 48 h in 3D collagen treated with IFNs. ( f ) Immunoblotting detection of Stat transcription factors Stat1, Stat2 and Stat3 activation after 1 h and 48 h IFN treatment in WM3629 cells. Scale bar 100 µm in both ( b ) and ( e ). R = round, E = elongated. p -values: *** p

    Journal: Cancers

    Article Title: Sustained Inflammatory Signalling through Stat1/Stat2/IRF9 Is Associated with Amoeboid Phenotype of Melanoma Cells

    doi: 10.3390/cancers12092450

    Figure Lengend Snippet: Role of IFN signalling in melanoma invasion plasticity. ( a ) Inhibition of Jak1/2 by Ruxolitinib promotes the elongated, mesenchymal phenotype of melanoma cells cultured in 3D collagen (48 h). ( b ) Representative image of WM3629 cells after 48 h in 3D collagen treated with DMSO or Ruxolitinib. ( c ) Quantification of morphology of WM3629 cells treated with IFNβ alone or IFNβ plus Ruxolitinib after 48 h in collagen. ( d ) Quantification of morphology of melanoma cells cultured in 3D collagen for 48 h after treatment with IFNs (overall exposure to IFNs took 96 h). ( e ) Representative images of WM3629 cells after 48 h in 3D collagen treated with IFNs. ( f ) Immunoblotting detection of Stat transcription factors Stat1, Stat2 and Stat3 activation after 1 h and 48 h IFN treatment in WM3629 cells. Scale bar 100 µm in both ( b ) and ( e ). R = round, E = elongated. p -values: *** p

    Article Snippet: The following primary antibodies were used: P-Stat1 (Thermo Fisher Scientific, Waltham, MA, USA; #MA5-15071), P-Stat2 (CST, Danvers, MA, USA; #88410), P-Stat3 (CST; #9145), Stat1 (Thermo Fisher Scientific; #MA5-15129), Stat2 (CST; #72604), Stat3 (CST; #12640), IRF9 (CST; #76684), Jak1 (CST; #3344), MX1 (CST; #37849) and GAPDH (Thermo Fisher Scientific; #MA5-15738).

    Techniques: Inhibition, Cell Culture, Activation Assay

    Effect of FGF23 on the phosphorylation of STAT1 induced by erythropoietin. Notes: mIMCD3 cells were stimulated with the following: 5 U/mL erythropoietin alone; 10 ng/mL FGF23 alone or 5 U/mL erythropoietin plus 10, 20 or 50 ng/mL FGF23. Phosphorylated STAT1 was detected by Western blotting. Abbreviations: PC, positive control for phosphorylated STAT1 (control cell extract); none, unstimulated cells; Epo, erythropoietin; p-STAT1, phosphorylated STAT1; GAPDH, control for total protein loading.

    Journal: International Journal of Nephrology and Renovascular Disease

    Article Title: FGF23 modulates the effects of erythropoietin on gene expression in renal epithelial cells

    doi: 10.2147/IJNRD.S158422

    Figure Lengend Snippet: Effect of FGF23 on the phosphorylation of STAT1 induced by erythropoietin. Notes: mIMCD3 cells were stimulated with the following: 5 U/mL erythropoietin alone; 10 ng/mL FGF23 alone or 5 U/mL erythropoietin plus 10, 20 or 50 ng/mL FGF23. Phosphorylated STAT1 was detected by Western blotting. Abbreviations: PC, positive control for phosphorylated STAT1 (control cell extract); none, unstimulated cells; Epo, erythropoietin; p-STAT1, phosphorylated STAT1; GAPDH, control for total protein loading.

    Article Snippet: The STAT1 knockdown sequence was inserted into the pcDNA3.1 vector (Thermo Fisher Scientific, Waltham, MA, USA). pcDNA3.1, either with or without the STAT1 knockdown sequence, was transferred into mIMCD3 cells.

    Techniques: Western Blot, Positive Control

    miR-146a-5p and miR-155-5p mechanism of action under normal conditions and early stages of inflammation ( 2 , 9 , 34 ). During the early stages of inflammation in GVHD, the TLR and Janus Kinase (JAK)-STAT1 pathway are activated by LPS and IFN-γ, respectively. Under such conditions, miR-146a-5p and miR-155-5p have reduced expression levels. This results in minor alterations of the mRNA target levels and may therefore represent subclinical early inflammatory GVHD.

    Journal: Frontiers in Immunology

    Article Title: miR-146a and miR-155 Expression Levels in Acute Graft-Versus-Host Disease Incidence

    doi: 10.3389/fimmu.2016.00056

    Figure Lengend Snippet: miR-146a-5p and miR-155-5p mechanism of action under normal conditions and early stages of inflammation ( 2 , 9 , 34 ). During the early stages of inflammation in GVHD, the TLR and Janus Kinase (JAK)-STAT1 pathway are activated by LPS and IFN-γ, respectively. Under such conditions, miR-146a-5p and miR-155-5p have reduced expression levels. This results in minor alterations of the mRNA target levels and may therefore represent subclinical early inflammatory GVHD.

    Article Snippet: The reaction mix was incubated at 37°C for 2 h and 10 min at 65°C. cDNA was used to run qPCR for the miRNA targets [TRAF6 (Hs00371512_g1), IRAK1 (Hs01018347_m1), STAT1-α (Hs01014003_m1), IRF5 (Hs00158114_m1), and SPI1 (Hs02786711_m1)].

    Techniques: Expressing