Structured Review

Santa Cruz Biotechnology stat1
Deletion of <t>STAT1</t> and STAT3 in LepRb expressing neurons. (A) Schematic diagram showing the cross of Lepr cre with Stat1 flox and Stat3 flox mice to generate STAT3 LepRb KO and STAT1STAT3 LepRb KO mice. pA: polyadenylation signal. (B) Representative images showing colocalization of STAT1-IR (red) with GFP-IR (green) in the arcuate nucleus of STAT3 LepR KO and STAT1STAT3 LepR KO (both of which are on the R26 eGFP-L10a background) mice. ( C – E ) Male STAT3 LepRb KO and STAT1STAT3 LepRb KO mice were placed on chow and body weight (C) and cumulative food intake (D) were measured weekly. (E) At 14–15 weeks of age, animals underwent body composition analysis by NMR spectroscopy. Mean, quartiles, and individual plots are shown; n = 8–14 per genotype. ANOVA analysis was performed for (C, D) ; unpaired t-test was performed for (E) . All comparisons p = not significant unless indicated.
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Images

1) Product Images from "Transcriptional and physiological roles for STAT proteins in leptin action"

Article Title: Transcriptional and physiological roles for STAT proteins in leptin action

Journal: Molecular Metabolism

doi: 10.1016/j.molmet.2019.01.007

Deletion of STAT1 and STAT3 in LepRb expressing neurons. (A) Schematic diagram showing the cross of Lepr cre with Stat1 flox and Stat3 flox mice to generate STAT3 LepRb KO and STAT1STAT3 LepRb KO mice. pA: polyadenylation signal. (B) Representative images showing colocalization of STAT1-IR (red) with GFP-IR (green) in the arcuate nucleus of STAT3 LepR KO and STAT1STAT3 LepR KO (both of which are on the R26 eGFP-L10a background) mice. ( C – E ) Male STAT3 LepRb KO and STAT1STAT3 LepRb KO mice were placed on chow and body weight (C) and cumulative food intake (D) were measured weekly. (E) At 14–15 weeks of age, animals underwent body composition analysis by NMR spectroscopy. Mean, quartiles, and individual plots are shown; n = 8–14 per genotype. ANOVA analysis was performed for (C, D) ; unpaired t-test was performed for (E) . All comparisons p = not significant unless indicated.
Figure Legend Snippet: Deletion of STAT1 and STAT3 in LepRb expressing neurons. (A) Schematic diagram showing the cross of Lepr cre with Stat1 flox and Stat3 flox mice to generate STAT3 LepRb KO and STAT1STAT3 LepRb KO mice. pA: polyadenylation signal. (B) Representative images showing colocalization of STAT1-IR (red) with GFP-IR (green) in the arcuate nucleus of STAT3 LepR KO and STAT1STAT3 LepR KO (both of which are on the R26 eGFP-L10a background) mice. ( C – E ) Male STAT3 LepRb KO and STAT1STAT3 LepRb KO mice were placed on chow and body weight (C) and cumulative food intake (D) were measured weekly. (E) At 14–15 weeks of age, animals underwent body composition analysis by NMR spectroscopy. Mean, quartiles, and individual plots are shown; n = 8–14 per genotype. ANOVA analysis was performed for (C, D) ; unpaired t-test was performed for (E) . All comparisons p = not significant unless indicated.

Techniques Used: Expressing, Mouse Assay, Nuclear Magnetic Resonance, Spectroscopy

Transcriptional response to the deletion of Stat3 in LepRb neurons. (A) Schematic diagram showing the Lepr cre -mediated deletion of Stat3 flox mice on the Rosa26 eGFP-L10a background to generate STAT3 LepRb KO-eGFP-L10a mice. (B – C) Expression values (fragments per million reads; FPM) for genes that were enriched in LepRb neurons under any condition, comparing STAT3 LepRb KO mice to control mice ( B ) or ob/ob mice ( C ) [16] were plotted. Black dots represent genes demonstrating statistically significant changes in gene expression for the comparison; some genes of interest are labeled. ( D ) Comparison of fold change in gene expression relative to control for STAT3 LepRb KO and ob/ob mice for all genes enriched in LepRb neurons. Dashed lines represent fold change values of 1.5 and 0.667 for both axes. Black dots represent regulated genes; red dots show genes that are regulated and known to be controlled by STAT1 [38] . Regions of the graph are denoted by Roman numerals for reference by the main text. (E) Representative images showing colocalization of STAT1-IR (red) and GFP-IR (green) in the ARC of Control and STAT3 LepRb KO mice.
Figure Legend Snippet: Transcriptional response to the deletion of Stat3 in LepRb neurons. (A) Schematic diagram showing the Lepr cre -mediated deletion of Stat3 flox mice on the Rosa26 eGFP-L10a background to generate STAT3 LepRb KO-eGFP-L10a mice. (B – C) Expression values (fragments per million reads; FPM) for genes that were enriched in LepRb neurons under any condition, comparing STAT3 LepRb KO mice to control mice ( B ) or ob/ob mice ( C ) [16] were plotted. Black dots represent genes demonstrating statistically significant changes in gene expression for the comparison; some genes of interest are labeled. ( D ) Comparison of fold change in gene expression relative to control for STAT3 LepRb KO and ob/ob mice for all genes enriched in LepRb neurons. Dashed lines represent fold change values of 1.5 and 0.667 for both axes. Black dots represent regulated genes; red dots show genes that are regulated and known to be controlled by STAT1 [38] . Regions of the graph are denoted by Roman numerals for reference by the main text. (E) Representative images showing colocalization of STAT1-IR (red) and GFP-IR (green) in the ARC of Control and STAT3 LepRb KO mice.

Techniques Used: Mouse Assay, Expressing, Labeling

2) Product Images from "Transcriptional and physiological roles for STAT proteins in leptin action"

Article Title: Transcriptional and physiological roles for STAT proteins in leptin action

Journal: Molecular Metabolism

doi: 10.1016/j.molmet.2019.01.007

Deletion of STAT1 and STAT3 in LepRb expressing neurons. (A) Schematic diagram showing the cross of Lepr cre with Stat1 flox and Stat3 flox mice to generate STAT3 LepRb KO and STAT1STAT3 LepRb KO mice. pA: polyadenylation signal. (B) Representative images showing colocalization of STAT1-IR (red) with GFP-IR (green) in the arcuate nucleus of STAT3 LepR KO and STAT1STAT3 LepR KO (both of which are on the R26 eGFP-L10a background) mice. ( C – E ) Male STAT3 LepRb KO and STAT1STAT3 LepRb KO mice were placed on chow and body weight (C) and cumulative food intake (D) were measured weekly. (E) At 14–15 weeks of age, animals underwent body composition analysis by NMR spectroscopy. Mean, quartiles, and individual plots are shown; n = 8–14 per genotype. ANOVA analysis was performed for (C, D) ; unpaired t-test was performed for (E) . All comparisons p = not significant unless indicated.
Figure Legend Snippet: Deletion of STAT1 and STAT3 in LepRb expressing neurons. (A) Schematic diagram showing the cross of Lepr cre with Stat1 flox and Stat3 flox mice to generate STAT3 LepRb KO and STAT1STAT3 LepRb KO mice. pA: polyadenylation signal. (B) Representative images showing colocalization of STAT1-IR (red) with GFP-IR (green) in the arcuate nucleus of STAT3 LepR KO and STAT1STAT3 LepR KO (both of which are on the R26 eGFP-L10a background) mice. ( C – E ) Male STAT3 LepRb KO and STAT1STAT3 LepRb KO mice were placed on chow and body weight (C) and cumulative food intake (D) were measured weekly. (E) At 14–15 weeks of age, animals underwent body composition analysis by NMR spectroscopy. Mean, quartiles, and individual plots are shown; n = 8–14 per genotype. ANOVA analysis was performed for (C, D) ; unpaired t-test was performed for (E) . All comparisons p = not significant unless indicated.

Techniques Used: Expressing, Mouse Assay, Nuclear Magnetic Resonance, Spectroscopy

Transcriptional response to the deletion of Stat3 in LepRb neurons. (A) Schematic diagram showing the Lepr cre -mediated deletion of Stat3 flox mice on the Rosa26 eGFP-L10a background to generate STAT3 LepRb KO-eGFP-L10a mice. (B – C) Expression values (fragments per million reads; FPM) for genes that were enriched in LepRb neurons under any condition, comparing STAT3 LepRb KO mice to control mice ( B ) or ob/ob mice ( C ) [16] were plotted. Black dots represent genes demonstrating statistically significant changes in gene expression for the comparison; some genes of interest are labeled. ( D ) Comparison of fold change in gene expression relative to control for STAT3 LepRb KO and ob/ob mice for all genes enriched in LepRb neurons. Dashed lines represent fold change values of 1.5 and 0.667 for both axes. Black dots represent regulated genes; red dots show genes that are regulated and known to be controlled by STAT1 [38] . Regions of the graph are denoted by Roman numerals for reference by the main text. (E) Representative images showing colocalization of STAT1-IR (red) and GFP-IR (green) in the ARC of Control and STAT3 LepRb KO mice.
Figure Legend Snippet: Transcriptional response to the deletion of Stat3 in LepRb neurons. (A) Schematic diagram showing the Lepr cre -mediated deletion of Stat3 flox mice on the Rosa26 eGFP-L10a background to generate STAT3 LepRb KO-eGFP-L10a mice. (B – C) Expression values (fragments per million reads; FPM) for genes that were enriched in LepRb neurons under any condition, comparing STAT3 LepRb KO mice to control mice ( B ) or ob/ob mice ( C ) [16] were plotted. Black dots represent genes demonstrating statistically significant changes in gene expression for the comparison; some genes of interest are labeled. ( D ) Comparison of fold change in gene expression relative to control for STAT3 LepRb KO and ob/ob mice for all genes enriched in LepRb neurons. Dashed lines represent fold change values of 1.5 and 0.667 for both axes. Black dots represent regulated genes; red dots show genes that are regulated and known to be controlled by STAT1 [38] . Regions of the graph are denoted by Roman numerals for reference by the main text. (E) Representative images showing colocalization of STAT1-IR (red) and GFP-IR (green) in the ARC of Control and STAT3 LepRb KO mice.

Techniques Used: Mouse Assay, Expressing, Labeling

3) Product Images from "Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation"

Article Title: Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation

Journal: Molecular Biology of the Cell

doi:

Role of Y724 in full-length FGFR3. 293T cells transfected with full-length derivatives of FGFR3, with or without the Y724F mutation, were lysed and analyzed by immunoblotting. (A) Immunoblotting with antisera against FGFR3 shows equivalent expression levels of full-length FGFR3 derivatives. (B) Stat1 is phosphorylated in response to full-length constitutively active FGFR3 but not if the Y724F mutation is present. Lysates were examined by immunoblotting with anti-phospho-Stat1 (Y701) sera. Equal amounts of Stat1 were present in each sample.
Figure Legend Snippet: Role of Y724 in full-length FGFR3. 293T cells transfected with full-length derivatives of FGFR3, with or without the Y724F mutation, were lysed and analyzed by immunoblotting. (A) Immunoblotting with antisera against FGFR3 shows equivalent expression levels of full-length FGFR3 derivatives. (B) Stat1 is phosphorylated in response to full-length constitutively active FGFR3 but not if the Y724F mutation is present. Lysates were examined by immunoblotting with anti-phospho-Stat1 (Y701) sera. Equal amounts of Stat1 were present in each sample.

Techniques Used: Transfection, Mutagenesis, Expressing

Phosphorylation of Stat1 and Stat3. (A) Phosphorylation of Stat1 in cells expressing FGFR3 derivatives. (B) Immunoblotting of Stat1 protein indicated equivalent expression in each sample. (C) Phosphorylation of Stat3 in response to FGFR3 constructs. (D) Immunoblotting of Stat3 protein indicated equal expression in each lane. (E) Levels of the FGFR3 Add-back and Single F mutants were examined by immunoblotting of whole-cell lysates with FGFR3 antisera.
Figure Legend Snippet: Phosphorylation of Stat1 and Stat3. (A) Phosphorylation of Stat1 in cells expressing FGFR3 derivatives. (B) Immunoblotting of Stat1 protein indicated equivalent expression in each sample. (C) Phosphorylation of Stat3 in response to FGFR3 constructs. (D) Immunoblotting of Stat3 protein indicated equal expression in each lane. (E) Levels of the FGFR3 Add-back and Single F mutants were examined by immunoblotting of whole-cell lysates with FGFR3 antisera.

Techniques Used: Expressing, Construct

4) Product Images from "Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation"

Article Title: Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation

Journal: Molecular Biology of the Cell

doi:

Role of Y724 in full-length FGFR3. 293T cells transfected with full-length derivatives of FGFR3, with or without the Y724F mutation, were lysed and analyzed by immunoblotting. (A) Immunoblotting with antisera against FGFR3 shows equivalent expression levels of full-length FGFR3 derivatives. (B) Stat1 is phosphorylated in response to full-length constitutively active FGFR3 but not if the Y724F mutation is present. Lysates were examined by immunoblotting with anti-phospho-Stat1 (Y701) sera. Equal amounts of Stat1 were present in each sample.
Figure Legend Snippet: Role of Y724 in full-length FGFR3. 293T cells transfected with full-length derivatives of FGFR3, with or without the Y724F mutation, were lysed and analyzed by immunoblotting. (A) Immunoblotting with antisera against FGFR3 shows equivalent expression levels of full-length FGFR3 derivatives. (B) Stat1 is phosphorylated in response to full-length constitutively active FGFR3 but not if the Y724F mutation is present. Lysates were examined by immunoblotting with anti-phospho-Stat1 (Y701) sera. Equal amounts of Stat1 were present in each sample.

Techniques Used: Transfection, Mutagenesis, Expressing

Phosphorylation of Stat1 and Stat3. (A) Phosphorylation of Stat1 in cells expressing FGFR3 derivatives. (B) Immunoblotting of Stat1 protein indicated equivalent expression in each sample. (C) Phosphorylation of Stat3 in response to FGFR3 constructs. (D) Immunoblotting of Stat3 protein indicated equal expression in each lane. (E) Levels of the FGFR3 Add-back and Single F mutants were examined by immunoblotting of whole-cell lysates with FGFR3 antisera.
Figure Legend Snippet: Phosphorylation of Stat1 and Stat3. (A) Phosphorylation of Stat1 in cells expressing FGFR3 derivatives. (B) Immunoblotting of Stat1 protein indicated equivalent expression in each sample. (C) Phosphorylation of Stat3 in response to FGFR3 constructs. (D) Immunoblotting of Stat3 protein indicated equal expression in each lane. (E) Levels of the FGFR3 Add-back and Single F mutants were examined by immunoblotting of whole-cell lysates with FGFR3 antisera.

Techniques Used: Expressing, Construct

5) Product Images from "Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation"

Article Title: Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation

Journal: Molecular Biology of the Cell

doi:

Role of Y724 in full-length FGFR3. 293T cells transfected with full-length derivatives of FGFR3, with or without the Y724F mutation, were lysed and analyzed by immunoblotting. (A) Immunoblotting with antisera against FGFR3 shows equivalent expression levels of full-length FGFR3 derivatives. (B) Stat1 is phosphorylated in response to full-length constitutively active FGFR3 but not if the Y724F mutation is present. Lysates were examined by immunoblotting with anti-phospho-Stat1 (Y701) sera. Equal amounts of Stat1 were present in each sample.
Figure Legend Snippet: Role of Y724 in full-length FGFR3. 293T cells transfected with full-length derivatives of FGFR3, with or without the Y724F mutation, were lysed and analyzed by immunoblotting. (A) Immunoblotting with antisera against FGFR3 shows equivalent expression levels of full-length FGFR3 derivatives. (B) Stat1 is phosphorylated in response to full-length constitutively active FGFR3 but not if the Y724F mutation is present. Lysates were examined by immunoblotting with anti-phospho-Stat1 (Y701) sera. Equal amounts of Stat1 were present in each sample.

Techniques Used: Transfection, Mutagenesis, Expressing

Phosphorylation of Stat1 and Stat3. (A) Phosphorylation of Stat1 in cells expressing FGFR3 derivatives. (B) Immunoblotting of Stat1 protein indicated equivalent expression in each sample. (C) Phosphorylation of Stat3 in response to FGFR3 constructs. (D) Immunoblotting of Stat3 protein indicated equal expression in each lane. (E) Levels of the FGFR3 Add-back and Single F mutants were examined by immunoblotting of whole-cell lysates with FGFR3 antisera.
Figure Legend Snippet: Phosphorylation of Stat1 and Stat3. (A) Phosphorylation of Stat1 in cells expressing FGFR3 derivatives. (B) Immunoblotting of Stat1 protein indicated equivalent expression in each sample. (C) Phosphorylation of Stat3 in response to FGFR3 constructs. (D) Immunoblotting of Stat3 protein indicated equal expression in each lane. (E) Levels of the FGFR3 Add-back and Single F mutants were examined by immunoblotting of whole-cell lysates with FGFR3 antisera.

Techniques Used: Expressing, Construct

6) Product Images from "A Point Mutation, E95D, in the Mumps Virus V Protein Disengages STAT3 Targeting from STAT1 Targeting ▿"

Article Title: A Point Mutation, E95D, in the Mumps Virus V Protein Disengages STAT3 Targeting from STAT1 Targeting ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00596-09

Differential STAT targeting by E95D mutant mumps virus V protein. (A) Indirect immunofluorescence used to visualize cellular STAT1 or STAT3 in mumps virus V protein-expressing cells. 2fTGH cells were transfected with expression vectors for FLAG-tagged WT or E95D or C189A mutant mumps virus V protein and treated with either 5 ng/ml IFN-γ or transfected with a v-Src expression vector. Cells were fixed, permeabilized, and stained with FLAG antibody (to detect V protein) and antiserum for either STAT1 or STAT3 24 h later. Images were captured with a Leica confocal microscope at ×63 magnification. Nuclei were stained with TOTO3. Cells expressing the mumps virus V protein are indicated by arrowheads. (B) Quantification of STAT targeting in V protein-expressing cells. 2fTGH cells were transfected and stained as described for panel A, and cells expressing both V protein and either STAT1 or STAT3 were counted ( n = 100).
Figure Legend Snippet: Differential STAT targeting by E95D mutant mumps virus V protein. (A) Indirect immunofluorescence used to visualize cellular STAT1 or STAT3 in mumps virus V protein-expressing cells. 2fTGH cells were transfected with expression vectors for FLAG-tagged WT or E95D or C189A mutant mumps virus V protein and treated with either 5 ng/ml IFN-γ or transfected with a v-Src expression vector. Cells were fixed, permeabilized, and stained with FLAG antibody (to detect V protein) and antiserum for either STAT1 or STAT3 24 h later. Images were captured with a Leica confocal microscope at ×63 magnification. Nuclei were stained with TOTO3. Cells expressing the mumps virus V protein are indicated by arrowheads. (B) Quantification of STAT targeting in V protein-expressing cells. 2fTGH cells were transfected and stained as described for panel A, and cells expressing both V protein and either STAT1 or STAT3 were counted ( n = 100).

Techniques Used: Mutagenesis, Immunofluorescence, Expressing, Transfection, Plasmid Preparation, Staining, Microscopy

Cytokine signaling interference by mutant mumps virus V proteins. (A) Schematic diagram of mumps virus V protein. The box illustrates the 224-residue V protein, the black box indicates the region between amino acids 92 and 100 that was targeted for site-directed mutagenesis, and the conserved CTD is hatched. (B) Comparison of mumps virus (MuV) and PIV5 in the targeted region. The indicated residues were mutated as shown. The position of PIV5 N100 and mumps virus E95 is indicated by the asterisk. (C) Effects of mutant mumps virus V proteins on STAT1-mediated IFN-γ signaling. 293T cells were transfected with a GAS-luciferase reporter gene along with the empty vector or expression vectors for WT mumps virus V protein (MuV WT) or mutant proteins as indicated. Cells were treated with 5 ng/ml IFN-γ for 8 h; this was followed by cell lysis and luciferase assay. Results represent values averaged from triplicate samples, normalized to Renilla luciferase. (D) Similar to panel C, but cells were cotransfected with a v-Src expression vector to activate STAT3 signaling. (E) Similar to panel D, but IL-6 was used to activate STAT3 signaling.
Figure Legend Snippet: Cytokine signaling interference by mutant mumps virus V proteins. (A) Schematic diagram of mumps virus V protein. The box illustrates the 224-residue V protein, the black box indicates the region between amino acids 92 and 100 that was targeted for site-directed mutagenesis, and the conserved CTD is hatched. (B) Comparison of mumps virus (MuV) and PIV5 in the targeted region. The indicated residues were mutated as shown. The position of PIV5 N100 and mumps virus E95 is indicated by the asterisk. (C) Effects of mutant mumps virus V proteins on STAT1-mediated IFN-γ signaling. 293T cells were transfected with a GAS-luciferase reporter gene along with the empty vector or expression vectors for WT mumps virus V protein (MuV WT) or mutant proteins as indicated. Cells were treated with 5 ng/ml IFN-γ for 8 h; this was followed by cell lysis and luciferase assay. Results represent values averaged from triplicate samples, normalized to Renilla luciferase. (D) Similar to panel C, but cells were cotransfected with a v-Src expression vector to activate STAT3 signaling. (E) Similar to panel D, but IL-6 was used to activate STAT3 signaling.

Techniques Used: Mutagenesis, Transfection, Luciferase, Plasmid Preparation, Expressing, Lysis

E95D mutant mumps virus V protein no longer associates with STAT3. 293T cells expressing FLAG-tagged WT or E95D mutant mumps virus V protein were lysed and subjected to FLAG affinity purification. Proteins were separated by SDS-PAGE and processed for immunoblotting with antiserum for STAT1, STAT2, STAT3, and DDB1. GFP, green fluorescent protein.
Figure Legend Snippet: E95D mutant mumps virus V protein no longer associates with STAT3. 293T cells expressing FLAG-tagged WT or E95D mutant mumps virus V protein were lysed and subjected to FLAG affinity purification. Proteins were separated by SDS-PAGE and processed for immunoblotting with antiserum for STAT1, STAT2, STAT3, and DDB1. GFP, green fluorescent protein.

Techniques Used: Mutagenesis, Expressing, Affinity Purification, SDS Page

7) Product Images from "A Point Mutation, E95D, in the Mumps Virus V Protein Disengages STAT3 Targeting from STAT1 Targeting ▿"

Article Title: A Point Mutation, E95D, in the Mumps Virus V Protein Disengages STAT3 Targeting from STAT1 Targeting ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00596-09

Differential STAT targeting by E95D mutant mumps virus V protein. (A) Indirect immunofluorescence used to visualize cellular STAT1 or STAT3 in mumps virus V protein-expressing cells. 2fTGH cells were transfected with expression vectors for FLAG-tagged WT or E95D or C189A mutant mumps virus V protein and treated with either 5 ng/ml IFN-γ or transfected with a v-Src expression vector. Cells were fixed, permeabilized, and stained with FLAG antibody (to detect V protein) and antiserum for either STAT1 or STAT3 24 h later. Images were captured with a Leica confocal microscope at ×63 magnification. Nuclei were stained with TOTO3. Cells expressing the mumps virus V protein are indicated by arrowheads. (B) Quantification of STAT targeting in V protein-expressing cells. 2fTGH cells were transfected and stained as described for panel A, and cells expressing both V protein and either STAT1 or STAT3 were counted ( n = 100).
Figure Legend Snippet: Differential STAT targeting by E95D mutant mumps virus V protein. (A) Indirect immunofluorescence used to visualize cellular STAT1 or STAT3 in mumps virus V protein-expressing cells. 2fTGH cells were transfected with expression vectors for FLAG-tagged WT or E95D or C189A mutant mumps virus V protein and treated with either 5 ng/ml IFN-γ or transfected with a v-Src expression vector. Cells were fixed, permeabilized, and stained with FLAG antibody (to detect V protein) and antiserum for either STAT1 or STAT3 24 h later. Images were captured with a Leica confocal microscope at ×63 magnification. Nuclei were stained with TOTO3. Cells expressing the mumps virus V protein are indicated by arrowheads. (B) Quantification of STAT targeting in V protein-expressing cells. 2fTGH cells were transfected and stained as described for panel A, and cells expressing both V protein and either STAT1 or STAT3 were counted ( n = 100).

Techniques Used: Mutagenesis, Immunofluorescence, Expressing, Transfection, Plasmid Preparation, Staining, Microscopy

Cytokine signaling interference by mutant mumps virus V proteins. (A) Schematic diagram of mumps virus V protein. The box illustrates the 224-residue V protein, the black box indicates the region between amino acids 92 and 100 that was targeted for site-directed mutagenesis, and the conserved CTD is hatched. (B) Comparison of mumps virus (MuV) and PIV5 in the targeted region. The indicated residues were mutated as shown. The position of PIV5 N100 and mumps virus E95 is indicated by the asterisk. (C) Effects of mutant mumps virus V proteins on STAT1-mediated IFN-γ signaling. 293T cells were transfected with a GAS-luciferase reporter gene along with the empty vector or expression vectors for WT mumps virus V protein (MuV WT) or mutant proteins as indicated. Cells were treated with 5 ng/ml IFN-γ for 8 h; this was followed by cell lysis and luciferase assay. Results represent values averaged from triplicate samples, normalized to Renilla luciferase. (D) Similar to panel C, but cells were cotransfected with a v-Src expression vector to activate STAT3 signaling. (E) Similar to panel D, but IL-6 was used to activate STAT3 signaling.
Figure Legend Snippet: Cytokine signaling interference by mutant mumps virus V proteins. (A) Schematic diagram of mumps virus V protein. The box illustrates the 224-residue V protein, the black box indicates the region between amino acids 92 and 100 that was targeted for site-directed mutagenesis, and the conserved CTD is hatched. (B) Comparison of mumps virus (MuV) and PIV5 in the targeted region. The indicated residues were mutated as shown. The position of PIV5 N100 and mumps virus E95 is indicated by the asterisk. (C) Effects of mutant mumps virus V proteins on STAT1-mediated IFN-γ signaling. 293T cells were transfected with a GAS-luciferase reporter gene along with the empty vector or expression vectors for WT mumps virus V protein (MuV WT) or mutant proteins as indicated. Cells were treated with 5 ng/ml IFN-γ for 8 h; this was followed by cell lysis and luciferase assay. Results represent values averaged from triplicate samples, normalized to Renilla luciferase. (D) Similar to panel C, but cells were cotransfected with a v-Src expression vector to activate STAT3 signaling. (E) Similar to panel D, but IL-6 was used to activate STAT3 signaling.

Techniques Used: Mutagenesis, Transfection, Luciferase, Plasmid Preparation, Expressing, Lysis

E95D mutant mumps virus V protein no longer associates with STAT3. 293T cells expressing FLAG-tagged WT or E95D mutant mumps virus V protein were lysed and subjected to FLAG affinity purification. Proteins were separated by SDS-PAGE and processed for immunoblotting with antiserum for STAT1, STAT2, STAT3, and DDB1. GFP, green fluorescent protein.
Figure Legend Snippet: E95D mutant mumps virus V protein no longer associates with STAT3. 293T cells expressing FLAG-tagged WT or E95D mutant mumps virus V protein were lysed and subjected to FLAG affinity purification. Proteins were separated by SDS-PAGE and processed for immunoblotting with antiserum for STAT1, STAT2, STAT3, and DDB1. GFP, green fluorescent protein.

Techniques Used: Mutagenesis, Expressing, Affinity Purification, SDS Page

8) Product Images from "A Point Mutation, E95D, in the Mumps Virus V Protein Disengages STAT3 Targeting from STAT1 Targeting ▿"

Article Title: A Point Mutation, E95D, in the Mumps Virus V Protein Disengages STAT3 Targeting from STAT1 Targeting ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00596-09

Differential STAT targeting by E95D mutant mumps virus V protein. (A) Indirect immunofluorescence used to visualize cellular STAT1 or STAT3 in mumps virus V protein-expressing cells. 2fTGH cells were transfected with expression vectors for FLAG-tagged WT or E95D or C189A mutant mumps virus V protein and treated with either 5 ng/ml IFN-γ or transfected with a v-Src expression vector. Cells were fixed, permeabilized, and stained with FLAG antibody (to detect V protein) and antiserum for either STAT1 or STAT3 24 h later. Images were captured with a Leica confocal microscope at ×63 magnification. Nuclei were stained with TOTO3. Cells expressing the mumps virus V protein are indicated by arrowheads. (B) Quantification of STAT targeting in V protein-expressing cells. 2fTGH cells were transfected and stained as described for panel A, and cells expressing both V protein and either STAT1 or STAT3 were counted ( n = 100).
Figure Legend Snippet: Differential STAT targeting by E95D mutant mumps virus V protein. (A) Indirect immunofluorescence used to visualize cellular STAT1 or STAT3 in mumps virus V protein-expressing cells. 2fTGH cells were transfected with expression vectors for FLAG-tagged WT or E95D or C189A mutant mumps virus V protein and treated with either 5 ng/ml IFN-γ or transfected with a v-Src expression vector. Cells were fixed, permeabilized, and stained with FLAG antibody (to detect V protein) and antiserum for either STAT1 or STAT3 24 h later. Images were captured with a Leica confocal microscope at ×63 magnification. Nuclei were stained with TOTO3. Cells expressing the mumps virus V protein are indicated by arrowheads. (B) Quantification of STAT targeting in V protein-expressing cells. 2fTGH cells were transfected and stained as described for panel A, and cells expressing both V protein and either STAT1 or STAT3 were counted ( n = 100).

Techniques Used: Mutagenesis, Immunofluorescence, Expressing, Transfection, Plasmid Preparation, Staining, Microscopy

Cytokine signaling interference by mutant mumps virus V proteins. (A) Schematic diagram of mumps virus V protein. The box illustrates the 224-residue V protein, the black box indicates the region between amino acids 92 and 100 that was targeted for site-directed mutagenesis, and the conserved CTD is hatched. (B) Comparison of mumps virus (MuV) and PIV5 in the targeted region. The indicated residues were mutated as shown. The position of PIV5 N100 and mumps virus E95 is indicated by the asterisk. (C) Effects of mutant mumps virus V proteins on STAT1-mediated IFN-γ signaling. 293T cells were transfected with a GAS-luciferase reporter gene along with the empty vector or expression vectors for WT mumps virus V protein (MuV WT) or mutant proteins as indicated. Cells were treated with 5 ng/ml IFN-γ for 8 h; this was followed by cell lysis and luciferase assay. Results represent values averaged from triplicate samples, normalized to Renilla luciferase. (D) Similar to panel C, but cells were cotransfected with a v-Src expression vector to activate STAT3 signaling. (E) Similar to panel D, but IL-6 was used to activate STAT3 signaling.
Figure Legend Snippet: Cytokine signaling interference by mutant mumps virus V proteins. (A) Schematic diagram of mumps virus V protein. The box illustrates the 224-residue V protein, the black box indicates the region between amino acids 92 and 100 that was targeted for site-directed mutagenesis, and the conserved CTD is hatched. (B) Comparison of mumps virus (MuV) and PIV5 in the targeted region. The indicated residues were mutated as shown. The position of PIV5 N100 and mumps virus E95 is indicated by the asterisk. (C) Effects of mutant mumps virus V proteins on STAT1-mediated IFN-γ signaling. 293T cells were transfected with a GAS-luciferase reporter gene along with the empty vector or expression vectors for WT mumps virus V protein (MuV WT) or mutant proteins as indicated. Cells were treated with 5 ng/ml IFN-γ for 8 h; this was followed by cell lysis and luciferase assay. Results represent values averaged from triplicate samples, normalized to Renilla luciferase. (D) Similar to panel C, but cells were cotransfected with a v-Src expression vector to activate STAT3 signaling. (E) Similar to panel D, but IL-6 was used to activate STAT3 signaling.

Techniques Used: Mutagenesis, Transfection, Luciferase, Plasmid Preparation, Expressing, Lysis

E95D mutant mumps virus V protein no longer associates with STAT3. 293T cells expressing FLAG-tagged WT or E95D mutant mumps virus V protein were lysed and subjected to FLAG affinity purification. Proteins were separated by SDS-PAGE and processed for immunoblotting with antiserum for STAT1, STAT2, STAT3, and DDB1. GFP, green fluorescent protein.
Figure Legend Snippet: E95D mutant mumps virus V protein no longer associates with STAT3. 293T cells expressing FLAG-tagged WT or E95D mutant mumps virus V protein were lysed and subjected to FLAG affinity purification. Proteins were separated by SDS-PAGE and processed for immunoblotting with antiserum for STAT1, STAT2, STAT3, and DDB1. GFP, green fluorescent protein.

Techniques Used: Mutagenesis, Expressing, Affinity Purification, SDS Page

9) Product Images from "Endothelial Cells Require STAT3 for Protection against Endotoxin-induced Inf lammation"

Article Title: Endothelial Cells Require STAT3 for Protection against Endotoxin-induced Inf lammation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20030077

Endothelial STAT3 deletion and endotoxin-induced lethality. (A) Peritoneal macrophages (p-Mac), T cells, B cells, and hepatocytes were isolated from WT and KO mice and total protein was extracted. SDS-PAGE fractionation and Western blotting using a STAT3-specific polyclonal antibody showed robust expression of STAT3 within these cell types. Total STAT1 expression was also unaltered. (B) Isolated ECs from TIE2e-Cre;STAT3 f/d , and TIE2e-Cre;STAT3 f/+ mice were stimulated with IL-6. Western blot analysis of phospho-specific STAT3 revealed significantly reduced phosphorylated STAT3 in conditional KOs. The membranes were stripped and reblotted with antibodies to STAT3 and STAT1. (C) STAT3 null and wild-type ECs were isolated and plated on Matrigel. Both genotypes formed normal tube structures in vitro, and no differences could be found between groups. (D) Peritoneal macrophages were isolated, stimulated with LPS, and assayed for TNF-α. There was no change in the production of TNF-α, indicating a normal response by macrophages. (E) TIE2e-Cre;STAT3 f/d and TIE2e-Cre;STAT3 f/+ mice were given an intraperitoneal dose of 5 mg/kg LPS. 60% of TIE2e-Cre;STAT3 f/d mice died, whereas all wild-type littermates survived. Lethality was initiated after 16 h. Both cardiomyocyte (αMHC-Cre;STAT3 f/f (F) and hepatocyte (TTR-Cre;STAT3 f/f ) STAT3 conditional KO (G) mice were tested for LPS-induced lethality.
Figure Legend Snippet: Endothelial STAT3 deletion and endotoxin-induced lethality. (A) Peritoneal macrophages (p-Mac), T cells, B cells, and hepatocytes were isolated from WT and KO mice and total protein was extracted. SDS-PAGE fractionation and Western blotting using a STAT3-specific polyclonal antibody showed robust expression of STAT3 within these cell types. Total STAT1 expression was also unaltered. (B) Isolated ECs from TIE2e-Cre;STAT3 f/d , and TIE2e-Cre;STAT3 f/+ mice were stimulated with IL-6. Western blot analysis of phospho-specific STAT3 revealed significantly reduced phosphorylated STAT3 in conditional KOs. The membranes were stripped and reblotted with antibodies to STAT3 and STAT1. (C) STAT3 null and wild-type ECs were isolated and plated on Matrigel. Both genotypes formed normal tube structures in vitro, and no differences could be found between groups. (D) Peritoneal macrophages were isolated, stimulated with LPS, and assayed for TNF-α. There was no change in the production of TNF-α, indicating a normal response by macrophages. (E) TIE2e-Cre;STAT3 f/d and TIE2e-Cre;STAT3 f/+ mice were given an intraperitoneal dose of 5 mg/kg LPS. 60% of TIE2e-Cre;STAT3 f/d mice died, whereas all wild-type littermates survived. Lethality was initiated after 16 h. Both cardiomyocyte (αMHC-Cre;STAT3 f/f (F) and hepatocyte (TTR-Cre;STAT3 f/f ) STAT3 conditional KO (G) mice were tested for LPS-induced lethality.

Techniques Used: Isolation, Mouse Assay, SDS Page, Fractionation, Western Blot, Expressing, In Vitro

10) Product Images from "STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis"

Article Title: STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0113318

CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Amplification, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.
Figure Legend Snippet: Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.

Techniques Used: Amplification, Isolation, Genome Wide, Expressing

IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Functional Assay, Activity Assay, Amplification, Isolation, Quantitative RT-PCR, Expressing, Migration

Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation

STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Isolation, Quantitative RT-PCR

11) Product Images from "STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis"

Article Title: STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0113318

CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Amplification, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.
Figure Legend Snippet: Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.

Techniques Used: Amplification, Isolation, Genome Wide, Expressing

IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Functional Assay, Activity Assay, Amplification, Isolation, Quantitative RT-PCR, Expressing, Migration

Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation

STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Isolation, Quantitative RT-PCR

12) Product Images from "STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis"

Article Title: STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0113318

CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Amplification, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.
Figure Legend Snippet: Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.

Techniques Used: Amplification, Isolation, Genome Wide, Expressing

IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Functional Assay, Activity Assay, Amplification, Isolation, Quantitative RT-PCR, Expressing, Migration

Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation

STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Isolation, Quantitative RT-PCR

13) Product Images from "Genkwadaphnin Induces IFN-γ via PKD1/NF-κB/STAT1 Dependent Pathway in NK-92 Cells"

Article Title: Genkwadaphnin Induces IFN-γ via PKD1/NF-κB/STAT1 Dependent Pathway in NK-92 Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0115146

STAT1 signaling pathway is activated by GD-1 treatment. A . The phosphorylation of STAT1 was induced within 1 hr after GD-1 treatment. B . The increase of phosphorylated STAT1 was detected at high concentration of GD-1 ( > 1 µg/ml). C . The addition of STAT1 inhibitor blocked IFN-γ production induced by GD-1 treatment on a concentration-dependent manner. All ELISA data are representative of at least three independent experiments. Triplicate samples in each time were tested and averaged. Error bars indicate standard deviation. * P
Figure Legend Snippet: STAT1 signaling pathway is activated by GD-1 treatment. A . The phosphorylation of STAT1 was induced within 1 hr after GD-1 treatment. B . The increase of phosphorylated STAT1 was detected at high concentration of GD-1 ( > 1 µg/ml). C . The addition of STAT1 inhibitor blocked IFN-γ production induced by GD-1 treatment on a concentration-dependent manner. All ELISA data are representative of at least three independent experiments. Triplicate samples in each time were tested and averaged. Error bars indicate standard deviation. * P

Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

14) Product Images from "DNA binding reduces the dissociation rate of STAT1 dimers and impairs the interdimeric exchange of protomers"

Article Title: DNA binding reduces the dissociation rate of STAT1 dimers and impairs the interdimeric exchange of protomers

Journal: BMC Biochemistry

doi: 10.1186/s12858-014-0028-z

(A) Demonstration of STAT1 heterotetrameric complexes occupying a double-stranded oligonucleotide containing two consensus GAS sites. Cellular extracts from U3A cells expressing exclusively either GFP-tagged or untagged STAT1 were incubated separately with [ 33 P]-labelled 2xGAS for 45 min (lanes 1 to 5) or, alternatively, mixed and co-incubated for 45 min (lane 6). Supershift reactions were performed by adding either an anti-STAT1 (lanes 2 and 4) or an unspecific anti-STAT3 antibody (lanes 1 and 3). Asterisks mark unspecific bands. (B) STAT1-GFP and STAT1 dimers compete for binding to 2xGAS. Different amounts of extracts from U3A cells expressing exclusively GFP-tagged or untagged STAT1 reacted either separately with [ 33 P]-2xGAS or were mixed and reacted with the probe immediately before being loaded onto the gel. (C) Absence of binding to GAS elements promotes interdimeric protomer exchange. STAT1-GFP- and STAT1-containing extracts were co-incubated for 45 min in the presence (lane 1) or absence of [ 33 P]-2xGAS (lane 2). Immediately before the reaction in lane 2 was loaded onto the gel, a similar amount of [ 33 P]-2xGAS was added to the reaction as in lane 1. (D) Accumulation of interchanged STAT1 complexes at tandem GAS sites. Mixed STAT1-GFP- and STAT1-containing cellular extracts were incubated with [ 33 P]-2xGAS for the indicated times, before being separated by gel electrophoresis (lanes 1 to 3). As controls, non-mixed extracts were used in lanes 4 and 5. (E) Time-dependent accumulation of tetrameric STAT1 at the expense of dimeric complexes. Histograms depict the ratio of tetrameric-to-total STAT1 binding activity presented as means and standard deviations from three independent experiments as shown in (D) . Bars and asterisks indicate significant differences in the pattern of tetrameric-to-total GAS occupancy.
Figure Legend Snippet: (A) Demonstration of STAT1 heterotetrameric complexes occupying a double-stranded oligonucleotide containing two consensus GAS sites. Cellular extracts from U3A cells expressing exclusively either GFP-tagged or untagged STAT1 were incubated separately with [ 33 P]-labelled 2xGAS for 45 min (lanes 1 to 5) or, alternatively, mixed and co-incubated for 45 min (lane 6). Supershift reactions were performed by adding either an anti-STAT1 (lanes 2 and 4) or an unspecific anti-STAT3 antibody (lanes 1 and 3). Asterisks mark unspecific bands. (B) STAT1-GFP and STAT1 dimers compete for binding to 2xGAS. Different amounts of extracts from U3A cells expressing exclusively GFP-tagged or untagged STAT1 reacted either separately with [ 33 P]-2xGAS or were mixed and reacted with the probe immediately before being loaded onto the gel. (C) Absence of binding to GAS elements promotes interdimeric protomer exchange. STAT1-GFP- and STAT1-containing extracts were co-incubated for 45 min in the presence (lane 1) or absence of [ 33 P]-2xGAS (lane 2). Immediately before the reaction in lane 2 was loaded onto the gel, a similar amount of [ 33 P]-2xGAS was added to the reaction as in lane 1. (D) Accumulation of interchanged STAT1 complexes at tandem GAS sites. Mixed STAT1-GFP- and STAT1-containing cellular extracts were incubated with [ 33 P]-2xGAS for the indicated times, before being separated by gel electrophoresis (lanes 1 to 3). As controls, non-mixed extracts were used in lanes 4 and 5. (E) Time-dependent accumulation of tetrameric STAT1 at the expense of dimeric complexes. Histograms depict the ratio of tetrameric-to-total STAT1 binding activity presented as means and standard deviations from three independent experiments as shown in (D) . Bars and asterisks indicate significant differences in the pattern of tetrameric-to-total GAS occupancy.

Techniques Used: Expressing, Incubation, Binding Assay, Nucleic Acid Electrophoresis, Activity Assay

Reciprocal aminoterminal interactions are not required for interdimeric protomer exchange. (A) Expression of tyrosine-phosphorylated GFP-tagged and untagged STAT1 and their corresponding F77A mutants in cellular extracts used for EMSA. A representative Western blot experiment using a STAT1-specific phospho-tyrosine antibody (top panel) and the corresponding re-blot after the stripping off of bound immunoreactivity and re-incubation with pan-STAT1 antibody C-24 (bottom panel) is shown. (B,C) Mutation of phenylalanine 77 to alanine does not interfere with the formation of heterodimeric STAT1 complexes. Extracts from U3A cells expressing wild-type or mutant STAT1 with and without the GFP-tag were co-incubated and the occupancy of the M67 element by heterodimers monitored over time using EMSA. A typical autoradiogram (B) and a quantification of three similar experiments (C) are shown. The histograms present means and standard deviations as well as significant differences over time. (D,E) Aminoterminal contacts between monomers are dispensable for the dissociation and re-association of STAT1 dimers. Extracts from U3A cells expressing either STAT1-GFP or untagged STAT1 were separately incubated in the presence of [ 33 P]-2xGAS before being loaded together onto the gel (lanes 7 and 10) or incubated as a mixture in the presence (lanes 8 and 11) or absence of [ 33 P]-2xGAS, which in the latter case was added immediately before gel electrophoresis (lanes 9 and 12). Reaction time was 45 min for all samples. An asterisk at the gel margin marks an unspecific band. (E) Quantification of band intensities corresponding to tetrameric STAT1 bound to tandem GAS sites across the indicated stoichiometry of GFP-tagged versus untagged STAT1 molecules. Numbers under each column give the ratio of STAT1-GFP/STAT1 molecules for each band. The protocol used for these experiments was similar to that shown in (D) .
Figure Legend Snippet: Reciprocal aminoterminal interactions are not required for interdimeric protomer exchange. (A) Expression of tyrosine-phosphorylated GFP-tagged and untagged STAT1 and their corresponding F77A mutants in cellular extracts used for EMSA. A representative Western blot experiment using a STAT1-specific phospho-tyrosine antibody (top panel) and the corresponding re-blot after the stripping off of bound immunoreactivity and re-incubation with pan-STAT1 antibody C-24 (bottom panel) is shown. (B,C) Mutation of phenylalanine 77 to alanine does not interfere with the formation of heterodimeric STAT1 complexes. Extracts from U3A cells expressing wild-type or mutant STAT1 with and without the GFP-tag were co-incubated and the occupancy of the M67 element by heterodimers monitored over time using EMSA. A typical autoradiogram (B) and a quantification of three similar experiments (C) are shown. The histograms present means and standard deviations as well as significant differences over time. (D,E) Aminoterminal contacts between monomers are dispensable for the dissociation and re-association of STAT1 dimers. Extracts from U3A cells expressing either STAT1-GFP or untagged STAT1 were separately incubated in the presence of [ 33 P]-2xGAS before being loaded together onto the gel (lanes 7 and 10) or incubated as a mixture in the presence (lanes 8 and 11) or absence of [ 33 P]-2xGAS, which in the latter case was added immediately before gel electrophoresis (lanes 9 and 12). Reaction time was 45 min for all samples. An asterisk at the gel margin marks an unspecific band. (E) Quantification of band intensities corresponding to tetrameric STAT1 bound to tandem GAS sites across the indicated stoichiometry of GFP-tagged versus untagged STAT1 molecules. Numbers under each column give the ratio of STAT1-GFP/STAT1 molecules for each band. The protocol used for these experiments was similar to that shown in (D) .

Techniques Used: Expressing, Western Blot, Stripping Membranes, Incubation, Mutagenesis, Nucleic Acid Electrophoresis

Graphic illustration of putative pathways for the interconversion of parallel and antiparallel STAT1 conformers. The domain structure of STAT1 is marked with different colours: the N-terminal domain (N, green) is added via a short flexible segment to the core fragment comprising the coiled-coil domain (C, yellow), the DNA-binding domain (D, blue), the linker domain (not shown), the SH2 domain (S, orange), and the carboxyterminal transactivation domain (not shown). The interface in the antiparallel dimer is formed by reciprocal binding between the coiled-coil domain of one protomer and the DNA-binding domain of the partner protomer, while in the parallel dimer there are reciprocal interactions between the phospho-tyrosine residue 701 (P, marked as red circles) and the SH2 domain. Model 1 (top left) assumes that the core domains of the two partner protomers rotate around each other facilitated by reciprocal N-terminal interactions [ 21 , 22 ]. In contrast, the dissociation/re-association model (model 2, bottom left) allows the formation of new dimer combinations and does not require the presence of the N-terminal domain [ 23 ]. Addition of specific DNA-binding elements termed gamma-activated sites (GAS, marked in red) results in the formation of STAT1 dimers bound to a single GAS site (top right) or STAT1 tetramers when complexed with two GAS sites arranged in a tandem orientation (bottom right).
Figure Legend Snippet: Graphic illustration of putative pathways for the interconversion of parallel and antiparallel STAT1 conformers. The domain structure of STAT1 is marked with different colours: the N-terminal domain (N, green) is added via a short flexible segment to the core fragment comprising the coiled-coil domain (C, yellow), the DNA-binding domain (D, blue), the linker domain (not shown), the SH2 domain (S, orange), and the carboxyterminal transactivation domain (not shown). The interface in the antiparallel dimer is formed by reciprocal binding between the coiled-coil domain of one protomer and the DNA-binding domain of the partner protomer, while in the parallel dimer there are reciprocal interactions between the phospho-tyrosine residue 701 (P, marked as red circles) and the SH2 domain. Model 1 (top left) assumes that the core domains of the two partner protomers rotate around each other facilitated by reciprocal N-terminal interactions [ 21 , 22 ]. In contrast, the dissociation/re-association model (model 2, bottom left) allows the formation of new dimer combinations and does not require the presence of the N-terminal domain [ 23 ]. Addition of specific DNA-binding elements termed gamma-activated sites (GAS, marked in red) results in the formation of STAT1 dimers bound to a single GAS site (top right) or STAT1 tetramers when complexed with two GAS sites arranged in a tandem orientation (bottom right).

Techniques Used: Binding Assay

(A) Electrophoretic mobility shift assay (EMSA) for the identification of green-fluorescent protein-tagged STAT1 (STAT1-GFP) and untagged STAT1. Extracts from reconstituted STAT1-negative U3A cells expressing recombinant GFP-tagged or untagged STAT1 were incubated at room temperature with a [ 33 P]-labelled double-stranded oligonucleotide containing two GAS sites in tandem orientation (2xGAS). Supershift reactions were performed by adding anti-STAT1 antibody C-24 (lanes 1 and 3), and, as control, an unspecific STAT3 antibody (lanes 2 and 4). For competition, a 750-fold molar excess of unlabelled GAS was added to the reaction (lane 5). Asterisks mark unspecific bands. (B,C) Similar dissociation kinetics of STAT1-GFP and STAT1 from a single consensus GAS site (M67). Cellular extracts from U3A cells expressing tagged or untagged STAT1 were incubated for 15 min with [ 33 P]-labelled M67 before on ice a 750-fold molar excess of unlabelled M67 was added for 0, 5, and 10 min, respectively. (B) Specific DNA-binding activity was detected by autoradiography in vacuum-dried gels. (C) The histograms demonstrate the decline in specific DNA-binding activity during challenge with unlabelled M67 for STAT1-GFP (black columns) and untagged STAT1 (grey columns). Bars and asterisks indicate significant differences between samples over time.
Figure Legend Snippet: (A) Electrophoretic mobility shift assay (EMSA) for the identification of green-fluorescent protein-tagged STAT1 (STAT1-GFP) and untagged STAT1. Extracts from reconstituted STAT1-negative U3A cells expressing recombinant GFP-tagged or untagged STAT1 were incubated at room temperature with a [ 33 P]-labelled double-stranded oligonucleotide containing two GAS sites in tandem orientation (2xGAS). Supershift reactions were performed by adding anti-STAT1 antibody C-24 (lanes 1 and 3), and, as control, an unspecific STAT3 antibody (lanes 2 and 4). For competition, a 750-fold molar excess of unlabelled GAS was added to the reaction (lane 5). Asterisks mark unspecific bands. (B,C) Similar dissociation kinetics of STAT1-GFP and STAT1 from a single consensus GAS site (M67). Cellular extracts from U3A cells expressing tagged or untagged STAT1 were incubated for 15 min with [ 33 P]-labelled M67 before on ice a 750-fold molar excess of unlabelled M67 was added for 0, 5, and 10 min, respectively. (B) Specific DNA-binding activity was detected by autoradiography in vacuum-dried gels. (C) The histograms demonstrate the decline in specific DNA-binding activity during challenge with unlabelled M67 for STAT1-GFP (black columns) and untagged STAT1 (grey columns). Bars and asterisks indicate significant differences between samples over time.

Techniques Used: Electrophoretic Mobility Shift Assay, Expressing, Recombinant, Incubation, Binding Assay, Activity Assay, Autoradiography

15) Product Images from "Impaired T-bet-pSTAT1α and perforin-mediated immune responses in the tumoral region of lung adenocarcinoma"

Article Title: Impaired T-bet-pSTAT1α and perforin-mediated immune responses in the tumoral region of lung adenocarcinoma

Journal: British Journal of Cancer

doi: 10.1038/bjc.2015.255

Reduced STAT1 phosphorylation in the tumoral region of NSCLC. ( A – F ) Western blot analysis of pSTAT1 and actin expression in lung tissue samples from the tumoral, peri-tumoral and control region of patients with adenocarcinoma (ADC) ( N Control =5; N Peri-tumoral =5, N Tumoral =5). Bar charts show mean values of the protein expression levels of pSTAT1 α -Isoform ( C and D ) and pSTAT1 β -Isoform ( E and F ) relative to actin levels in ADC and in SCC, respectively. ( G ) Flow cytometry analysis of pSTAT1 + cells in the tumoral, the peri-tumoral and the control lung region of one representative patient with NSCLC. pSTAT1 staining was performed with the lung cell suspensions after 20 min of incubation at 37 °C in the presence or absence of IFN- γ . pSTAT1 + cells were gated on CD4 + , CD8 + or CD11b + cells, respectively. ( H ) Flow cytometry analysis of pSTAT1 + CD4 + and pSTAT1 + CD8 + T cells gated on lymphocytes as well as pSTAT1 + CD11b + cells gated on big cells in the control, the peri-tumoral and the tumoral lung region of one representative patient with NSCLC. Data are shown as mean values±s.e.m. using Student's t -test * P =0.05.
Figure Legend Snippet: Reduced STAT1 phosphorylation in the tumoral region of NSCLC. ( A – F ) Western blot analysis of pSTAT1 and actin expression in lung tissue samples from the tumoral, peri-tumoral and control region of patients with adenocarcinoma (ADC) ( N Control =5; N Peri-tumoral =5, N Tumoral =5). Bar charts show mean values of the protein expression levels of pSTAT1 α -Isoform ( C and D ) and pSTAT1 β -Isoform ( E and F ) relative to actin levels in ADC and in SCC, respectively. ( G ) Flow cytometry analysis of pSTAT1 + cells in the tumoral, the peri-tumoral and the control lung region of one representative patient with NSCLC. pSTAT1 staining was performed with the lung cell suspensions after 20 min of incubation at 37 °C in the presence or absence of IFN- γ . pSTAT1 + cells were gated on CD4 + , CD8 + or CD11b + cells, respectively. ( H ) Flow cytometry analysis of pSTAT1 + CD4 + and pSTAT1 + CD8 + T cells gated on lymphocytes as well as pSTAT1 + CD11b + cells gated on big cells in the control, the peri-tumoral and the tumoral lung region of one representative patient with NSCLC. Data are shown as mean values±s.e.m. using Student's t -test * P =0.05.

Techniques Used: Western Blot, Expressing, Flow Cytometry, Cytometry, Staining, Incubation

STAT1 RNA and protein expression in NSCLC lung samples. ( A and B ) qPCR-based analysis of STAT1A mRNA expression in lung tissue samples from the tumoral, peri-tumoral and control region of patients with ADC and SCC (ADC: N Control =16, N Peri-tumoral =15, N Tumoral =16; SCC: N Control =17, N Peri-tumoral =17, N Tumoral =17). ( C and D ) qPCR-based analysis of STAT1B mRNA expression in lung tissue samples from the tumoral, peri-tumoral and control region of patients with ADC and SCC (ADC: N Control =15, N Peri-tumoral =13, N Tumoral =15; SCC: N Control =16, N Peri-tumoral =14, N Tumoral =16). ( E ) IHC staining of STAT1 + cells was performed on paraffin-embedded tissue sections from the control and the tumoral region of the lungs of patients with ADC and SCC as well as from the lungs of control subjects without lung tumour. ( F ) IHC staining was performed on cryospins of A549 human adenocarcinoma cell line. Data are shown as mean values±s.e.m. using Student's t -test * P =0.05.
Figure Legend Snippet: STAT1 RNA and protein expression in NSCLC lung samples. ( A and B ) qPCR-based analysis of STAT1A mRNA expression in lung tissue samples from the tumoral, peri-tumoral and control region of patients with ADC and SCC (ADC: N Control =16, N Peri-tumoral =15, N Tumoral =16; SCC: N Control =17, N Peri-tumoral =17, N Tumoral =17). ( C and D ) qPCR-based analysis of STAT1B mRNA expression in lung tissue samples from the tumoral, peri-tumoral and control region of patients with ADC and SCC (ADC: N Control =15, N Peri-tumoral =13, N Tumoral =15; SCC: N Control =16, N Peri-tumoral =14, N Tumoral =16). ( E ) IHC staining of STAT1 + cells was performed on paraffin-embedded tissue sections from the control and the tumoral region of the lungs of patients with ADC and SCC as well as from the lungs of control subjects without lung tumour. ( F ) IHC staining was performed on cryospins of A549 human adenocarcinoma cell line. Data are shown as mean values±s.e.m. using Student's t -test * P =0.05.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining

16) Product Images from "Porcine bocavirus NP1 negatively regulates interferon signaling pathway by targeting the DNA-binding domain of IRF9"

Article Title: Porcine bocavirus NP1 negatively regulates interferon signaling pathway by targeting the DNA-binding domain of IRF9

Journal: Virology

doi: 10.1016/j.virol.2015.08.005

PBoV NP1 does not impair ISGF3 complex formation but inhibits its DNA-binding activity. (A) NP1 did not affect formation of the ISGF3 complex. 293T cells were transfected with FLAG-IRF9, FLAG-STAT1, FLAG-STAT2 or empty vector plasmid and HA-tagged NP1 or empty vector plasmid. At 24-h post-transfection, the cells were incubated with IFN-α for 6 h. Cells were lysed and subjected to immunoprecipitation (IP) using rabbit anti-IRF9 antibody. The whole-cell lysates and immunoprecipitation complexes were analyzed by immunoblotting using mouse anti-Flag or mouse anti-HA antibodies. (B) ChIP analysis of ISGF3 binding to the IFN-β promoter. HEK-293T cells were transfected with NP1 or empty expression vector (10 μg each). At 24 h post-transfection, the cells were incubated with IFN-α for 6 h. Cells were lysed and subjected to immunoprecipitation (IP) using rabbit anti-IRF9 antibody. ChIP assays were then performed as described in the Materials and methods section. Real-time PCR analysis of the relative binding of ISGF3 to the ISG56 promoter was performed. The results were expressed as a signal ratio, which represents the signal to the background (IgG) ratio. One of three experiments is shown.
Figure Legend Snippet: PBoV NP1 does not impair ISGF3 complex formation but inhibits its DNA-binding activity. (A) NP1 did not affect formation of the ISGF3 complex. 293T cells were transfected with FLAG-IRF9, FLAG-STAT1, FLAG-STAT2 or empty vector plasmid and HA-tagged NP1 or empty vector plasmid. At 24-h post-transfection, the cells were incubated with IFN-α for 6 h. Cells were lysed and subjected to immunoprecipitation (IP) using rabbit anti-IRF9 antibody. The whole-cell lysates and immunoprecipitation complexes were analyzed by immunoblotting using mouse anti-Flag or mouse anti-HA antibodies. (B) ChIP analysis of ISGF3 binding to the IFN-β promoter. HEK-293T cells were transfected with NP1 or empty expression vector (10 μg each). At 24 h post-transfection, the cells were incubated with IFN-α for 6 h. Cells were lysed and subjected to immunoprecipitation (IP) using rabbit anti-IRF9 antibody. ChIP assays were then performed as described in the Materials and methods section. Real-time PCR analysis of the relative binding of ISGF3 to the ISG56 promoter was performed. The results were expressed as a signal ratio, which represents the signal to the background (IgG) ratio. One of three experiments is shown.

Techniques Used: Binding Assay, Activity Assay, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, Chromatin Immunoprecipitation, Expressing, Real-time Polymerase Chain Reaction

PBoV NP1 does not degrade or prevent phosphorylation and nuclear translocation of STAT1/STAT2. (A) Effects of NP1 on STAT1/2 phosphorylation and expression. HEK-293T cells were mock-transfected or transfected with HA-tagged NP1 expression plasmid. At 24-h post-transfection, the cells were treated with IFN-α (1000 IU/ml) for 30 min. Cell lysates were collected for immunoblot analysis with antibodies directed against phosphorylated STAT1 (Y701), phosphorylated STAT2 (Y690), STAT1, STAT2, IRF9, HA, or β-actin. (B) NP1 did not prevent STAT1 or STAT2 translocation. HeLa cells were transfected with HA-tagged NP1 expression plasmid. At 24-h post-transfection, the cells were treated with IFN-α (1000 IU/ml) for 30 min. Cells were fixed and stained with mouse anti-HA specific for NP1 (green) and rabbit antibody for STAT1 or STAT2 (red). Cells were viewed under the confocal microscope (Olympus Fluoview ver. 3.1). One of three experiments is shown.
Figure Legend Snippet: PBoV NP1 does not degrade or prevent phosphorylation and nuclear translocation of STAT1/STAT2. (A) Effects of NP1 on STAT1/2 phosphorylation and expression. HEK-293T cells were mock-transfected or transfected with HA-tagged NP1 expression plasmid. At 24-h post-transfection, the cells were treated with IFN-α (1000 IU/ml) for 30 min. Cell lysates were collected for immunoblot analysis with antibodies directed against phosphorylated STAT1 (Y701), phosphorylated STAT2 (Y690), STAT1, STAT2, IRF9, HA, or β-actin. (B) NP1 did not prevent STAT1 or STAT2 translocation. HeLa cells were transfected with HA-tagged NP1 expression plasmid. At 24-h post-transfection, the cells were treated with IFN-α (1000 IU/ml) for 30 min. Cells were fixed and stained with mouse anti-HA specific for NP1 (green) and rabbit antibody for STAT1 or STAT2 (red). Cells were viewed under the confocal microscope (Olympus Fluoview ver. 3.1). One of three experiments is shown.

Techniques Used: Translocation Assay, Expressing, Transfection, Plasmid Preparation, Staining, Microscopy

17) Product Images from "Coordinated regulation of IFITM1, 2 and 3 genes by an IFN-responsive enhancer through long-range chromatin interactions"

Article Title: Coordinated regulation of IFITM1, 2 and 3 genes by an IFN-responsive enhancer through long-range chromatin interactions

Journal: Biochimica et Biophysica Acta. Gene Regulatory Mechanisms

doi: 10.1016/j.bbagrm.2017.05.003

STAT1 binds to E2-3 with the treatment of IFNβ. (A) ChIP-qPCR using antibody against STAT1 in HEK293 cells treated with 1000 U/mL IFNβ or PBS for 4 h. The promoter of IFITM1 served as positive control. A locus near R fragment served as negative control. Statistical analyses were performed with unpaired Student's t -tests (* P
Figure Legend Snippet: STAT1 binds to E2-3 with the treatment of IFNβ. (A) ChIP-qPCR using antibody against STAT1 in HEK293 cells treated with 1000 U/mL IFNβ or PBS for 4 h. The promoter of IFITM1 served as positive control. A locus near R fragment served as negative control. Statistical analyses were performed with unpaired Student's t -tests (* P

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control

E2-3 is an IFNβ-responsive enhancer. (A) UCSC Genome Browser output (hg19) of the IFITM genomic region, illustrating the location of the putative enhancer regions. In addition, the ENCODE integrated regulation track containing K562 Pol II ChIA-PET interactions, DNaseI hypersensitivity clusters, H3K4Me1 marks, H3K27Ac marks, transcription factor ChIP-seq data of P300, STAT1 and STAT2, and conservation are displayed. (B) Six fragments, all contained in the two regions interacting with IFITM locus, was used to enhance expression of an SV40 promoter-driven Firefly luciferase reporter gene vector (pGL3-promoter). R fragment, outside the two interacting region, was used as a negative control. Following transient co-transfection, HEK293 cells were treated with PBS or 1000 U/mL IFNβ for 24 h before collected. Results are displayed as ratios of Firefly to SV40 promoter-driven Renilla luciferase activities from three independent experiments. Statistical analyses were performed with unpaired Student's t -tests (* P
Figure Legend Snippet: E2-3 is an IFNβ-responsive enhancer. (A) UCSC Genome Browser output (hg19) of the IFITM genomic region, illustrating the location of the putative enhancer regions. In addition, the ENCODE integrated regulation track containing K562 Pol II ChIA-PET interactions, DNaseI hypersensitivity clusters, H3K4Me1 marks, H3K27Ac marks, transcription factor ChIP-seq data of P300, STAT1 and STAT2, and conservation are displayed. (B) Six fragments, all contained in the two regions interacting with IFITM locus, was used to enhance expression of an SV40 promoter-driven Firefly luciferase reporter gene vector (pGL3-promoter). R fragment, outside the two interacting region, was used as a negative control. Following transient co-transfection, HEK293 cells were treated with PBS or 1000 U/mL IFNβ for 24 h before collected. Results are displayed as ratios of Firefly to SV40 promoter-driven Renilla luciferase activities from three independent experiments. Statistical analyses were performed with unpaired Student's t -tests (* P

Techniques Used: ChIA Pet Assay, Chromatin Immunoprecipitation, Expressing, Luciferase, Plasmid Preparation, Negative Control, Cotransfection

18) Product Images from "STAT1 is a sex‐specific tumor suppressor in colitis‐associated colorectal cancer"

Article Title: STAT1 is a sex‐specific tumor suppressor in colitis‐associated colorectal cancer

Journal: Molecular Oncology

doi: 10.1002/1878-0261.12178

Epithelial STAT1 is a sex‐specific suppressor of colitis‐associated CRC. (A) Tumor load (% tumor area per total colon area) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (B) Tumor multiplicity (number of tumors per cm 2 colon) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (C) Mean tumor size in male and female STAT1 flox/flox and STAT1 ∆IEC mice (automated quantitative histomorphometry of ≥ 75 tumors per genotype in ≥ 13 animals per genotype). Bars represent data ± SEM. (D) Histopathological grading of colon tumors in male and female STAT1 flox/flox and STAT1 ∆IEC mice (≥ 75 tumors per genotype in ≥ 13 animals per genotype). (E) IHC for STAT1 in tumors of STAT1 flox/flox and STAT1 ∆IEC mice (insets show high magnifications; positive epithelial cells are indicated by arrowheads; positive stroma cells are indicated by arrows; scale bar indicates 50 μm). (F,G) BrdU IHC stainings for cell proliferation (F) and cleaved caspase‐3 IHC stainings for apoptosis (G) in colon tumors of male and female STAT1 flox/flox and STAT1 ∆IEC mice (images; scale bar indicates 50 μm; positive cells are indicated by arrowheads and shown in detail by insets). Positive cells were quantified by automated quantitative histomorphometry (≥ 8 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. (H) IHC staining for granzyme B (images; scale bar indicates 100 μm; positive cells are indicated by arrowheads) and histomorphometric quantitation of granzyme B + cells in colon tumors (≥ 9 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. ns: not significant.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific suppressor of colitis‐associated CRC. (A) Tumor load (% tumor area per total colon area) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (B) Tumor multiplicity (number of tumors per cm 2 colon) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (C) Mean tumor size in male and female STAT1 flox/flox and STAT1 ∆IEC mice (automated quantitative histomorphometry of ≥ 75 tumors per genotype in ≥ 13 animals per genotype). Bars represent data ± SEM. (D) Histopathological grading of colon tumors in male and female STAT1 flox/flox and STAT1 ∆IEC mice (≥ 75 tumors per genotype in ≥ 13 animals per genotype). (E) IHC for STAT1 in tumors of STAT1 flox/flox and STAT1 ∆IEC mice (insets show high magnifications; positive epithelial cells are indicated by arrowheads; positive stroma cells are indicated by arrows; scale bar indicates 50 μm). (F,G) BrdU IHC stainings for cell proliferation (F) and cleaved caspase‐3 IHC stainings for apoptosis (G) in colon tumors of male and female STAT1 flox/flox and STAT1 ∆IEC mice (images; scale bar indicates 50 μm; positive cells are indicated by arrowheads and shown in detail by insets). Positive cells were quantified by automated quantitative histomorphometry (≥ 8 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. (H) IHC staining for granzyme B (images; scale bar indicates 100 μm; positive cells are indicated by arrowheads) and histomorphometric quantitation of granzyme B + cells in colon tumors (≥ 9 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. ns: not significant.

Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Quantitation Assay

Epithelial STAT1 is a sex‐specific promoter of chemokine expression in acute colitis. (A–D) qRT‐PCR analysis for mRNA expression of IL‐6 (A), CXCL‐9 (B), CXCL‐10 (C), and CXCL‐11 (D) in isolated IECs of untreated versus DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice (4–6 biological replicates per treatment, sex, and genotype). Bars represent data ± SEM. ns: not significant. (E,F) Representative IHC stainings for IL‐6 (images; positive epithelial cells are indicated by arrowheads; scale bars indicate 50 μm) in DSS‐treated male (E) and female (F) STAT1 flox/flox and STAT1 ∆IEC mice. (G) Quantification of positive epithelial cells with different staining intensities (bar diagrams; automated quantitative histomorphometry of four animals per genotype and sex) in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Bars represent data ± SEM.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific promoter of chemokine expression in acute colitis. (A–D) qRT‐PCR analysis for mRNA expression of IL‐6 (A), CXCL‐9 (B), CXCL‐10 (C), and CXCL‐11 (D) in isolated IECs of untreated versus DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice (4–6 biological replicates per treatment, sex, and genotype). Bars represent data ± SEM. ns: not significant. (E,F) Representative IHC stainings for IL‐6 (images; positive epithelial cells are indicated by arrowheads; scale bars indicate 50 μm) in DSS‐treated male (E) and female (F) STAT1 flox/flox and STAT1 ∆IEC mice. (G) Quantification of positive epithelial cells with different staining intensities (bar diagrams; automated quantitative histomorphometry of four animals per genotype and sex) in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Bars represent data ± SEM.

Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Mouse Assay, Immunohistochemistry, Staining

Epithelial STAT1 is a sex‐specific promoter of intraepithelial CD8 + T‐cell infiltration in acute colitis. (A–C) Flow cytometry data for intraepithelial infiltration of TCRαß + T cells (A), CD8 + TCRαß + T cells (B), and CD8 + TCRαß + granzyme B + T cells (C) into the mucosa of male and female STAT1 flox/flox and STAT1 ∆IEC mice during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. (D) Mean fluorescence intensity for granzyme B expression in intraepithelial CD8 + T cells during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. ns: not significant.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific promoter of intraepithelial CD8 + T‐cell infiltration in acute colitis. (A–C) Flow cytometry data for intraepithelial infiltration of TCRαß + T cells (A), CD8 + TCRαß + T cells (B), and CD8 + TCRαß + granzyme B + T cells (C) into the mucosa of male and female STAT1 flox/flox and STAT1 ∆IEC mice during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. (D) Mean fluorescence intensity for granzyme B expression in intraepithelial CD8 + T cells during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. ns: not significant.

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

Tumor cell‐intrinsic nuclear STAT1 is a sex‐specific prognostic marker for human CRC. (A) Survival curves of male and female CRC patients with or without tumor cell‐intrinsic nuclear STAT1 expression. The analysis is based on published survival data (Gordziel et al ., 2013 ) that were used for sex stratification. (B) STAT1 expression‐ and sex‐based stratification of CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) . (C,D) STAT1 (C) and CXCL‐11 (D) log 2 expression within CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) in all patients and after sex stratification. (E) CIBERSORT analysis for CD8 + T‐cell infiltration in STAT1 high and STAT1 low CMS1—four subtypes of CRC without (all patients) and with sex stratification. (F‐I) CIBERSORT analysis for CD8 + T‐cell infiltration in sex‐stratified STAT1 high and STAT1 low CMS1 (F), CMS2 (G), CMS3 (H), and CMS4 (I) subtypes of CRC. Note that high STAT1 expression is indicative of CD8 + T‐cell infiltration in CMS4 CRC of male but not of female patients.
Figure Legend Snippet: Tumor cell‐intrinsic nuclear STAT1 is a sex‐specific prognostic marker for human CRC. (A) Survival curves of male and female CRC patients with or without tumor cell‐intrinsic nuclear STAT1 expression. The analysis is based on published survival data (Gordziel et al ., 2013 ) that were used for sex stratification. (B) STAT1 expression‐ and sex‐based stratification of CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) . (C,D) STAT1 (C) and CXCL‐11 (D) log 2 expression within CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) in all patients and after sex stratification. (E) CIBERSORT analysis for CD8 + T‐cell infiltration in STAT1 high and STAT1 low CMS1—four subtypes of CRC without (all patients) and with sex stratification. (F‐I) CIBERSORT analysis for CD8 + T‐cell infiltration in sex‐stratified STAT1 high and STAT1 low CMS1 (F), CMS2 (G), CMS3 (H), and CMS4 (I) subtypes of CRC. Note that high STAT1 expression is indicative of CD8 + T‐cell infiltration in CMS4 CRC of male but not of female patients.

Techniques Used: Marker, Expressing

Epithelial STAT1 is a sex‐specific promoter of acute colitis. (A) Weight loss of DSS‐treated male (nine STAT1 flox/flox , nine STAT1 ∆IEC ) and female (eight STAT1 flox/flox , nine STAT1 ∆IEC ) mice. (B) Colitis score of DSS‐treated male (18 STAT1 flox/flox , 18 STAT1 ∆IEC ) and female (10 STAT1 flox/flox , 15 STAT1 ∆IEC ) mice. (C,D) H E‐stained images for evaluation of colitis in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Scale bar indicates 100 μm (C) or 20 μm (D). Arrow: complete erosion of epithelial surface; arrowhead: immune infiltration into the mucosa; double arrow: epithelial regenerative atypia simulating dysplasia; ӿ: immune infiltration into the submucosa; ӿӿ: immune infiltration into the subserosa. (E,F) Colon shortening in DSS‐treated male (nine STAT1 flox/flox , 12 STAT1 ∆IEC ) and female (eight STAT1 flox/flox , seven STAT1 ∆IEC ) mice (≥ 5 control mice per sex and genotype). Bars represent data ± SEM. ns: not significant.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific promoter of acute colitis. (A) Weight loss of DSS‐treated male (nine STAT1 flox/flox , nine STAT1 ∆IEC ) and female (eight STAT1 flox/flox , nine STAT1 ∆IEC ) mice. (B) Colitis score of DSS‐treated male (18 STAT1 flox/flox , 18 STAT1 ∆IEC ) and female (10 STAT1 flox/flox , 15 STAT1 ∆IEC ) mice. (C,D) H E‐stained images for evaluation of colitis in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Scale bar indicates 100 μm (C) or 20 μm (D). Arrow: complete erosion of epithelial surface; arrowhead: immune infiltration into the mucosa; double arrow: epithelial regenerative atypia simulating dysplasia; ӿ: immune infiltration into the submucosa; ӿӿ: immune infiltration into the subserosa. (E,F) Colon shortening in DSS‐treated male (nine STAT1 flox/flox , 12 STAT1 ∆IEC ) and female (eight STAT1 flox/flox , seven STAT1 ∆IEC ) mice (≥ 5 control mice per sex and genotype). Bars represent data ± SEM. ns: not significant.

Techniques Used: Mouse Assay, Staining

19) Product Images from "STAT1 is a sex‐specific tumor suppressor in colitis‐associated colorectal cancer"

Article Title: STAT1 is a sex‐specific tumor suppressor in colitis‐associated colorectal cancer

Journal: Molecular Oncology

doi: 10.1002/1878-0261.12178

Epithelial STAT1 is a sex‐specific suppressor of colitis‐associated CRC. (A) Tumor load (% tumor area per total colon area) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (B) Tumor multiplicity (number of tumors per cm 2 colon) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (C) Mean tumor size in male and female STAT1 flox/flox and STAT1 ∆IEC mice (automated quantitative histomorphometry of ≥ 75 tumors per genotype in ≥ 13 animals per genotype). Bars represent data ± SEM. (D) Histopathological grading of colon tumors in male and female STAT1 flox/flox and STAT1 ∆IEC mice (≥ 75 tumors per genotype in ≥ 13 animals per genotype). (E) IHC for STAT1 in tumors of STAT1 flox/flox and STAT1 ∆IEC mice (insets show high magnifications; positive epithelial cells are indicated by arrowheads; positive stroma cells are indicated by arrows; scale bar indicates 50 μm). (F,G) BrdU IHC stainings for cell proliferation (F) and cleaved caspase‐3 IHC stainings for apoptosis (G) in colon tumors of male and female STAT1 flox/flox and STAT1 ∆IEC mice (images; scale bar indicates 50 μm; positive cells are indicated by arrowheads and shown in detail by insets). Positive cells were quantified by automated quantitative histomorphometry (≥ 8 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. (H) IHC staining for granzyme B (images; scale bar indicates 100 μm; positive cells are indicated by arrowheads) and histomorphometric quantitation of granzyme B + cells in colon tumors (≥ 9 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. ns: not significant.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific suppressor of colitis‐associated CRC. (A) Tumor load (% tumor area per total colon area) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (B) Tumor multiplicity (number of tumors per cm 2 colon) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (C) Mean tumor size in male and female STAT1 flox/flox and STAT1 ∆IEC mice (automated quantitative histomorphometry of ≥ 75 tumors per genotype in ≥ 13 animals per genotype). Bars represent data ± SEM. (D) Histopathological grading of colon tumors in male and female STAT1 flox/flox and STAT1 ∆IEC mice (≥ 75 tumors per genotype in ≥ 13 animals per genotype). (E) IHC for STAT1 in tumors of STAT1 flox/flox and STAT1 ∆IEC mice (insets show high magnifications; positive epithelial cells are indicated by arrowheads; positive stroma cells are indicated by arrows; scale bar indicates 50 μm). (F,G) BrdU IHC stainings for cell proliferation (F) and cleaved caspase‐3 IHC stainings for apoptosis (G) in colon tumors of male and female STAT1 flox/flox and STAT1 ∆IEC mice (images; scale bar indicates 50 μm; positive cells are indicated by arrowheads and shown in detail by insets). Positive cells were quantified by automated quantitative histomorphometry (≥ 8 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. (H) IHC staining for granzyme B (images; scale bar indicates 100 μm; positive cells are indicated by arrowheads) and histomorphometric quantitation of granzyme B + cells in colon tumors (≥ 9 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. ns: not significant.

Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Quantitation Assay

Epithelial STAT1 is a sex‐specific promoter of chemokine expression in acute colitis. (A–D) qRT‐PCR analysis for mRNA expression of IL‐6 (A), CXCL‐9 (B), CXCL‐10 (C), and CXCL‐11 (D) in isolated IECs of untreated versus DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice (4–6 biological replicates per treatment, sex, and genotype). Bars represent data ± SEM. ns: not significant. (E,F) Representative IHC stainings for IL‐6 (images; positive epithelial cells are indicated by arrowheads; scale bars indicate 50 μm) in DSS‐treated male (E) and female (F) STAT1 flox/flox and STAT1 ∆IEC mice. (G) Quantification of positive epithelial cells with different staining intensities (bar diagrams; automated quantitative histomorphometry of four animals per genotype and sex) in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Bars represent data ± SEM.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific promoter of chemokine expression in acute colitis. (A–D) qRT‐PCR analysis for mRNA expression of IL‐6 (A), CXCL‐9 (B), CXCL‐10 (C), and CXCL‐11 (D) in isolated IECs of untreated versus DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice (4–6 biological replicates per treatment, sex, and genotype). Bars represent data ± SEM. ns: not significant. (E,F) Representative IHC stainings for IL‐6 (images; positive epithelial cells are indicated by arrowheads; scale bars indicate 50 μm) in DSS‐treated male (E) and female (F) STAT1 flox/flox and STAT1 ∆IEC mice. (G) Quantification of positive epithelial cells with different staining intensities (bar diagrams; automated quantitative histomorphometry of four animals per genotype and sex) in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Bars represent data ± SEM.

Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Mouse Assay, Immunohistochemistry, Staining

Epithelial STAT1 is a sex‐specific promoter of intraepithelial CD8 + T‐cell infiltration in acute colitis. (A–C) Flow cytometry data for intraepithelial infiltration of TCRαß + T cells (A), CD8 + TCRαß + T cells (B), and CD8 + TCRαß + granzyme B + T cells (C) into the mucosa of male and female STAT1 flox/flox and STAT1 ∆IEC mice during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. (D) Mean fluorescence intensity for granzyme B expression in intraepithelial CD8 + T cells during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. ns: not significant.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific promoter of intraepithelial CD8 + T‐cell infiltration in acute colitis. (A–C) Flow cytometry data for intraepithelial infiltration of TCRαß + T cells (A), CD8 + TCRαß + T cells (B), and CD8 + TCRαß + granzyme B + T cells (C) into the mucosa of male and female STAT1 flox/flox and STAT1 ∆IEC mice during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. (D) Mean fluorescence intensity for granzyme B expression in intraepithelial CD8 + T cells during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. ns: not significant.

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

Tumor cell‐intrinsic nuclear STAT1 is a sex‐specific prognostic marker for human CRC. (A) Survival curves of male and female CRC patients with or without tumor cell‐intrinsic nuclear STAT1 expression. The analysis is based on published survival data (Gordziel et al ., 2013 ) that were used for sex stratification. (B) STAT1 expression‐ and sex‐based stratification of CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) . (C,D) STAT1 (C) and CXCL‐11 (D) log 2 expression within CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) in all patients and after sex stratification. (E) CIBERSORT analysis for CD8 + T‐cell infiltration in STAT1 high and STAT1 low CMS1—four subtypes of CRC without (all patients) and with sex stratification. (F‐I) CIBERSORT analysis for CD8 + T‐cell infiltration in sex‐stratified STAT1 high and STAT1 low CMS1 (F), CMS2 (G), CMS3 (H), and CMS4 (I) subtypes of CRC. Note that high STAT1 expression is indicative of CD8 + T‐cell infiltration in CMS4 CRC of male but not of female patients.
Figure Legend Snippet: Tumor cell‐intrinsic nuclear STAT1 is a sex‐specific prognostic marker for human CRC. (A) Survival curves of male and female CRC patients with or without tumor cell‐intrinsic nuclear STAT1 expression. The analysis is based on published survival data (Gordziel et al ., 2013 ) that were used for sex stratification. (B) STAT1 expression‐ and sex‐based stratification of CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) . (C,D) STAT1 (C) and CXCL‐11 (D) log 2 expression within CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) in all patients and after sex stratification. (E) CIBERSORT analysis for CD8 + T‐cell infiltration in STAT1 high and STAT1 low CMS1—four subtypes of CRC without (all patients) and with sex stratification. (F‐I) CIBERSORT analysis for CD8 + T‐cell infiltration in sex‐stratified STAT1 high and STAT1 low CMS1 (F), CMS2 (G), CMS3 (H), and CMS4 (I) subtypes of CRC. Note that high STAT1 expression is indicative of CD8 + T‐cell infiltration in CMS4 CRC of male but not of female patients.

Techniques Used: Marker, Expressing

Epithelial STAT1 is a sex‐specific promoter of acute colitis. (A) Weight loss of DSS‐treated male (nine STAT1 flox/flox , nine STAT1 ∆IEC ) and female (eight STAT1 flox/flox , nine STAT1 ∆IEC ) mice. (B) Colitis score of DSS‐treated male (18 STAT1 flox/flox , 18 STAT1 ∆IEC ) and female (10 STAT1 flox/flox , 15 STAT1 ∆IEC ) mice. (C,D) H E‐stained images for evaluation of colitis in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Scale bar indicates 100 μm (C) or 20 μm (D). Arrow: complete erosion of epithelial surface; arrowhead: immune infiltration into the mucosa; double arrow: epithelial regenerative atypia simulating dysplasia; ӿ: immune infiltration into the submucosa; ӿӿ: immune infiltration into the subserosa. (E,F) Colon shortening in DSS‐treated male (nine STAT1 flox/flox , 12 STAT1 ∆IEC ) and female (eight STAT1 flox/flox , seven STAT1 ∆IEC ) mice (≥ 5 control mice per sex and genotype). Bars represent data ± SEM. ns: not significant.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific promoter of acute colitis. (A) Weight loss of DSS‐treated male (nine STAT1 flox/flox , nine STAT1 ∆IEC ) and female (eight STAT1 flox/flox , nine STAT1 ∆IEC ) mice. (B) Colitis score of DSS‐treated male (18 STAT1 flox/flox , 18 STAT1 ∆IEC ) and female (10 STAT1 flox/flox , 15 STAT1 ∆IEC ) mice. (C,D) H E‐stained images for evaluation of colitis in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Scale bar indicates 100 μm (C) or 20 μm (D). Arrow: complete erosion of epithelial surface; arrowhead: immune infiltration into the mucosa; double arrow: epithelial regenerative atypia simulating dysplasia; ӿ: immune infiltration into the submucosa; ӿӿ: immune infiltration into the subserosa. (E,F) Colon shortening in DSS‐treated male (nine STAT1 flox/flox , 12 STAT1 ∆IEC ) and female (eight STAT1 flox/flox , seven STAT1 ∆IEC ) mice (≥ 5 control mice per sex and genotype). Bars represent data ± SEM. ns: not significant.

Techniques Used: Mouse Assay, Staining

20) Product Images from "STAT1 is a sex‐specific tumor suppressor in colitis‐associated colorectal cancer"

Article Title: STAT1 is a sex‐specific tumor suppressor in colitis‐associated colorectal cancer

Journal: Molecular Oncology

doi: 10.1002/1878-0261.12178

Epithelial STAT1 is a sex‐specific suppressor of colitis‐associated CRC. (A) Tumor load (% tumor area per total colon area) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (B) Tumor multiplicity (number of tumors per cm 2 colon) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (C) Mean tumor size in male and female STAT1 flox/flox and STAT1 ∆IEC mice (automated quantitative histomorphometry of ≥ 75 tumors per genotype in ≥ 13 animals per genotype). Bars represent data ± SEM. (D) Histopathological grading of colon tumors in male and female STAT1 flox/flox and STAT1 ∆IEC mice (≥ 75 tumors per genotype in ≥ 13 animals per genotype). (E) IHC for STAT1 in tumors of STAT1 flox/flox and STAT1 ∆IEC mice (insets show high magnifications; positive epithelial cells are indicated by arrowheads; positive stroma cells are indicated by arrows; scale bar indicates 50 μm). (F,G) BrdU IHC stainings for cell proliferation (F) and cleaved caspase‐3 IHC stainings for apoptosis (G) in colon tumors of male and female STAT1 flox/flox and STAT1 ∆IEC mice (images; scale bar indicates 50 μm; positive cells are indicated by arrowheads and shown in detail by insets). Positive cells were quantified by automated quantitative histomorphometry (≥ 8 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. (H) IHC staining for granzyme B (images; scale bar indicates 100 μm; positive cells are indicated by arrowheads) and histomorphometric quantitation of granzyme B + cells in colon tumors (≥ 9 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. ns: not significant.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific suppressor of colitis‐associated CRC. (A) Tumor load (% tumor area per total colon area) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (B) Tumor multiplicity (number of tumors per cm 2 colon) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (C) Mean tumor size in male and female STAT1 flox/flox and STAT1 ∆IEC mice (automated quantitative histomorphometry of ≥ 75 tumors per genotype in ≥ 13 animals per genotype). Bars represent data ± SEM. (D) Histopathological grading of colon tumors in male and female STAT1 flox/flox and STAT1 ∆IEC mice (≥ 75 tumors per genotype in ≥ 13 animals per genotype). (E) IHC for STAT1 in tumors of STAT1 flox/flox and STAT1 ∆IEC mice (insets show high magnifications; positive epithelial cells are indicated by arrowheads; positive stroma cells are indicated by arrows; scale bar indicates 50 μm). (F,G) BrdU IHC stainings for cell proliferation (F) and cleaved caspase‐3 IHC stainings for apoptosis (G) in colon tumors of male and female STAT1 flox/flox and STAT1 ∆IEC mice (images; scale bar indicates 50 μm; positive cells are indicated by arrowheads and shown in detail by insets). Positive cells were quantified by automated quantitative histomorphometry (≥ 8 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. (H) IHC staining for granzyme B (images; scale bar indicates 100 μm; positive cells are indicated by arrowheads) and histomorphometric quantitation of granzyme B + cells in colon tumors (≥ 9 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. ns: not significant.

Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Quantitation Assay

Epithelial STAT1 is a sex‐specific promoter of chemokine expression in acute colitis. (A–D) qRT‐PCR analysis for mRNA expression of IL‐6 (A), CXCL‐9 (B), CXCL‐10 (C), and CXCL‐11 (D) in isolated IECs of untreated versus DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice (4–6 biological replicates per treatment, sex, and genotype). Bars represent data ± SEM. ns: not significant. (E,F) Representative IHC stainings for IL‐6 (images; positive epithelial cells are indicated by arrowheads; scale bars indicate 50 μm) in DSS‐treated male (E) and female (F) STAT1 flox/flox and STAT1 ∆IEC mice. (G) Quantification of positive epithelial cells with different staining intensities (bar diagrams; automated quantitative histomorphometry of four animals per genotype and sex) in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Bars represent data ± SEM.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific promoter of chemokine expression in acute colitis. (A–D) qRT‐PCR analysis for mRNA expression of IL‐6 (A), CXCL‐9 (B), CXCL‐10 (C), and CXCL‐11 (D) in isolated IECs of untreated versus DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice (4–6 biological replicates per treatment, sex, and genotype). Bars represent data ± SEM. ns: not significant. (E,F) Representative IHC stainings for IL‐6 (images; positive epithelial cells are indicated by arrowheads; scale bars indicate 50 μm) in DSS‐treated male (E) and female (F) STAT1 flox/flox and STAT1 ∆IEC mice. (G) Quantification of positive epithelial cells with different staining intensities (bar diagrams; automated quantitative histomorphometry of four animals per genotype and sex) in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Bars represent data ± SEM.

Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Mouse Assay, Immunohistochemistry, Staining

Epithelial STAT1 is a sex‐specific promoter of intraepithelial CD8 + T‐cell infiltration in acute colitis. (A–C) Flow cytometry data for intraepithelial infiltration of TCRαß + T cells (A), CD8 + TCRαß + T cells (B), and CD8 + TCRαß + granzyme B + T cells (C) into the mucosa of male and female STAT1 flox/flox and STAT1 ∆IEC mice during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. (D) Mean fluorescence intensity for granzyme B expression in intraepithelial CD8 + T cells during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. ns: not significant.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific promoter of intraepithelial CD8 + T‐cell infiltration in acute colitis. (A–C) Flow cytometry data for intraepithelial infiltration of TCRαß + T cells (A), CD8 + TCRαß + T cells (B), and CD8 + TCRαß + granzyme B + T cells (C) into the mucosa of male and female STAT1 flox/flox and STAT1 ∆IEC mice during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. (D) Mean fluorescence intensity for granzyme B expression in intraepithelial CD8 + T cells during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. ns: not significant.

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

Tumor cell‐intrinsic nuclear STAT1 is a sex‐specific prognostic marker for human CRC. (A) Survival curves of male and female CRC patients with or without tumor cell‐intrinsic nuclear STAT1 expression. The analysis is based on published survival data (Gordziel et al ., 2013 ) that were used for sex stratification. (B) STAT1 expression‐ and sex‐based stratification of CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) . (C,D) STAT1 (C) and CXCL‐11 (D) log 2 expression within CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) in all patients and after sex stratification. (E) CIBERSORT analysis for CD8 + T‐cell infiltration in STAT1 high and STAT1 low CMS1—four subtypes of CRC without (all patients) and with sex stratification. (F‐I) CIBERSORT analysis for CD8 + T‐cell infiltration in sex‐stratified STAT1 high and STAT1 low CMS1 (F), CMS2 (G), CMS3 (H), and CMS4 (I) subtypes of CRC. Note that high STAT1 expression is indicative of CD8 + T‐cell infiltration in CMS4 CRC of male but not of female patients.
Figure Legend Snippet: Tumor cell‐intrinsic nuclear STAT1 is a sex‐specific prognostic marker for human CRC. (A) Survival curves of male and female CRC patients with or without tumor cell‐intrinsic nuclear STAT1 expression. The analysis is based on published survival data (Gordziel et al ., 2013 ) that were used for sex stratification. (B) STAT1 expression‐ and sex‐based stratification of CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) . (C,D) STAT1 (C) and CXCL‐11 (D) log 2 expression within CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) in all patients and after sex stratification. (E) CIBERSORT analysis for CD8 + T‐cell infiltration in STAT1 high and STAT1 low CMS1—four subtypes of CRC without (all patients) and with sex stratification. (F‐I) CIBERSORT analysis for CD8 + T‐cell infiltration in sex‐stratified STAT1 high and STAT1 low CMS1 (F), CMS2 (G), CMS3 (H), and CMS4 (I) subtypes of CRC. Note that high STAT1 expression is indicative of CD8 + T‐cell infiltration in CMS4 CRC of male but not of female patients.

Techniques Used: Marker, Expressing

Epithelial STAT1 is a sex‐specific promoter of acute colitis. (A) Weight loss of DSS‐treated male (nine STAT1 flox/flox , nine STAT1 ∆IEC ) and female (eight STAT1 flox/flox , nine STAT1 ∆IEC ) mice. (B) Colitis score of DSS‐treated male (18 STAT1 flox/flox , 18 STAT1 ∆IEC ) and female (10 STAT1 flox/flox , 15 STAT1 ∆IEC ) mice. (C,D) H E‐stained images for evaluation of colitis in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Scale bar indicates 100 μm (C) or 20 μm (D). Arrow: complete erosion of epithelial surface; arrowhead: immune infiltration into the mucosa; double arrow: epithelial regenerative atypia simulating dysplasia; ӿ: immune infiltration into the submucosa; ӿӿ: immune infiltration into the subserosa. (E,F) Colon shortening in DSS‐treated male (nine STAT1 flox/flox , 12 STAT1 ∆IEC ) and female (eight STAT1 flox/flox , seven STAT1 ∆IEC ) mice (≥ 5 control mice per sex and genotype). Bars represent data ± SEM. ns: not significant.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific promoter of acute colitis. (A) Weight loss of DSS‐treated male (nine STAT1 flox/flox , nine STAT1 ∆IEC ) and female (eight STAT1 flox/flox , nine STAT1 ∆IEC ) mice. (B) Colitis score of DSS‐treated male (18 STAT1 flox/flox , 18 STAT1 ∆IEC ) and female (10 STAT1 flox/flox , 15 STAT1 ∆IEC ) mice. (C,D) H E‐stained images for evaluation of colitis in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Scale bar indicates 100 μm (C) or 20 μm (D). Arrow: complete erosion of epithelial surface; arrowhead: immune infiltration into the mucosa; double arrow: epithelial regenerative atypia simulating dysplasia; ӿ: immune infiltration into the submucosa; ӿӿ: immune infiltration into the subserosa. (E,F) Colon shortening in DSS‐treated male (nine STAT1 flox/flox , 12 STAT1 ∆IEC ) and female (eight STAT1 flox/flox , seven STAT1 ∆IEC ) mice (≥ 5 control mice per sex and genotype). Bars represent data ± SEM. ns: not significant.

Techniques Used: Mouse Assay, Staining

21) Product Images from "STAT1 is a sex‐specific tumor suppressor in colitis‐associated colorectal cancer"

Article Title: STAT1 is a sex‐specific tumor suppressor in colitis‐associated colorectal cancer

Journal: Molecular Oncology

doi: 10.1002/1878-0261.12178

Epithelial STAT1 is a sex‐specific suppressor of colitis‐associated CRC. (A) Tumor load (% tumor area per total colon area) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (B) Tumor multiplicity (number of tumors per cm 2 colon) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (C) Mean tumor size in male and female STAT1 flox/flox and STAT1 ∆IEC mice (automated quantitative histomorphometry of ≥ 75 tumors per genotype in ≥ 13 animals per genotype). Bars represent data ± SEM. (D) Histopathological grading of colon tumors in male and female STAT1 flox/flox and STAT1 ∆IEC mice (≥ 75 tumors per genotype in ≥ 13 animals per genotype). (E) IHC for STAT1 in tumors of STAT1 flox/flox and STAT1 ∆IEC mice (insets show high magnifications; positive epithelial cells are indicated by arrowheads; positive stroma cells are indicated by arrows; scale bar indicates 50 μm). (F,G) BrdU IHC stainings for cell proliferation (F) and cleaved caspase‐3 IHC stainings for apoptosis (G) in colon tumors of male and female STAT1 flox/flox and STAT1 ∆IEC mice (images; scale bar indicates 50 μm; positive cells are indicated by arrowheads and shown in detail by insets). Positive cells were quantified by automated quantitative histomorphometry (≥ 8 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. (H) IHC staining for granzyme B (images; scale bar indicates 100 μm; positive cells are indicated by arrowheads) and histomorphometric quantitation of granzyme B + cells in colon tumors (≥ 9 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. ns: not significant.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific suppressor of colitis‐associated CRC. (A) Tumor load (% tumor area per total colon area) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (B) Tumor multiplicity (number of tumors per cm 2 colon) in male and female STAT1 flox/flox and STAT1 ∆IEC mice. (C) Mean tumor size in male and female STAT1 flox/flox and STAT1 ∆IEC mice (automated quantitative histomorphometry of ≥ 75 tumors per genotype in ≥ 13 animals per genotype). Bars represent data ± SEM. (D) Histopathological grading of colon tumors in male and female STAT1 flox/flox and STAT1 ∆IEC mice (≥ 75 tumors per genotype in ≥ 13 animals per genotype). (E) IHC for STAT1 in tumors of STAT1 flox/flox and STAT1 ∆IEC mice (insets show high magnifications; positive epithelial cells are indicated by arrowheads; positive stroma cells are indicated by arrows; scale bar indicates 50 μm). (F,G) BrdU IHC stainings for cell proliferation (F) and cleaved caspase‐3 IHC stainings for apoptosis (G) in colon tumors of male and female STAT1 flox/flox and STAT1 ∆IEC mice (images; scale bar indicates 50 μm; positive cells are indicated by arrowheads and shown in detail by insets). Positive cells were quantified by automated quantitative histomorphometry (≥ 8 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. (H) IHC staining for granzyme B (images; scale bar indicates 100 μm; positive cells are indicated by arrowheads) and histomorphometric quantitation of granzyme B + cells in colon tumors (≥ 9 tumors per genotype in ≥ 3 animals per genotype). Bars represent data ± SEM. ns: not significant.

Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Quantitation Assay

Epithelial STAT1 is a sex‐specific promoter of chemokine expression in acute colitis. (A–D) qRT‐PCR analysis for mRNA expression of IL‐6 (A), CXCL‐9 (B), CXCL‐10 (C), and CXCL‐11 (D) in isolated IECs of untreated versus DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice (4–6 biological replicates per treatment, sex, and genotype). Bars represent data ± SEM. ns: not significant. (E,F) Representative IHC stainings for IL‐6 (images; positive epithelial cells are indicated by arrowheads; scale bars indicate 50 μm) in DSS‐treated male (E) and female (F) STAT1 flox/flox and STAT1 ∆IEC mice. (G) Quantification of positive epithelial cells with different staining intensities (bar diagrams; automated quantitative histomorphometry of four animals per genotype and sex) in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Bars represent data ± SEM.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific promoter of chemokine expression in acute colitis. (A–D) qRT‐PCR analysis for mRNA expression of IL‐6 (A), CXCL‐9 (B), CXCL‐10 (C), and CXCL‐11 (D) in isolated IECs of untreated versus DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice (4–6 biological replicates per treatment, sex, and genotype). Bars represent data ± SEM. ns: not significant. (E,F) Representative IHC stainings for IL‐6 (images; positive epithelial cells are indicated by arrowheads; scale bars indicate 50 μm) in DSS‐treated male (E) and female (F) STAT1 flox/flox and STAT1 ∆IEC mice. (G) Quantification of positive epithelial cells with different staining intensities (bar diagrams; automated quantitative histomorphometry of four animals per genotype and sex) in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Bars represent data ± SEM.

Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Mouse Assay, Immunohistochemistry, Staining

Epithelial STAT1 is a sex‐specific promoter of intraepithelial CD8 + T‐cell infiltration in acute colitis. (A–C) Flow cytometry data for intraepithelial infiltration of TCRαß + T cells (A), CD8 + TCRαß + T cells (B), and CD8 + TCRαß + granzyme B + T cells (C) into the mucosa of male and female STAT1 flox/flox and STAT1 ∆IEC mice during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. (D) Mean fluorescence intensity for granzyme B expression in intraepithelial CD8 + T cells during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. ns: not significant.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific promoter of intraepithelial CD8 + T‐cell infiltration in acute colitis. (A–C) Flow cytometry data for intraepithelial infiltration of TCRαß + T cells (A), CD8 + TCRαß + T cells (B), and CD8 + TCRαß + granzyme B + T cells (C) into the mucosa of male and female STAT1 flox/flox and STAT1 ∆IEC mice during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. (D) Mean fluorescence intensity for granzyme B expression in intraepithelial CD8 + T cells during DSS‐induced acute colitis. Each data point represents a biological replicate with cells pooled from three mice. ns: not significant.

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

Tumor cell‐intrinsic nuclear STAT1 is a sex‐specific prognostic marker for human CRC. (A) Survival curves of male and female CRC patients with or without tumor cell‐intrinsic nuclear STAT1 expression. The analysis is based on published survival data (Gordziel et al ., 2013 ) that were used for sex stratification. (B) STAT1 expression‐ and sex‐based stratification of CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) . (C,D) STAT1 (C) and CXCL‐11 (D) log 2 expression within CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) in all patients and after sex stratification. (E) CIBERSORT analysis for CD8 + T‐cell infiltration in STAT1 high and STAT1 low CMS1—four subtypes of CRC without (all patients) and with sex stratification. (F‐I) CIBERSORT analysis for CD8 + T‐cell infiltration in sex‐stratified STAT1 high and STAT1 low CMS1 (F), CMS2 (G), CMS3 (H), and CMS4 (I) subtypes of CRC. Note that high STAT1 expression is indicative of CD8 + T‐cell infiltration in CMS4 CRC of male but not of female patients.
Figure Legend Snippet: Tumor cell‐intrinsic nuclear STAT1 is a sex‐specific prognostic marker for human CRC. (A) Survival curves of male and female CRC patients with or without tumor cell‐intrinsic nuclear STAT1 expression. The analysis is based on published survival data (Gordziel et al ., 2013 ) that were used for sex stratification. (B) STAT1 expression‐ and sex‐based stratification of CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) . (C,D) STAT1 (C) and CXCL‐11 (D) log 2 expression within CMS1‐4 subtypes of CRC (Guinney et al ., 2015 ) in all patients and after sex stratification. (E) CIBERSORT analysis for CD8 + T‐cell infiltration in STAT1 high and STAT1 low CMS1—four subtypes of CRC without (all patients) and with sex stratification. (F‐I) CIBERSORT analysis for CD8 + T‐cell infiltration in sex‐stratified STAT1 high and STAT1 low CMS1 (F), CMS2 (G), CMS3 (H), and CMS4 (I) subtypes of CRC. Note that high STAT1 expression is indicative of CD8 + T‐cell infiltration in CMS4 CRC of male but not of female patients.

Techniques Used: Marker, Expressing

Epithelial STAT1 is a sex‐specific promoter of acute colitis. (A) Weight loss of DSS‐treated male (nine STAT1 flox/flox , nine STAT1 ∆IEC ) and female (eight STAT1 flox/flox , nine STAT1 ∆IEC ) mice. (B) Colitis score of DSS‐treated male (18 STAT1 flox/flox , 18 STAT1 ∆IEC ) and female (10 STAT1 flox/flox , 15 STAT1 ∆IEC ) mice. (C,D) H E‐stained images for evaluation of colitis in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Scale bar indicates 100 μm (C) or 20 μm (D). Arrow: complete erosion of epithelial surface; arrowhead: immune infiltration into the mucosa; double arrow: epithelial regenerative atypia simulating dysplasia; ӿ: immune infiltration into the submucosa; ӿӿ: immune infiltration into the subserosa. (E,F) Colon shortening in DSS‐treated male (nine STAT1 flox/flox , 12 STAT1 ∆IEC ) and female (eight STAT1 flox/flox , seven STAT1 ∆IEC ) mice (≥ 5 control mice per sex and genotype). Bars represent data ± SEM. ns: not significant.
Figure Legend Snippet: Epithelial STAT1 is a sex‐specific promoter of acute colitis. (A) Weight loss of DSS‐treated male (nine STAT1 flox/flox , nine STAT1 ∆IEC ) and female (eight STAT1 flox/flox , nine STAT1 ∆IEC ) mice. (B) Colitis score of DSS‐treated male (18 STAT1 flox/flox , 18 STAT1 ∆IEC ) and female (10 STAT1 flox/flox , 15 STAT1 ∆IEC ) mice. (C,D) H E‐stained images for evaluation of colitis in DSS‐treated male and female STAT1 flox/flox and STAT1 ∆IEC mice. Scale bar indicates 100 μm (C) or 20 μm (D). Arrow: complete erosion of epithelial surface; arrowhead: immune infiltration into the mucosa; double arrow: epithelial regenerative atypia simulating dysplasia; ӿ: immune infiltration into the submucosa; ӿӿ: immune infiltration into the subserosa. (E,F) Colon shortening in DSS‐treated male (nine STAT1 flox/flox , 12 STAT1 ∆IEC ) and female (eight STAT1 flox/flox , seven STAT1 ∆IEC ) mice (≥ 5 control mice per sex and genotype). Bars represent data ± SEM. ns: not significant.

Techniques Used: Mouse Assay, Staining

22) Product Images from "Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms"

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

Journal: Redox Biology

doi: 10.1016/j.redox.2016.12.027

α-Lipoic acid prevents zinc deficiency-induced impaired STAT1 and STAT3 nuclear import. Nuclear fraction and cytosolic fraction were isolated from IMR-32 cells incubated for 24 h in control (C), or zinc depleted (1.5Zn) medium with or without supplementation with 0.5mM α-lipoic acid (LA). Western blots for: (A) STAT1 and hnRNP as housekeeping protein in the nuclear fraction (top left), and STAT1 and β-actin as housekeeping protein in the cytosolic fraction (top right), and band quantification expressed as the ratio of total STAT1 nuclear/cytosolic (bottom); (B) STAT3 and hnRNP in the nuclear fraction (top left) and STAT3 and β-actin in the cytosolic fraction (top right), and band quantification expressed as the ratio total STAT3 nuclear/cytosolic fractions (bottom). Results were normalized to controls values, and shown as means±S.E.M. of three independent experiments. *, ** Significantly different compared to C groups ( P
Figure Legend Snippet: α-Lipoic acid prevents zinc deficiency-induced impaired STAT1 and STAT3 nuclear import. Nuclear fraction and cytosolic fraction were isolated from IMR-32 cells incubated for 24 h in control (C), or zinc depleted (1.5Zn) medium with or without supplementation with 0.5mM α-lipoic acid (LA). Western blots for: (A) STAT1 and hnRNP as housekeeping protein in the nuclear fraction (top left), and STAT1 and β-actin as housekeeping protein in the cytosolic fraction (top right), and band quantification expressed as the ratio of total STAT1 nuclear/cytosolic (bottom); (B) STAT3 and hnRNP in the nuclear fraction (top left) and STAT3 and β-actin in the cytosolic fraction (top right), and band quantification expressed as the ratio total STAT3 nuclear/cytosolic fractions (bottom). Results were normalized to controls values, and shown as means±S.E.M. of three independent experiments. *, ** Significantly different compared to C groups ( P

Techniques Used: Isolation, Incubation, Western Blot

Zinc deficiency affects total protein content and phosphorylation patterns of STAT1 and STAT3 in IMR-32 cell nuclear fractions. Nuclear fractions were isolated from IMR-32 cells incubated for 24 h in control (C) or 1.5Zn medium. STAT1 and STAT3 content and phosphorylations were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and hnRNP as the nuclear housekeeping protein (top), and their quantification expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/hnRNP (bottom). (B) Representative images for pY 705 -STAT3, pS 727 -STAT3, STAT3, and hnRNP (top), and their quantification expressed as pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/hnRNP (bottom). Results were normalized to control values (dotted lined) and shown as means±S.E.M. of four independent experiments. *, ** Significantly different compared to C groups ( P
Figure Legend Snippet: Zinc deficiency affects total protein content and phosphorylation patterns of STAT1 and STAT3 in IMR-32 cell nuclear fractions. Nuclear fractions were isolated from IMR-32 cells incubated for 24 h in control (C) or 1.5Zn medium. STAT1 and STAT3 content and phosphorylations were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and hnRNP as the nuclear housekeeping protein (top), and their quantification expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/hnRNP (bottom). (B) Representative images for pY 705 -STAT3, pS 727 -STAT3, STAT3, and hnRNP (top), and their quantification expressed as pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/hnRNP (bottom). Results were normalized to control values (dotted lined) and shown as means±S.E.M. of four independent experiments. *, ** Significantly different compared to C groups ( P

Techniques Used: Isolation, Incubation, Western Blot

Gestational MZD affects phosphorylations of STAT1 and STAT3 in E19 brain total fractions. From gestation day 0 through 19, dams were fed ad libitum control (C) or MZD diets. STAT1 and STAT3 contents and phosphorylations in E19 brain total homogenates were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and β-actin (top); and band quantifications expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and total STAT1/β-actin (bottom). (B) Representative images for pY 705 -STAT3, pS 727 -STAT3, STAT3, and β-actin (top), and band quantifications expressed as pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3 and STAT3/β-actin (bottom). Results were normalized to control values and are shown as means±S.E.M. of 6 animals per group. *, ** Significantly different compared to C groups ( P
Figure Legend Snippet: Gestational MZD affects phosphorylations of STAT1 and STAT3 in E19 brain total fractions. From gestation day 0 through 19, dams were fed ad libitum control (C) or MZD diets. STAT1 and STAT3 contents and phosphorylations in E19 brain total homogenates were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and β-actin (top); and band quantifications expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and total STAT1/β-actin (bottom). (B) Representative images for pY 705 -STAT3, pS 727 -STAT3, STAT3, and β-actin (top), and band quantifications expressed as pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3 and STAT3/β-actin (bottom). Results were normalized to control values and are shown as means±S.E.M. of 6 animals per group. *, ** Significantly different compared to C groups ( P

Techniques Used: Western Blot

Zinc deficiency affects total protein content and phosphorylation patterns of STAT1 and STAT3 in IMR-32 total fractions. Total cell fractions were isolated from IMR-32 cells incubated for 24 h in control (C) or zinc -depleted (1.5 μM zinc (1.5Zn)) medium. STAT1 and STAT3 contents and phosphorylations were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and β-tubulin as the housekeeping protein (top), and their quantification expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/β-tubulin (bottom). (B) Representative images for pY 705 -STAT3, pS 727 -STAT3, STAT3, and β-tubulin (top), and their quantification expressed as pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/β-tubulin (bottom). Results were normalized to control values (dotted lined) and shown as means±S.E.M. of four independent experiments. *, ** Significantly different compared to C groups ( P
Figure Legend Snippet: Zinc deficiency affects total protein content and phosphorylation patterns of STAT1 and STAT3 in IMR-32 total fractions. Total cell fractions were isolated from IMR-32 cells incubated for 24 h in control (C) or zinc -depleted (1.5 μM zinc (1.5Zn)) medium. STAT1 and STAT3 contents and phosphorylations were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and β-tubulin as the housekeeping protein (top), and their quantification expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/β-tubulin (bottom). (B) Representative images for pY 705 -STAT3, pS 727 -STAT3, STAT3, and β-tubulin (top), and their quantification expressed as pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/β-tubulin (bottom). Results were normalized to control values (dotted lined) and shown as means±S.E.M. of four independent experiments. *, ** Significantly different compared to C groups ( P

Techniques Used: Isolation, Incubation, Western Blot

Gestational MZD affects DNA binding and content of STAT1 and STAT3 in nuclear fractions isolated from E19 brains. From gestation day 0 through 19, dams were fed ad libitum control (C) or MZD diets. Nuclear and cytosolic fractions were prepared from E19 brains as described in the Materials and Methods section. (A) EMSA for STAT1 and STAT3 in nuclear and cytosolic fractions. To determine the specificity of each transcription factor-DNA complex, a control nuclear fraction was incubated in the presence of a 100-fold molar excess of unlabeled oligonucleotide containing the consensus sequence for either the specific (S) or an unspecific (U) transcription factor before the binding assay. Bands were quantified and the ratio nuclear/cytosolic DNA binding (NF/CF) was calculated. (B) Western blots for pY 701 -STAT1, pS 727 -STAT1, and STAT1 (left); and pY 705 -STAT3, pS 727 -STAT3, and STAT3 (right); and hnRNP as the nuclear housekeeping protein. After quantification, results were expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/hnRNP (bottom left), or pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/hnRNP (bottom right). Results were normalized to control values and are shown as means±S.E.M. of 6 animals per group. *, ** Significantly different compared to C groups ( P
Figure Legend Snippet: Gestational MZD affects DNA binding and content of STAT1 and STAT3 in nuclear fractions isolated from E19 brains. From gestation day 0 through 19, dams were fed ad libitum control (C) or MZD diets. Nuclear and cytosolic fractions were prepared from E19 brains as described in the Materials and Methods section. (A) EMSA for STAT1 and STAT3 in nuclear and cytosolic fractions. To determine the specificity of each transcription factor-DNA complex, a control nuclear fraction was incubated in the presence of a 100-fold molar excess of unlabeled oligonucleotide containing the consensus sequence for either the specific (S) or an unspecific (U) transcription factor before the binding assay. Bands were quantified and the ratio nuclear/cytosolic DNA binding (NF/CF) was calculated. (B) Western blots for pY 701 -STAT1, pS 727 -STAT1, and STAT1 (left); and pY 705 -STAT3, pS 727 -STAT3, and STAT3 (right); and hnRNP as the nuclear housekeeping protein. After quantification, results were expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/hnRNP (bottom left), or pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/hnRNP (bottom right). Results were normalized to control values and are shown as means±S.E.M. of 6 animals per group. *, ** Significantly different compared to C groups ( P

Techniques Used: Binding Assay, Isolation, Incubation, Sequencing, Western Blot

Zinc deficiency affects nuclear STAT1- and STAT3-DNA binding, and transactivation of STAT1 and STAT3-dependent genes in IMR-32 cells . Nuclear and total cell fractions were isolated after incubating IMR-32 cells for 24 h in control medium (C), or in chelated medium containing 1.5 μM zinc (1.5Zn), or 15 μM zinc (15Zn). (A) Representative image of an EMSA for STAT1 and STAT3 in nuclear fractions (top). A control nuclear fraction was incubated in the presence of a 100-fold molar excess of unlabeled oligonucleotide containing the consensus sequence for aspecific (S) transcription factor before the binding assay. Bands were quantified, results expressed as the ratio nuclear/total fraction binding (NF/TF) for STAT1 (white bars) and STAT3 (grey bars), and normalized to control values. Results are shown as means±S.E.M. of three independent experiments. (B) Transactivation of STAT1- and STAT3-driven luciferase was measured as described in Materials and Methods in cells incubated for 24 h in control, 1.5Zn or 15Zn medium. Data are expressed as the ratio luciferase activity/β-galactosidase activity. Results are shown as means±S.E.M of three independent experiments. *Significantly different compared to the C groups ( P
Figure Legend Snippet: Zinc deficiency affects nuclear STAT1- and STAT3-DNA binding, and transactivation of STAT1 and STAT3-dependent genes in IMR-32 cells . Nuclear and total cell fractions were isolated after incubating IMR-32 cells for 24 h in control medium (C), or in chelated medium containing 1.5 μM zinc (1.5Zn), or 15 μM zinc (15Zn). (A) Representative image of an EMSA for STAT1 and STAT3 in nuclear fractions (top). A control nuclear fraction was incubated in the presence of a 100-fold molar excess of unlabeled oligonucleotide containing the consensus sequence for aspecific (S) transcription factor before the binding assay. Bands were quantified, results expressed as the ratio nuclear/total fraction binding (NF/TF) for STAT1 (white bars) and STAT3 (grey bars), and normalized to control values. Results are shown as means±S.E.M. of three independent experiments. (B) Transactivation of STAT1- and STAT3-driven luciferase was measured as described in Materials and Methods in cells incubated for 24 h in control, 1.5Zn or 15Zn medium. Data are expressed as the ratio luciferase activity/β-galactosidase activity. Results are shown as means±S.E.M of three independent experiments. *Significantly different compared to the C groups ( P

Techniques Used: Binding Assay, Isolation, Incubation, Sequencing, Luciferase, Activity Assay

α-Lipoic acid differentially affects STAT1 and STAT3 phosphorylation in zinc deficient IMR-32 cells. Total fractions were prepared from IMR-32 cells incubated for 24 h in control (C), or zinc deficient (1.5Zn) medium with or without supplementation with 0.5mM α-lipoic acid (LA). STAT1 and STAT3 content and phosphorylation were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and β-actin and their quantification expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1, and STAT1/β-actin (bottom). (B) Representative images for pY 705 -STAT3, STAT3 and β-actin, and their quantification expressed as pY 705 -STAT3/STAT3, and STAT3/β-actin. Results were normalized to control values and shown as means±S.E.M. of four independent experiments. *, **, *** Significantly different compared to C groups ( P
Figure Legend Snippet: α-Lipoic acid differentially affects STAT1 and STAT3 phosphorylation in zinc deficient IMR-32 cells. Total fractions were prepared from IMR-32 cells incubated for 24 h in control (C), or zinc deficient (1.5Zn) medium with or without supplementation with 0.5mM α-lipoic acid (LA). STAT1 and STAT3 content and phosphorylation were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and β-actin and their quantification expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1, and STAT1/β-actin (bottom). (B) Representative images for pY 705 -STAT3, STAT3 and β-actin, and their quantification expressed as pY 705 -STAT3/STAT3, and STAT3/β-actin. Results were normalized to control values and shown as means±S.E.M. of four independent experiments. *, **, *** Significantly different compared to C groups ( P

Techniques Used: Incubation, Western Blot

STAT1 and STAT3 require a functional cytoskeleton for nuclear translocation in IMR-32 cells. Nuclear fractions and total fractions were prepared from IMR-32 cells incubated for 24 h in control medium (C) with or without the addition of 0.5 μM vinblastine (Vb), 0.5 μM colchicine (Col), or 0.5 μM cytochalasin D (Cyt D). (A) EMSA for STAT1 in nuclear and total fractions (top), and band quantifications (bottom). (B) EMSA for STAT3 in nuclear and total fractions (top), and band quantifications (bottom). Results were expressed as the ratio nuclear/total fractions of DNA binding (NF/TF) and normalized to their control levels. Results are shown as means±S.E.M. of three independent experiments. *Significantly different compared to C without inhibitors ( P
Figure Legend Snippet: STAT1 and STAT3 require a functional cytoskeleton for nuclear translocation in IMR-32 cells. Nuclear fractions and total fractions were prepared from IMR-32 cells incubated for 24 h in control medium (C) with or without the addition of 0.5 μM vinblastine (Vb), 0.5 μM colchicine (Col), or 0.5 μM cytochalasin D (Cyt D). (A) EMSA for STAT1 in nuclear and total fractions (top), and band quantifications (bottom). (B) EMSA for STAT3 in nuclear and total fractions (top), and band quantifications (bottom). Results were expressed as the ratio nuclear/total fractions of DNA binding (NF/TF) and normalized to their control levels. Results are shown as means±S.E.M. of three independent experiments. *Significantly different compared to C without inhibitors ( P

Techniques Used: Functional Assay, Translocation Assay, Incubation, Binding Assay

23) Product Images from "Enhanced Immunosuppressive Properties of Human Mesenchymal Stem Cells Primed by Interferon-γ"

Article Title: Enhanced Immunosuppressive Properties of Human Mesenchymal Stem Cells Primed by Interferon-γ

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2018.01.002

IDO expression in IFN-γ-primed MSCs via a JAK-STAT1 signaling pathway. MSCs derived from four different tissues (BM-, AT-, CB-, and WJ-MSC) were used. (a) MSCs were incubated with 200 IU/mL IFN-γ for the indicated amounts of time. The expression levels of phospho-JAK1/2, phospho-STAT1, STAT1, and IRF-1 in these MSCs were detected by immunoblotting. (b) To inhibit the activity of JAK, an intracellular domain of the IFN-γ receptor, MSCs were incubated with 1 μM AG490 (a JAK inhibitor) for 24 h before IFN-γ priming. The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in AG490-treated MSCs were detected by immunoblotting. AG490 treatment induced the down-regulation of STAT1 activity and IDO expression. (c) To down-regulate STAT1 activity, MSCs were transfected with a scrambled siRNA or with an siRNA targeting STAT1 . The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in these transfected MSCs were detected by immunoblotting. Down-regulation of STAT1 activity effectively induced a decrease in IDO expression in IFN-γ-primed MSCs. (d) MSCs were treated with 200 IU/mL IFN-γ or 100 μg/mL poly I:C for 24 h. The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in these MSCs were detected by immunoblotting. β-Actin was used as a loading control for all western blots.
Figure Legend Snippet: IDO expression in IFN-γ-primed MSCs via a JAK-STAT1 signaling pathway. MSCs derived from four different tissues (BM-, AT-, CB-, and WJ-MSC) were used. (a) MSCs were incubated with 200 IU/mL IFN-γ for the indicated amounts of time. The expression levels of phospho-JAK1/2, phospho-STAT1, STAT1, and IRF-1 in these MSCs were detected by immunoblotting. (b) To inhibit the activity of JAK, an intracellular domain of the IFN-γ receptor, MSCs were incubated with 1 μM AG490 (a JAK inhibitor) for 24 h before IFN-γ priming. The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in AG490-treated MSCs were detected by immunoblotting. AG490 treatment induced the down-regulation of STAT1 activity and IDO expression. (c) To down-regulate STAT1 activity, MSCs were transfected with a scrambled siRNA or with an siRNA targeting STAT1 . The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in these transfected MSCs were detected by immunoblotting. Down-regulation of STAT1 activity effectively induced a decrease in IDO expression in IFN-γ-primed MSCs. (d) MSCs were treated with 200 IU/mL IFN-γ or 100 μg/mL poly I:C for 24 h. The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in these MSCs were detected by immunoblotting. β-Actin was used as a loading control for all western blots.

Techniques Used: Expressing, Derivative Assay, Incubation, Activity Assay, Transfection, Western Blot

Enhanced immunosuppressive properties of IFN-γ-primed MSCs. (a–b) 200 IU/mL IFN-γ was added to MSCs, and the cells were incubated for 24 h. PHA-stimulated hPBMCs were incubated in the absence or presence of PBS-treated, or IFN-γ-primed MSCs. (a) Pretreatment with an anti-IFN-γ antibody (#1, once; #2, twice) before IFN-γ priming significantly decreased the suppressive effect of MSCs on PHA-induced T-cell proliferation. (b) Down-regulation of STAT1 activity using an siRNA before IFN-γ priming significantly decreased the suppressive effect of MSCs on PHA-induced T-cell proliferation. hPBMC proliferation was evaluated on day 3 and is expressed as the percentage of BrdU + cells. Data are expressed as the percentage of hPBMC proliferation in the absence of MSCs and represent the mean ± SD of three separate experiments. **P
Figure Legend Snippet: Enhanced immunosuppressive properties of IFN-γ-primed MSCs. (a–b) 200 IU/mL IFN-γ was added to MSCs, and the cells were incubated for 24 h. PHA-stimulated hPBMCs were incubated in the absence or presence of PBS-treated, or IFN-γ-primed MSCs. (a) Pretreatment with an anti-IFN-γ antibody (#1, once; #2, twice) before IFN-γ priming significantly decreased the suppressive effect of MSCs on PHA-induced T-cell proliferation. (b) Down-regulation of STAT1 activity using an siRNA before IFN-γ priming significantly decreased the suppressive effect of MSCs on PHA-induced T-cell proliferation. hPBMC proliferation was evaluated on day 3 and is expressed as the percentage of BrdU + cells. Data are expressed as the percentage of hPBMC proliferation in the absence of MSCs and represent the mean ± SD of three separate experiments. **P

Techniques Used: Incubation, Activity Assay

24) Product Images from "STAT3 Ubiquitylation and Degradation by Mumps Virus Suppress Cytokine and Oncogene Signaling"

Article Title: STAT3 Ubiquitylation and Degradation by Mumps Virus Suppress Cytokine and Oncogene Signaling

Journal: Journal of Virology

doi: 10.1128/JVI.77.11.6385-6393.2003

Analysis of STAT interference by mumps virus. (A) Mumps virus V protein blocks IFN signal transduction. 293T cells were transfected with an ISRE-luciferase reporter gene (left panel) and either empty vector or mumps virus V protein expression vector, as indicated. Cells were treated with 1,000 U of IFN-α per ml or not treated for 6 h prior to lysis and luciferase assays. The same experiment was carried out with a GAS-luciferase reporter gene (right panel) and treatment with 5 ng of IFN-γ per ml. All bars represent average values from triplicate samples, normalized to cotransfected CMV-LacZ ± standard deviation. (B) Mumps virus V protein targets both STAT1 and STAT3 . 2fTGH cells were transfected with empty vector or Flag-tagged mumps virus V expression plasmid and subjected to indirect immunofluorescence staining 24 h later. Cells were fixed, permeabilized, and stained sequentially for Flag and then STAT1, STAT2, or STAT3, and analyzed by confocal microscopy. Nuclei were visualized by staining with TOTO3. Arrows point to the location of V-expressing cells. (C) Mumps virus V interferes with cytokine responses. Similar to panel B, except that cells were treated with IFN-α, IFN-γ, or IL-6 for 30 min prior to fixation. (D) Mumps virus infection causes STAT degradation and relocalization. Cells were infected with mumps virus (2 PFU/cell) and 20 h later processed for indirect immunofluorescence as described above, except that an antibody specific for mumps virus nucleocapsid protein (NP) was used to detect infected cells.
Figure Legend Snippet: Analysis of STAT interference by mumps virus. (A) Mumps virus V protein blocks IFN signal transduction. 293T cells were transfected with an ISRE-luciferase reporter gene (left panel) and either empty vector or mumps virus V protein expression vector, as indicated. Cells were treated with 1,000 U of IFN-α per ml or not treated for 6 h prior to lysis and luciferase assays. The same experiment was carried out with a GAS-luciferase reporter gene (right panel) and treatment with 5 ng of IFN-γ per ml. All bars represent average values from triplicate samples, normalized to cotransfected CMV-LacZ ± standard deviation. (B) Mumps virus V protein targets both STAT1 and STAT3 . 2fTGH cells were transfected with empty vector or Flag-tagged mumps virus V expression plasmid and subjected to indirect immunofluorescence staining 24 h later. Cells were fixed, permeabilized, and stained sequentially for Flag and then STAT1, STAT2, or STAT3, and analyzed by confocal microscopy. Nuclei were visualized by staining with TOTO3. Arrows point to the location of V-expressing cells. (C) Mumps virus V interferes with cytokine responses. Similar to panel B, except that cells were treated with IFN-α, IFN-γ, or IL-6 for 30 min prior to fixation. (D) Mumps virus infection causes STAT degradation and relocalization. Cells were infected with mumps virus (2 PFU/cell) and 20 h later processed for indirect immunofluorescence as described above, except that an antibody specific for mumps virus nucleocapsid protein (NP) was used to detect infected cells.

Techniques Used: Transduction, Transfection, Luciferase, Plasmid Preparation, Expressing, Lysis, Standard Deviation, Immunofluorescence, Staining, Confocal Microscopy, Infection

25) Product Images from "Endothelial Cells Require STAT3 for Protection against Endotoxin-induced Inf lammation"

Article Title: Endothelial Cells Require STAT3 for Protection against Endotoxin-induced Inf lammation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20030077

Endothelial STAT3 deletion and endotoxin-induced lethality. (A) Peritoneal macrophages (p-Mac), T cells, B cells, and hepatocytes were isolated from WT and KO mice and total protein was extracted. SDS-PAGE fractionation and Western blotting using a STAT3-specific polyclonal antibody showed robust expression of STAT3 within these cell types. Total STAT1 expression was also unaltered. (B) Isolated ECs from TIE2e-Cre;STAT3 f/d , and TIE2e-Cre;STAT3 f/+ mice were stimulated with IL-6. Western blot analysis of phospho-specific STAT3 revealed significantly reduced phosphorylated STAT3 in conditional KOs. The membranes were stripped and reblotted with antibodies to STAT3 and STAT1. (C) STAT3 null and wild-type ECs were isolated and plated on Matrigel. Both genotypes formed normal tube structures in vitro, and no differences could be found between groups. (D) Peritoneal macrophages were isolated, stimulated with LPS, and assayed for TNF-α. There was no change in the production of TNF-α, indicating a normal response by macrophages. (E) TIE2e-Cre;STAT3 f/d and TIE2e-Cre;STAT3 f/+ mice were given an intraperitoneal dose of 5 mg/kg LPS. 60% of TIE2e-Cre;STAT3 f/d mice died, whereas all wild-type littermates survived. Lethality was initiated after 16 h. Both cardiomyocyte (αMHC-Cre;STAT3 f/f (F) and hepatocyte (TTR-Cre;STAT3 f/f ) STAT3 conditional KO (G) mice were tested for LPS-induced lethality.
Figure Legend Snippet: Endothelial STAT3 deletion and endotoxin-induced lethality. (A) Peritoneal macrophages (p-Mac), T cells, B cells, and hepatocytes were isolated from WT and KO mice and total protein was extracted. SDS-PAGE fractionation and Western blotting using a STAT3-specific polyclonal antibody showed robust expression of STAT3 within these cell types. Total STAT1 expression was also unaltered. (B) Isolated ECs from TIE2e-Cre;STAT3 f/d , and TIE2e-Cre;STAT3 f/+ mice were stimulated with IL-6. Western blot analysis of phospho-specific STAT3 revealed significantly reduced phosphorylated STAT3 in conditional KOs. The membranes were stripped and reblotted with antibodies to STAT3 and STAT1. (C) STAT3 null and wild-type ECs were isolated and plated on Matrigel. Both genotypes formed normal tube structures in vitro, and no differences could be found between groups. (D) Peritoneal macrophages were isolated, stimulated with LPS, and assayed for TNF-α. There was no change in the production of TNF-α, indicating a normal response by macrophages. (E) TIE2e-Cre;STAT3 f/d and TIE2e-Cre;STAT3 f/+ mice were given an intraperitoneal dose of 5 mg/kg LPS. 60% of TIE2e-Cre;STAT3 f/d mice died, whereas all wild-type littermates survived. Lethality was initiated after 16 h. Both cardiomyocyte (αMHC-Cre;STAT3 f/f (F) and hepatocyte (TTR-Cre;STAT3 f/f ) STAT3 conditional KO (G) mice were tested for LPS-induced lethality.

Techniques Used: Isolation, Mouse Assay, SDS Page, Fractionation, Western Blot, Expressing, In Vitro

26) Product Images from "Mumps virus Enders strain is sensitive to interferon (IFN) despite encoding a functional IFN antagonist"

Article Title: Mumps virus Enders strain is sensitive to interferon (IFN) despite encoding a functional IFN antagonist

Journal: The Journal of General Virology

doi: 10.1099/vir.0.013722-0

MuV Enders V protein targets STAT1 and STAT3 for degradation and interacts with mda-5. (a) Naïve Hep2 cells were infected with MuV Enders for 8 h, or mock infected, prior to addition of IFN- α [Roferon A (Roche) 1000 IU ml −1 ] to the culture medium. At 8 and 24 h p.i., cells were harvested and the presence of STAT1 and STAT3 was detected by immunoblot analysis. β -Actin acted as a loading control. (b) 293 cells were transfected with a control plasmid (empty vector) or plasmids expressing V5-tagged V proteins of MuV Enders or JL5, which has previously been shown to bind mda-5 ( Andrejeva et al. , 2004 ; Childs et al. , 2007 ), together with a plasmid that expresses the FLAG-tagged helicase domain of mda-5. At 48 h post-transfection, the V proteins were immunoprecipitated and the presence of the mda-5 helicase domain was detected by immunoblot analysis. Immunoglobulin heavy chain (IgH) was also detected in the immunoblot.
Figure Legend Snippet: MuV Enders V protein targets STAT1 and STAT3 for degradation and interacts with mda-5. (a) Naïve Hep2 cells were infected with MuV Enders for 8 h, or mock infected, prior to addition of IFN- α [Roferon A (Roche) 1000 IU ml −1 ] to the culture medium. At 8 and 24 h p.i., cells were harvested and the presence of STAT1 and STAT3 was detected by immunoblot analysis. β -Actin acted as a loading control. (b) 293 cells were transfected with a control plasmid (empty vector) or plasmids expressing V5-tagged V proteins of MuV Enders or JL5, which has previously been shown to bind mda-5 ( Andrejeva et al. , 2004 ; Childs et al. , 2007 ), together with a plasmid that expresses the FLAG-tagged helicase domain of mda-5. At 48 h post-transfection, the V proteins were immunoprecipitated and the presence of the mda-5 helicase domain was detected by immunoblot analysis. Immunoglobulin heavy chain (IgH) was also detected in the immunoblot.

Techniques Used: Multiple Displacement Amplification, Infection, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation

Characterization of Hep2 cells that constitutively express the V protein of MuV Enders. (a) STAT1 is degraded in cells that constitutively express MuV-V. Naïve Hep2, Hep2/BVDV-Npro and Hep2/MuV-V cells were or were not treated with IFN- α [Roferon A (Roche) 1000 IU ml −1 ] for 18 h and STAT1 was detected by immunoblot analysis. BVDV-Npro and MuV-V had N-terminal or C-terminal V5 tags, respectively, and their presence was detected by an anti-V5 tag antibody; β -actin acted as a loading control. (b) Unlike wild-type Bunyamwera virus (BUNV wt), which forms plaques in naïve Hep2, Hep2/MuV-V and Hep2/BVDV cells, a recombinant Bunyamwera virus that does not encode the NSs protein, termed rBUNVΔNSs, does not form plaques in naïve Hep2 cells but does plaque Hep2/MuV-V and Hep2/BVDV cells. Plaques shown are 4 days p.i. (c) The ability of naïve Hep2, Hep2/MuV-V, Hep2/PIV5-V and Hep2/BVDV-Npro cells to support the replication of MuV was compared. Cells were infected at an m.o.i. of 0.01 p.f.u. per cell and the amount of infectious virus in the culture medium was titrated at 2, 4, 6 and 8 days p.i. (light, medium and dark grey, and black, respectively).
Figure Legend Snippet: Characterization of Hep2 cells that constitutively express the V protein of MuV Enders. (a) STAT1 is degraded in cells that constitutively express MuV-V. Naïve Hep2, Hep2/BVDV-Npro and Hep2/MuV-V cells were or were not treated with IFN- α [Roferon A (Roche) 1000 IU ml −1 ] for 18 h and STAT1 was detected by immunoblot analysis. BVDV-Npro and MuV-V had N-terminal or C-terminal V5 tags, respectively, and their presence was detected by an anti-V5 tag antibody; β -actin acted as a loading control. (b) Unlike wild-type Bunyamwera virus (BUNV wt), which forms plaques in naïve Hep2, Hep2/MuV-V and Hep2/BVDV cells, a recombinant Bunyamwera virus that does not encode the NSs protein, termed rBUNVΔNSs, does not form plaques in naïve Hep2 cells but does plaque Hep2/MuV-V and Hep2/BVDV cells. Plaques shown are 4 days p.i. (c) The ability of naïve Hep2, Hep2/MuV-V, Hep2/PIV5-V and Hep2/BVDV-Npro cells to support the replication of MuV was compared. Cells were infected at an m.o.i. of 0.01 p.f.u. per cell and the amount of infectious virus in the culture medium was titrated at 2, 4, 6 and 8 days p.i. (light, medium and dark grey, and black, respectively).

Techniques Used: Recombinant, Infection

27) Product Images from "Local and Systemic Alterations in Signal Transducers and Activators of Transcription (STAT) Associated with Human Abdominal Aortic Aneurysms"

Article Title: Local and Systemic Alterations in Signal Transducers and Activators of Transcription (STAT) Associated with Human Abdominal Aortic Aneurysms

Journal: The Journal of Surgical Research

doi: 10.1016/j.jss.2011.05.041

Representative samples from aortic tissue obtained from non-aneurysmal aorta ( A and C ) and aneurysmal aorta ( B and D ). Immunohistochemical analysis failed to show any staining for STAT1 in non-aneurysmal tissue ( A (40x) and C (100x)), but was present
Figure Legend Snippet: Representative samples from aortic tissue obtained from non-aneurysmal aorta ( A and C ) and aneurysmal aorta ( B and D ). Immunohistochemical analysis failed to show any staining for STAT1 in non-aneurysmal tissue ( A (40x) and C (100x)), but was present

Techniques Used: Immunohistochemistry, Staining

Graphic representation of aortic STAT mRNA obtained from AAA (black bars) or NA (gray bars). STAT mRNA expression was assessed by real time RT-PCR and normalized to β-actin and is expressed in arbitrary units (AU). STAT1, 2, and 4 mRNA were significantly
Figure Legend Snippet: Graphic representation of aortic STAT mRNA obtained from AAA (black bars) or NA (gray bars). STAT mRNA expression was assessed by real time RT-PCR and normalized to β-actin and is expressed in arbitrary units (AU). STAT1, 2, and 4 mRNA were significantly

Techniques Used: Expressing, Quantitative RT-PCR

28) Product Images from "Role of Host Protein Tyrosine Phosphatase SHP-1 in Leishmania donovani-Induced Inhibition of Nitric Oxide Production "

Article Title: Role of Host Protein Tyrosine Phosphatase SHP-1 in Leishmania donovani-Induced Inhibition of Nitric Oxide Production

Journal:

doi: 10.1128/IAI.00853-05

STAT1 nuclear translocation upon IFN-γ stimulation in infected and uninfected SHP-1-deficient macrophages and littermate control cells. Uninfected (Nil) or L. donovani -infected (Ld) SHP-1-deficient or littermate macrophages were stimulated with
Figure Legend Snippet: STAT1 nuclear translocation upon IFN-γ stimulation in infected and uninfected SHP-1-deficient macrophages and littermate control cells. Uninfected (Nil) or L. donovani -infected (Ld) SHP-1-deficient or littermate macrophages were stimulated with

Techniques Used: Translocation Assay, Infection

29) Product Images from "STAT1 gene expression is enhanced by nuclear EGFR and HER2 via cooperation with STAT3"

Article Title: STAT1 gene expression is enhanced by nuclear EGFR and HER2 via cooperation with STAT3

Journal: Molecular carcinogenesis

doi: 10.1002/mc.21936

Endogenous associations between STAT1, EGFR and p-STAT3 in breast cancer cells
Figure Legend Snippet: Endogenous associations between STAT1, EGFR and p-STAT3 in breast cancer cells

Techniques Used:

EGFR synergizes with STAT3 to activate the STAT1 gene promoter
Figure Legend Snippet: EGFR synergizes with STAT3 to activate the STAT1 gene promoter

Techniques Used:

Structural characterization of the human STAT1 gene promoter for activation by the EGFR-STAT3 signaling axis
Figure Legend Snippet: Structural characterization of the human STAT1 gene promoter for activation by the EGFR-STAT3 signaling axis

Techniques Used: Activation Assay

EGFRvIII cooperates with STAT3 to activate the STAT1 gene promoter
Figure Legend Snippet: EGFRvIII cooperates with STAT3 to activate the STAT1 gene promoter

Techniques Used:

HER2 and STAT3 synergize to upregulate STAT1 gene expression in breast cancer cells
Figure Legend Snippet: HER2 and STAT3 synergize to upregulate STAT1 gene expression in breast cancer cells

Techniques Used: Expressing

STAT1 gene expression is enhanced by nuclear EGFR
Figure Legend Snippet: STAT1 gene expression is enhanced by nuclear EGFR

Techniques Used: Expressing

30) Product Images from "STAT1 gene expression is enhanced by nuclear EGFR and HER2 via cooperation with STAT3"

Article Title: STAT1 gene expression is enhanced by nuclear EGFR and HER2 via cooperation with STAT3

Journal: Molecular carcinogenesis

doi: 10.1002/mc.21936

Endogenous associations between STAT1, EGFR and p-STAT3 in breast cancer cells
Figure Legend Snippet: Endogenous associations between STAT1, EGFR and p-STAT3 in breast cancer cells

Techniques Used:

EGFR synergizes with STAT3 to activate the STAT1 gene promoter
Figure Legend Snippet: EGFR synergizes with STAT3 to activate the STAT1 gene promoter

Techniques Used:

Structural characterization of the human STAT1 gene promoter for activation by the EGFR-STAT3 signaling axis
Figure Legend Snippet: Structural characterization of the human STAT1 gene promoter for activation by the EGFR-STAT3 signaling axis

Techniques Used: Activation Assay

EGFRvIII cooperates with STAT3 to activate the STAT1 gene promoter
Figure Legend Snippet: EGFRvIII cooperates with STAT3 to activate the STAT1 gene promoter

Techniques Used:

HER2 and STAT3 synergize to upregulate STAT1 gene expression in breast cancer cells
Figure Legend Snippet: HER2 and STAT3 synergize to upregulate STAT1 gene expression in breast cancer cells

Techniques Used: Expressing

STAT1 gene expression is enhanced by nuclear EGFR
Figure Legend Snippet: STAT1 gene expression is enhanced by nuclear EGFR

Techniques Used: Expressing

31) Product Images from "STAT1 gene expression is enhanced by nuclear EGFR and HER2 via cooperation with STAT3"

Article Title: STAT1 gene expression is enhanced by nuclear EGFR and HER2 via cooperation with STAT3

Journal: Molecular carcinogenesis

doi: 10.1002/mc.21936

Endogenous associations between STAT1, EGFR and p-STAT3 in breast cancer cells
Figure Legend Snippet: Endogenous associations between STAT1, EGFR and p-STAT3 in breast cancer cells

Techniques Used:

EGFR synergizes with STAT3 to activate the STAT1 gene promoter
Figure Legend Snippet: EGFR synergizes with STAT3 to activate the STAT1 gene promoter

Techniques Used:

Structural characterization of the human STAT1 gene promoter for activation by the EGFR-STAT3 signaling axis
Figure Legend Snippet: Structural characterization of the human STAT1 gene promoter for activation by the EGFR-STAT3 signaling axis

Techniques Used: Activation Assay

EGFRvIII cooperates with STAT3 to activate the STAT1 gene promoter
Figure Legend Snippet: EGFRvIII cooperates with STAT3 to activate the STAT1 gene promoter

Techniques Used:

HER2 and STAT3 synergize to upregulate STAT1 gene expression in breast cancer cells
Figure Legend Snippet: HER2 and STAT3 synergize to upregulate STAT1 gene expression in breast cancer cells

Techniques Used: Expressing

STAT1 gene expression is enhanced by nuclear EGFR
Figure Legend Snippet: STAT1 gene expression is enhanced by nuclear EGFR

Techniques Used: Expressing

32) Product Images from "STAT1 gene expression is enhanced by nuclear EGFR and HER2 via cooperation with STAT3"

Article Title: STAT1 gene expression is enhanced by nuclear EGFR and HER2 via cooperation with STAT3

Journal: Molecular carcinogenesis

doi: 10.1002/mc.21936

Endogenous associations between STAT1, EGFR and p-STAT3 in breast cancer cells
Figure Legend Snippet: Endogenous associations between STAT1, EGFR and p-STAT3 in breast cancer cells

Techniques Used:

EGFR synergizes with STAT3 to activate the STAT1 gene promoter
Figure Legend Snippet: EGFR synergizes with STAT3 to activate the STAT1 gene promoter

Techniques Used:

Structural characterization of the human STAT1 gene promoter for activation by the EGFR-STAT3 signaling axis
Figure Legend Snippet: Structural characterization of the human STAT1 gene promoter for activation by the EGFR-STAT3 signaling axis

Techniques Used: Activation Assay

EGFRvIII cooperates with STAT3 to activate the STAT1 gene promoter
Figure Legend Snippet: EGFRvIII cooperates with STAT3 to activate the STAT1 gene promoter

Techniques Used:

HER2 and STAT3 synergize to upregulate STAT1 gene expression in breast cancer cells
Figure Legend Snippet: HER2 and STAT3 synergize to upregulate STAT1 gene expression in breast cancer cells

Techniques Used: Expressing

STAT1 gene expression is enhanced by nuclear EGFR
Figure Legend Snippet: STAT1 gene expression is enhanced by nuclear EGFR

Techniques Used: Expressing

33) Product Images from "Chikungunya Virus Nonstructural Protein 2 Inhibits Type I/II Interferon-Stimulated JAK-STAT Signaling ▿Chikungunya Virus Nonstructural Protein 2 Inhibits Type I/II Interferon-Stimulated JAK-STAT Signaling ▿ †"

Article Title: Chikungunya Virus Nonstructural Protein 2 Inhibits Type I/II Interferon-Stimulated JAK-STAT Signaling ▿Chikungunya Virus Nonstructural Protein 2 Inhibits Type I/II Interferon-Stimulated JAK-STAT Signaling ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.00949-10

Mutation of a conserved proline in nsP2 abolishes the inhibitory effect of CHIKV and SINV replicons on JAK-STAT signaling. (A) Schematic representation of the CHIKrep-pac2AEGFP and SINrepLuc replicons. nsP2 mutations P718S and P726S are indicated with asterisks; pac, puromycin acetyltransferase. (B) Partial amino acid alignment of alphavirus nsP2s. RRV, Ross River virus; VEEV, Venezuelan equine encephalitis virus. The conserved proline and amino acid numbers within nsP2 proteins are indicated. (C) pSTAT1 nuclear translocation upon IFN-β induction in SINrepGFP (wild type and mutant nsP2-P726S)-transfected Vero cells. Cells were immunostained with an anti-pSTAT1 antibody. Open arrowheads indicate replicon-positive cells lacking nuclear pSTAT1; solid arrowheads indicate replicon-positive cells with nuclear pSTAT1. (D) Nuclear translocation of phospho-STAT1 upon IFN-β induction in CHIKrep-pac2AEGFP (wild type and mutant nsP2-P718S)-transfected Vero cells. Cells were immunostained with an anti-pSTAT1 antibody.
Figure Legend Snippet: Mutation of a conserved proline in nsP2 abolishes the inhibitory effect of CHIKV and SINV replicons on JAK-STAT signaling. (A) Schematic representation of the CHIKrep-pac2AEGFP and SINrepLuc replicons. nsP2 mutations P718S and P726S are indicated with asterisks; pac, puromycin acetyltransferase. (B) Partial amino acid alignment of alphavirus nsP2s. RRV, Ross River virus; VEEV, Venezuelan equine encephalitis virus. The conserved proline and amino acid numbers within nsP2 proteins are indicated. (C) pSTAT1 nuclear translocation upon IFN-β induction in SINrepGFP (wild type and mutant nsP2-P726S)-transfected Vero cells. Cells were immunostained with an anti-pSTAT1 antibody. Open arrowheads indicate replicon-positive cells lacking nuclear pSTAT1; solid arrowheads indicate replicon-positive cells with nuclear pSTAT1. (D) Nuclear translocation of phospho-STAT1 upon IFN-β induction in CHIKrep-pac2AEGFP (wild type and mutant nsP2-P718S)-transfected Vero cells. Cells were immunostained with an anti-pSTAT1 antibody.

Techniques Used: Mutagenesis, Translocation Assay, Transfection

Inhibition of IFN-β-induced STAT1 nuclear translocation by individual CHIKV nsPs. (A) Schematic representation of the pCMV-nsP1, -2, -3, and -4 expression plasmids and the CHIKrep-mCherry replicon, expressing mCherry. CMV, cytomegalovirus immediate-early promoter; 2A, foot-and-mouth disease virus 2A autoprotease. The bacteriophage SP6 and CHIKV 26S promoters are indicated. (B) pSTAT1 nuclear translocation upon IFN-β induction in Vero cells transfected with pCMV-nsP1, -2, -3, or -4. Cells were immunostained with an anti-pSTAT1 antibody. (C) pSTAT1 nuclear translocation upon IFN-β induction in CHIKrep-mCherry-transfected Vero cells. Open arrowheads indicate cells positive for nsP1, -2, -3, or -4- or for the CHIKV replicon that lack nuclear pSTAT1; solid arrowheads indicate nsP1- to nsP4-positive cells with nuclear pSTAT1.
Figure Legend Snippet: Inhibition of IFN-β-induced STAT1 nuclear translocation by individual CHIKV nsPs. (A) Schematic representation of the pCMV-nsP1, -2, -3, and -4 expression plasmids and the CHIKrep-mCherry replicon, expressing mCherry. CMV, cytomegalovirus immediate-early promoter; 2A, foot-and-mouth disease virus 2A autoprotease. The bacteriophage SP6 and CHIKV 26S promoters are indicated. (B) pSTAT1 nuclear translocation upon IFN-β induction in Vero cells transfected with pCMV-nsP1, -2, -3, or -4. Cells were immunostained with an anti-pSTAT1 antibody. (C) pSTAT1 nuclear translocation upon IFN-β induction in CHIKrep-mCherry-transfected Vero cells. Open arrowheads indicate cells positive for nsP1, -2, -3, or -4- or for the CHIKV replicon that lack nuclear pSTAT1; solid arrowheads indicate nsP1- to nsP4-positive cells with nuclear pSTAT1.

Techniques Used: Inhibition, Translocation Assay, Expressing, Transfection

(A to C) CHIKV infection blocks STAT1/STAT2 nuclear translocation without depleting endogenous STAT1 levels. Vero cells were infected by CHIKV and were treated with IFN-α (A and B) or IFN-γ (C) for 30 min. Cells were fixed and stained with monoclonal antibodies specific for CHIKV envelope protein and STAT1 (A and C) or STAT2 (B). (C) Block in nuclear translocation of STAT1 in CHIKV infection in response to treatment with IFN-γ. Arrowheads indicate cells negatively infected with CHIKV but with nuclear STAT1/2. (D) CHIKV infection blocks STAT1 phosphorylation in Vero cells in response to IFN treatment. pSTAT1, STAT1, and tubulin were detected by Western blotting in CHIKV-infected or mock-infected Vero cells that were either left untreated or induced with type I or type II IFNs. Lane 1, protein size marker (in kilodaltons).
Figure Legend Snippet: (A to C) CHIKV infection blocks STAT1/STAT2 nuclear translocation without depleting endogenous STAT1 levels. Vero cells were infected by CHIKV and were treated with IFN-α (A and B) or IFN-γ (C) for 30 min. Cells were fixed and stained with monoclonal antibodies specific for CHIKV envelope protein and STAT1 (A and C) or STAT2 (B). (C) Block in nuclear translocation of STAT1 in CHIKV infection in response to treatment with IFN-γ. Arrowheads indicate cells negatively infected with CHIKV but with nuclear STAT1/2. (D) CHIKV infection blocks STAT1 phosphorylation in Vero cells in response to IFN treatment. pSTAT1, STAT1, and tubulin were detected by Western blotting in CHIKV-infected or mock-infected Vero cells that were either left untreated or induced with type I or type II IFNs. Lane 1, protein size marker (in kilodaltons).

Techniques Used: Infection, Translocation Assay, Staining, Blocking Assay, Western Blot, Marker

A CHIKV replicon efficiently inhibits type I/II IFN-induced JAK-STAT signaling independently of host shutoff. (A) Schematic representation of CHIKrepEGFP, expressing EGFP. (B) pSTAT1 nuclear translocation in Vero cells upon induction with type I and type II IFNs. (C) A CHIKV replicon blocks pSTAT1 nuclear translocation upon type I/II IFN induction. Vero cells were immunostained with an anti-pSTAT1 antibody 24 h p.t. (D) CHIKV RNA replication, but not translational shutoff, blocks STAT1 nuclear translocation. Vero cells were transfected with CHIKrep-EGFP replicon RNA in the absence or presence of cycloheximide (Chx). Cells were induced for 30 min with IFN-β at 12 h p.t. and were stained with an anti-STAT1 antibody. Open arrowheads indicate CHIKV replicon-positive cells lacking nuclear STAT1.
Figure Legend Snippet: A CHIKV replicon efficiently inhibits type I/II IFN-induced JAK-STAT signaling independently of host shutoff. (A) Schematic representation of CHIKrepEGFP, expressing EGFP. (B) pSTAT1 nuclear translocation in Vero cells upon induction with type I and type II IFNs. (C) A CHIKV replicon blocks pSTAT1 nuclear translocation upon type I/II IFN induction. Vero cells were immunostained with an anti-pSTAT1 antibody 24 h p.t. (D) CHIKV RNA replication, but not translational shutoff, blocks STAT1 nuclear translocation. Vero cells were transfected with CHIKrep-EGFP replicon RNA in the absence or presence of cycloheximide (Chx). Cells were induced for 30 min with IFN-β at 12 h p.t. and were stained with an anti-STAT1 antibody. Open arrowheads indicate CHIKV replicon-positive cells lacking nuclear STAT1.

Techniques Used: Expressing, Translocation Assay, Transfection, Staining

34) Product Images from "Cell-intrinsic role for IFN-?-STAT1 signals in regulating murine Peyer patch plasmacytoid dendritic cells and conditioning an inflammatory response"

Article Title: Cell-intrinsic role for IFN-?-STAT1 signals in regulating murine Peyer patch plasmacytoid dendritic cells and conditioning an inflammatory response

Journal: Blood

doi: 10.1182/blood-2011-04-349761

Role for STAT1 in IFN-α–mediated pDC development. (A) STAT activation and expression in IFN-α–, GM-CSF-, or IFN-α + GM-CSF-treated (30 minutes) DC progenitors (lin − Flt3 + cells), determined by immunoblotting, as indicated. (B) Proportion of pDCs (top left quadrant) and cDCs (bottom right quadrant) in total BM cultures from Stat1 −/− or wild-type mice in GM-CSF or GM-CSF + IFN-α for 4 days. Results were gated on the CD11c + population (not shown); B220 and CD11b analysis is shown. n = 5. (C-D) Proportion (C) and absolute numbers (D) of pDCs and cDCs in BM and spleen of Stat1 −/− or wild-type mice treated by HGT, as indicated. Results were analyzed as indicated in panel B. n = 5. Error bars represent SEM. (E) Gene expression in D2SC/1 cells stimulated with GM-CSF, IFN-α, or both for 2 hours or left unstimulated, as indicated. Error bars represent SEM. (F) EMSAs with nuclear extracts from D2SC/1 cells stimulated with or without IFN-α for 30 minutes. Some samples contained STAT1 competitor antibody or competitor oligonucleotides, as indicated. d indicates Irf8 STAT site; m, mutated Irf8 STAT site; and s, STAT1-consensus oligonucleotide. (G) Luciferase assays in IFN-α–stimulated (IFN) or untreated (NT) D2SC/1 cells transfected with empty vector (pGL4.12) or an Irf8 reporter (pGL4.12/IRF8) with an intact (WT) or mutated (MU) STAT element, plus pMNC/mSTAT1, pMNC/mSTAT2, and phRL-TK plasmids. Error bars represent SEM. (H) ChIPs from D2SC/1 cells with or without IFN-α stimulation (1 hour) with STAT1 antibodies or IgG controls, as indicated. PCR reactions were performed with total cell lysates (input) or immunoprecipitated samples, as shown. (A-H) Results represent 3 independent experiments.
Figure Legend Snippet: Role for STAT1 in IFN-α–mediated pDC development. (A) STAT activation and expression in IFN-α–, GM-CSF-, or IFN-α + GM-CSF-treated (30 minutes) DC progenitors (lin − Flt3 + cells), determined by immunoblotting, as indicated. (B) Proportion of pDCs (top left quadrant) and cDCs (bottom right quadrant) in total BM cultures from Stat1 −/− or wild-type mice in GM-CSF or GM-CSF + IFN-α for 4 days. Results were gated on the CD11c + population (not shown); B220 and CD11b analysis is shown. n = 5. (C-D) Proportion (C) and absolute numbers (D) of pDCs and cDCs in BM and spleen of Stat1 −/− or wild-type mice treated by HGT, as indicated. Results were analyzed as indicated in panel B. n = 5. Error bars represent SEM. (E) Gene expression in D2SC/1 cells stimulated with GM-CSF, IFN-α, or both for 2 hours or left unstimulated, as indicated. Error bars represent SEM. (F) EMSAs with nuclear extracts from D2SC/1 cells stimulated with or without IFN-α for 30 minutes. Some samples contained STAT1 competitor antibody or competitor oligonucleotides, as indicated. d indicates Irf8 STAT site; m, mutated Irf8 STAT site; and s, STAT1-consensus oligonucleotide. (G) Luciferase assays in IFN-α–stimulated (IFN) or untreated (NT) D2SC/1 cells transfected with empty vector (pGL4.12) or an Irf8 reporter (pGL4.12/IRF8) with an intact (WT) or mutated (MU) STAT element, plus pMNC/mSTAT1, pMNC/mSTAT2, and phRL-TK plasmids. Error bars represent SEM. (H) ChIPs from D2SC/1 cells with or without IFN-α stimulation (1 hour) with STAT1 antibodies or IgG controls, as indicated. PCR reactions were performed with total cell lysates (input) or immunoprecipitated samples, as shown. (A-H) Results represent 3 independent experiments.

Techniques Used: Activation Assay, Expressing, Mouse Assay, Luciferase, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Immunoprecipitation

Characterization of PP pDCs and their requirement for IFN-STAT1 signaling. (A) Gating strategy of PP pDCs. Surface expression of PDCA-1, B220, CCR9, CD86, and TLR4 and intracellular TLR7 and TLR9 expression in pDCs from BM, spleen, and PP. Shaded area represents isotype control staining. (B) Gene expression of Irf7 , Irf8 , and Tcf4 in pDCs from BM, spleen, and PP, determined by quantitative PCR. (C-D) Proportion (C) and absolute number (D) of PP pDCs from Stat1 −/− , Ifnar −/− , and wild-type (WT) controls. Error bars represent SEM of results from 4-8 mice. (E) PP pDC proportions in mice receiving HGT with pORF vector (NT) or pORF encoding Flt3L (10 μg), 4 days after treatment. (F) PP pDC proportions in Stat1 −/− mice at 8 days after transfer of 10 5 congenic CD45.1 + DC progenitors (BM lin − Flt3 + CD115 + c-kit int cells) intravenously. Similar results were obtained after transfer into Ifnar1 −/− mice (not shown). (G) PP pDC proportions in Stat1 −/− or Ifnar1 −/− mice 8 days after transfer of 10 5 wild-type DC progenitors (BM lin − Flt3 + CD115 + c-kit int cells) intravenously (CDP) or in animals left untreated (NT), as indicated. Some mice received Flt3L HGT 2 days before DC progenitor transfer (CDP + Flt3L) or Flt3L HGT alone (Flt3L), as shown. (B-G) Data represent 2 or 3 independent experiments. N = 4-10.
Figure Legend Snippet: Characterization of PP pDCs and their requirement for IFN-STAT1 signaling. (A) Gating strategy of PP pDCs. Surface expression of PDCA-1, B220, CCR9, CD86, and TLR4 and intracellular TLR7 and TLR9 expression in pDCs from BM, spleen, and PP. Shaded area represents isotype control staining. (B) Gene expression of Irf7 , Irf8 , and Tcf4 in pDCs from BM, spleen, and PP, determined by quantitative PCR. (C-D) Proportion (C) and absolute number (D) of PP pDCs from Stat1 −/− , Ifnar −/− , and wild-type (WT) controls. Error bars represent SEM of results from 4-8 mice. (E) PP pDC proportions in mice receiving HGT with pORF vector (NT) or pORF encoding Flt3L (10 μg), 4 days after treatment. (F) PP pDC proportions in Stat1 −/− mice at 8 days after transfer of 10 5 congenic CD45.1 + DC progenitors (BM lin − Flt3 + CD115 + c-kit int cells) intravenously. Similar results were obtained after transfer into Ifnar1 −/− mice (not shown). (G) PP pDC proportions in Stat1 −/− or Ifnar1 −/− mice 8 days after transfer of 10 5 wild-type DC progenitors (BM lin − Flt3 + CD115 + c-kit int cells) intravenously (CDP) or in animals left untreated (NT), as indicated. Some mice received Flt3L HGT 2 days before DC progenitor transfer (CDP + Flt3L) or Flt3L HGT alone (Flt3L), as shown. (B-G) Data represent 2 or 3 independent experiments. N = 4-10.

Techniques Used: Expressing, Staining, Real-time Polymerase Chain Reaction, Mouse Assay, Plasmid Preparation

35) Product Images from "Cell-intrinsic role for IFN-?-STAT1 signals in regulating murine Peyer patch plasmacytoid dendritic cells and conditioning an inflammatory response"

Article Title: Cell-intrinsic role for IFN-?-STAT1 signals in regulating murine Peyer patch plasmacytoid dendritic cells and conditioning an inflammatory response

Journal: Blood

doi: 10.1182/blood-2011-04-349761

Role for STAT1 in IFN-α–mediated pDC development. (A) STAT activation and expression in IFN-α–, GM-CSF-, or IFN-α + GM-CSF-treated (30 minutes) DC progenitors (lin − Flt3 + cells), determined by immunoblotting, as indicated. (B) Proportion of pDCs (top left quadrant) and cDCs (bottom right quadrant) in total BM cultures from Stat1 −/− or wild-type mice in GM-CSF or GM-CSF + IFN-α for 4 days. Results were gated on the CD11c + population (not shown); B220 and CD11b analysis is shown. n = 5. (C-D) Proportion (C) and absolute numbers (D) of pDCs and cDCs in BM and spleen of Stat1 −/− or wild-type mice treated by HGT, as indicated. Results were analyzed as indicated in panel B. n = 5. Error bars represent SEM. (E) Gene expression in D2SC/1 cells stimulated with GM-CSF, IFN-α, or both for 2 hours or left unstimulated, as indicated. Error bars represent SEM. (F) EMSAs with nuclear extracts from D2SC/1 cells stimulated with or without IFN-α for 30 minutes. Some samples contained STAT1 competitor antibody or competitor oligonucleotides, as indicated. d indicates Irf8 STAT site; m, mutated Irf8 STAT site; and s, STAT1-consensus oligonucleotide. (G) Luciferase assays in IFN-α–stimulated (IFN) or untreated (NT) D2SC/1 cells transfected with empty vector (pGL4.12) or an Irf8 reporter (pGL4.12/IRF8) with an intact (WT) or mutated (MU) STAT element, plus pMNC/mSTAT1, pMNC/mSTAT2, and phRL-TK plasmids. Error bars represent SEM. (H) ChIPs from D2SC/1 cells with or without IFN-α stimulation (1 hour) with STAT1 antibodies or IgG controls, as indicated. PCR reactions were performed with total cell lysates (input) or immunoprecipitated samples, as shown. (A-H) Results represent 3 independent experiments.
Figure Legend Snippet: Role for STAT1 in IFN-α–mediated pDC development. (A) STAT activation and expression in IFN-α–, GM-CSF-, or IFN-α + GM-CSF-treated (30 minutes) DC progenitors (lin − Flt3 + cells), determined by immunoblotting, as indicated. (B) Proportion of pDCs (top left quadrant) and cDCs (bottom right quadrant) in total BM cultures from Stat1 −/− or wild-type mice in GM-CSF or GM-CSF + IFN-α for 4 days. Results were gated on the CD11c + population (not shown); B220 and CD11b analysis is shown. n = 5. (C-D) Proportion (C) and absolute numbers (D) of pDCs and cDCs in BM and spleen of Stat1 −/− or wild-type mice treated by HGT, as indicated. Results were analyzed as indicated in panel B. n = 5. Error bars represent SEM. (E) Gene expression in D2SC/1 cells stimulated with GM-CSF, IFN-α, or both for 2 hours or left unstimulated, as indicated. Error bars represent SEM. (F) EMSAs with nuclear extracts from D2SC/1 cells stimulated with or without IFN-α for 30 minutes. Some samples contained STAT1 competitor antibody or competitor oligonucleotides, as indicated. d indicates Irf8 STAT site; m, mutated Irf8 STAT site; and s, STAT1-consensus oligonucleotide. (G) Luciferase assays in IFN-α–stimulated (IFN) or untreated (NT) D2SC/1 cells transfected with empty vector (pGL4.12) or an Irf8 reporter (pGL4.12/IRF8) with an intact (WT) or mutated (MU) STAT element, plus pMNC/mSTAT1, pMNC/mSTAT2, and phRL-TK plasmids. Error bars represent SEM. (H) ChIPs from D2SC/1 cells with or without IFN-α stimulation (1 hour) with STAT1 antibodies or IgG controls, as indicated. PCR reactions were performed with total cell lysates (input) or immunoprecipitated samples, as shown. (A-H) Results represent 3 independent experiments.

Techniques Used: Activation Assay, Expressing, Mouse Assay, Luciferase, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Immunoprecipitation

Characterization of PP pDCs and their requirement for IFN-STAT1 signaling. (A) Gating strategy of PP pDCs. Surface expression of PDCA-1, B220, CCR9, CD86, and TLR4 and intracellular TLR7 and TLR9 expression in pDCs from BM, spleen, and PP. Shaded area represents isotype control staining. (B) Gene expression of Irf7 , Irf8 , and Tcf4 in pDCs from BM, spleen, and PP, determined by quantitative PCR. (C-D) Proportion (C) and absolute number (D) of PP pDCs from Stat1 −/− , Ifnar −/− , and wild-type (WT) controls. Error bars represent SEM of results from 4-8 mice. (E) PP pDC proportions in mice receiving HGT with pORF vector (NT) or pORF encoding Flt3L (10 μg), 4 days after treatment. (F) PP pDC proportions in Stat1 −/− mice at 8 days after transfer of 10 5 congenic CD45.1 + DC progenitors (BM lin − Flt3 + CD115 + c-kit int cells) intravenously. Similar results were obtained after transfer into Ifnar1 −/− mice (not shown). (G) PP pDC proportions in Stat1 −/− or Ifnar1 −/− mice 8 days after transfer of 10 5 wild-type DC progenitors (BM lin − Flt3 + CD115 + c-kit int cells) intravenously (CDP) or in animals left untreated (NT), as indicated. Some mice received Flt3L HGT 2 days before DC progenitor transfer (CDP + Flt3L) or Flt3L HGT alone (Flt3L), as shown. (B-G) Data represent 2 or 3 independent experiments. N = 4-10.
Figure Legend Snippet: Characterization of PP pDCs and their requirement for IFN-STAT1 signaling. (A) Gating strategy of PP pDCs. Surface expression of PDCA-1, B220, CCR9, CD86, and TLR4 and intracellular TLR7 and TLR9 expression in pDCs from BM, spleen, and PP. Shaded area represents isotype control staining. (B) Gene expression of Irf7 , Irf8 , and Tcf4 in pDCs from BM, spleen, and PP, determined by quantitative PCR. (C-D) Proportion (C) and absolute number (D) of PP pDCs from Stat1 −/− , Ifnar −/− , and wild-type (WT) controls. Error bars represent SEM of results from 4-8 mice. (E) PP pDC proportions in mice receiving HGT with pORF vector (NT) or pORF encoding Flt3L (10 μg), 4 days after treatment. (F) PP pDC proportions in Stat1 −/− mice at 8 days after transfer of 10 5 congenic CD45.1 + DC progenitors (BM lin − Flt3 + CD115 + c-kit int cells) intravenously. Similar results were obtained after transfer into Ifnar1 −/− mice (not shown). (G) PP pDC proportions in Stat1 −/− or Ifnar1 −/− mice 8 days after transfer of 10 5 wild-type DC progenitors (BM lin − Flt3 + CD115 + c-kit int cells) intravenously (CDP) or in animals left untreated (NT), as indicated. Some mice received Flt3L HGT 2 days before DC progenitor transfer (CDP + Flt3L) or Flt3L HGT alone (Flt3L), as shown. (B-G) Data represent 2 or 3 independent experiments. N = 4-10.

Techniques Used: Expressing, Staining, Real-time Polymerase Chain Reaction, Mouse Assay, Plasmid Preparation

36) Product Images from "Regulation of iNOS Gene Transcription by IL-1β and IFN-γ Requires a Coactivator Exchange Mechanism"

Article Title: Regulation of iNOS Gene Transcription by IL-1β and IFN-γ Requires a Coactivator Exchange Mechanism

Journal: Molecular Endocrinology

doi: 10.1210/me.2013-1159

Phosphorylation of STAT1 at Ser727 is critical for IL-1β-stimulated transcription of the iNOS gene. A, 832/13 cells were cotransfected with WT or cGAS −1-kb constructs and either an siRNA duplex containing a control sequence (siScramble)
Figure Legend Snippet: Phosphorylation of STAT1 at Ser727 is critical for IL-1β-stimulated transcription of the iNOS gene. A, 832/13 cells were cotransfected with WT or cGAS −1-kb constructs and either an siRNA duplex containing a control sequence (siScramble)

Techniques Used: Construct, Sequencing

37) Product Images from "Regulation of iNOS Gene Transcription by IL-1β and IFN-γ Requires a Coactivator Exchange Mechanism"

Article Title: Regulation of iNOS Gene Transcription by IL-1β and IFN-γ Requires a Coactivator Exchange Mechanism

Journal: Molecular Endocrinology

doi: 10.1210/me.2013-1159

Phosphorylation of STAT1 at Ser727 is critical for IL-1β-stimulated transcription of the iNOS gene. A, 832/13 cells were cotransfected with WT or cGAS −1-kb constructs and either an siRNA duplex containing a control sequence (siScramble)
Figure Legend Snippet: Phosphorylation of STAT1 at Ser727 is critical for IL-1β-stimulated transcription of the iNOS gene. A, 832/13 cells were cotransfected with WT or cGAS −1-kb constructs and either an siRNA duplex containing a control sequence (siScramble)

Techniques Used: Construct, Sequencing

38) Product Images from "Intestinal Myofibroblasts Produce Nitric Oxide in Response to Combinatorial Cytokine Stimulation"

Article Title: Intestinal Myofibroblasts Produce Nitric Oxide in Response to Combinatorial Cytokine Stimulation

Journal: Journal of cellular physiology

doi: 10.1002/jcp.24164

Combinatorial cytokine stimulation of IMF induces iNOS expression and NO production through a signaling pathway that requires activation of Akt,STAT1, and NF-κB. A: Western blots with antibodies specific to phosphorylated Ser473 on Akt, total
Figure Legend Snippet: Combinatorial cytokine stimulation of IMF induces iNOS expression and NO production through a signaling pathway that requires activation of Akt,STAT1, and NF-κB. A: Western blots with antibodies specific to phosphorylated Ser473 on Akt, total

Techniques Used: Expressing, Activation Assay, Western Blot

39) Product Images from "Differential Lymphocyte and Antibody Responses in Deer Mice Infected with Sin Nombre Hantavirus or Andes Hantavirus"

Article Title: Differential Lymphocyte and Antibody Responses in Deer Mice Infected with Sin Nombre Hantavirus or Andes Hantavirus

Journal: Journal of Virology

doi: 10.1128/JVI.00004-14

Detection of phosphorylated STAT1 and IRF8 in LNC from SNV-infected and ANDV-infected deer mice. All cultures were responsive to stimulation, as indicated by increased expression of IRF8 and STAT1 and by STAT1 activation (phospho-Y701). Cells were harvested
Figure Legend Snippet: Detection of phosphorylated STAT1 and IRF8 in LNC from SNV-infected and ANDV-infected deer mice. All cultures were responsive to stimulation, as indicated by increased expression of IRF8 and STAT1 and by STAT1 activation (phospho-Y701). Cells were harvested

Techniques Used: Infection, Mouse Assay, Expressing, Activation Assay

40) Product Images from "JUNB/AP-1 controls IFN-? during inflammatory liver disease"

Article Title: JUNB/AP-1 controls IFN-? during inflammatory liver disease

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI70405

STAT1 targets, IRF1 and iNos, are altered in Junb Δ li* mice.
Figure Legend Snippet: STAT1 targets, IRF1 and iNos, are altered in Junb Δ li* mice.

Techniques Used: Mouse Assay

STAT1 pathway is altered in Junb Δ li* mice.
Figure Legend Snippet: STAT1 pathway is altered in Junb Δ li* mice.

Techniques Used: Mouse Assay

Related Articles

In Vivo:

Article Title: A Point Mutation, E95D, in the Mumps Virus V Protein Disengages STAT3 Targeting from STAT1 Targeting ▿
Article Snippet: .. Considering that STAT1 targeting in the absence of STAT3 targeting would create an unusual situation for mumps virus, it seems reasonable to predict that more dramatic consequences would result from the E95D mutant virus in vivo if an appropriate model system were available. ..

Mutagenesis:

Article Title: A Point Mutation, E95D, in the Mumps Virus V Protein Disengages STAT3 Targeting from STAT1 Targeting ▿
Article Snippet: .. Considering that STAT1 targeting in the absence of STAT3 targeting would create an unusual situation for mumps virus, it seems reasonable to predict that more dramatic consequences would result from the E95D mutant virus in vivo if an appropriate model system were available. ..

Article Title: Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation
Article Snippet: .. Examination of individual FGFR3 signaling effectors showed that phosphorylation of Shp2, MAPK, Stat1, and Stat3 was also stimulated by both 4Y and the 3F-724Y Add-back mutant. .. Mutation of Y724 to F abolished both FGFR3-dependent transformation and activation of signaling pathways, further demonstrating that Y724 functions as the critical regulatory tyrosine residue for the panel of mutants examined here.

Construct:

Article Title: Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation
Article Snippet: .. Expression of either 4Y or the 3F-724Y Add-back construct led to phosphorylation of Stat1 and Stat3 proteins in 293T cells (Figure , A and C, lanes 3 and 5). .. Removal of Y724 in the Single F mutant 3Y-724F abrogated the ability of this FGFR3 derivative to mediate phosphorylation of Stat1 or Stat3 (Figure , A and C, lane 13).

Mouse Assay:

Article Title: Transcriptional and physiological roles for STAT proteins in leptin action
Article Snippet: .. 3.2 STAT1 fails to compensate for the lack of STAT3 in LepRb neurons To determine whether STAT1 might play a role in LepRb signaling and the control of energy balance by leptin, we bred Stat1 flox onto the Lepr cre background to generate Lepr cre/cre ;Stat1 flox/flox (STAT1LepRb KO) mice null for Stat1 in LepRb neurons. .. The deletion of Stat1 from LepRb neurons failed to alter body weight, food intake, adiposity, or glucose homeostasis ( ).

Article Title: Transcriptional and physiological roles for STAT proteins in leptin action
Article Snippet: .. The disruption of Stat1 in LepRb neurons fails to alter energy balance in normal mice, or in STAT3LepRb KO mice, however. .. Thus, STAT1 is unable to compensate, even in part, for the lack of STAT3 in leptin action.

other:

Article Title: A Point Mutation, E95D, in the Mumps Virus V Protein Disengages STAT3 Targeting from STAT1 Targeting ▿
Article Snippet: When activated together (e.g., by IL-6 or IFN-α signaling), STAT1 and STAT3 can form heterodimers.

Article Title: Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation
Article Snippet: Levels of Stat1 and Stat3 were equivalent in each sample (Figure , B and D).

Article Title: A Point Mutation, E95D, in the Mumps Virus V Protein Disengages STAT3 Targeting from STAT1 Targeting ▿
Article Snippet: The mumps virus V protein can associate with and induce the polyubiquitylation and proteasome-mediated degradation of cellular STAT1 and STAT3 proteins to modulate host innate and adaptive antiviral responses ( ).

Expressing:

Article Title: Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation
Article Snippet: .. Expression of either 4Y or the 3F-724Y Add-back construct led to phosphorylation of Stat1 and Stat3 proteins in 293T cells (Figure , A and C, lanes 3 and 5). .. Removal of Y724 in the Single F mutant 3Y-724F abrogated the ability of this FGFR3 derivative to mediate phosphorylation of Stat1 or Stat3 (Figure , A and C, lane 13).

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    Santa Cruz Biotechnology stat1
    Deletion of <t>STAT1</t> and STAT3 in LepRb expressing neurons. (A) Schematic diagram showing the cross of Lepr cre with Stat1 flox and Stat3 flox mice to generate STAT3 LepRb KO and STAT1STAT3 LepRb KO mice. pA: polyadenylation signal. (B) Representative images showing colocalization of STAT1-IR (red) with GFP-IR (green) in the arcuate nucleus of STAT3 LepR KO and STAT1STAT3 LepR KO (both of which are on the R26 eGFP-L10a background) mice. ( C – E ) Male STAT3 LepRb KO and STAT1STAT3 LepRb KO mice were placed on chow and body weight (C) and cumulative food intake (D) were measured weekly. (E) At 14–15 weeks of age, animals underwent body composition analysis by NMR spectroscopy. Mean, quartiles, and individual plots are shown; n = 8–14 per genotype. ANOVA analysis was performed for (C, D) ; unpaired t-test was performed for (E) . All comparisons p = not significant unless indicated.
    Stat1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Deletion of STAT1 and STAT3 in LepRb expressing neurons. (A) Schematic diagram showing the cross of Lepr cre with Stat1 flox and Stat3 flox mice to generate STAT3 LepRb KO and STAT1STAT3 LepRb KO mice. pA: polyadenylation signal. (B) Representative images showing colocalization of STAT1-IR (red) with GFP-IR (green) in the arcuate nucleus of STAT3 LepR KO and STAT1STAT3 LepR KO (both of which are on the R26 eGFP-L10a background) mice. ( C – E ) Male STAT3 LepRb KO and STAT1STAT3 LepRb KO mice were placed on chow and body weight (C) and cumulative food intake (D) were measured weekly. (E) At 14–15 weeks of age, animals underwent body composition analysis by NMR spectroscopy. Mean, quartiles, and individual plots are shown; n = 8–14 per genotype. ANOVA analysis was performed for (C, D) ; unpaired t-test was performed for (E) . All comparisons p = not significant unless indicated.

    Journal: Molecular Metabolism

    Article Title: Transcriptional and physiological roles for STAT proteins in leptin action

    doi: 10.1016/j.molmet.2019.01.007

    Figure Lengend Snippet: Deletion of STAT1 and STAT3 in LepRb expressing neurons. (A) Schematic diagram showing the cross of Lepr cre with Stat1 flox and Stat3 flox mice to generate STAT3 LepRb KO and STAT1STAT3 LepRb KO mice. pA: polyadenylation signal. (B) Representative images showing colocalization of STAT1-IR (red) with GFP-IR (green) in the arcuate nucleus of STAT3 LepR KO and STAT1STAT3 LepR KO (both of which are on the R26 eGFP-L10a background) mice. ( C – E ) Male STAT3 LepRb KO and STAT1STAT3 LepRb KO mice were placed on chow and body weight (C) and cumulative food intake (D) were measured weekly. (E) At 14–15 weeks of age, animals underwent body composition analysis by NMR spectroscopy. Mean, quartiles, and individual plots are shown; n = 8–14 per genotype. ANOVA analysis was performed for (C, D) ; unpaired t-test was performed for (E) . All comparisons p = not significant unless indicated.

    Article Snippet: 3.2 STAT1 fails to compensate for the lack of STAT3 in LepRb neurons To determine whether STAT1 might play a role in LepRb signaling and the control of energy balance by leptin, we bred Stat1 flox onto the Lepr cre background to generate Lepr cre/cre ;Stat1 flox/flox (STAT1LepRb KO) mice null for Stat1 in LepRb neurons.

    Techniques: Expressing, Mouse Assay, Nuclear Magnetic Resonance, Spectroscopy

    Transcriptional response to the deletion of Stat3 in LepRb neurons. (A) Schematic diagram showing the Lepr cre -mediated deletion of Stat3 flox mice on the Rosa26 eGFP-L10a background to generate STAT3 LepRb KO-eGFP-L10a mice. (B – C) Expression values (fragments per million reads; FPM) for genes that were enriched in LepRb neurons under any condition, comparing STAT3 LepRb KO mice to control mice ( B ) or ob/ob mice ( C ) [16] were plotted. Black dots represent genes demonstrating statistically significant changes in gene expression for the comparison; some genes of interest are labeled. ( D ) Comparison of fold change in gene expression relative to control for STAT3 LepRb KO and ob/ob mice for all genes enriched in LepRb neurons. Dashed lines represent fold change values of 1.5 and 0.667 for both axes. Black dots represent regulated genes; red dots show genes that are regulated and known to be controlled by STAT1 [38] . Regions of the graph are denoted by Roman numerals for reference by the main text. (E) Representative images showing colocalization of STAT1-IR (red) and GFP-IR (green) in the ARC of Control and STAT3 LepRb KO mice.

    Journal: Molecular Metabolism

    Article Title: Transcriptional and physiological roles for STAT proteins in leptin action

    doi: 10.1016/j.molmet.2019.01.007

    Figure Lengend Snippet: Transcriptional response to the deletion of Stat3 in LepRb neurons. (A) Schematic diagram showing the Lepr cre -mediated deletion of Stat3 flox mice on the Rosa26 eGFP-L10a background to generate STAT3 LepRb KO-eGFP-L10a mice. (B – C) Expression values (fragments per million reads; FPM) for genes that were enriched in LepRb neurons under any condition, comparing STAT3 LepRb KO mice to control mice ( B ) or ob/ob mice ( C ) [16] were plotted. Black dots represent genes demonstrating statistically significant changes in gene expression for the comparison; some genes of interest are labeled. ( D ) Comparison of fold change in gene expression relative to control for STAT3 LepRb KO and ob/ob mice for all genes enriched in LepRb neurons. Dashed lines represent fold change values of 1.5 and 0.667 for both axes. Black dots represent regulated genes; red dots show genes that are regulated and known to be controlled by STAT1 [38] . Regions of the graph are denoted by Roman numerals for reference by the main text. (E) Representative images showing colocalization of STAT1-IR (red) and GFP-IR (green) in the ARC of Control and STAT3 LepRb KO mice.

    Article Snippet: 3.2 STAT1 fails to compensate for the lack of STAT3 in LepRb neurons To determine whether STAT1 might play a role in LepRb signaling and the control of energy balance by leptin, we bred Stat1 flox onto the Lepr cre background to generate Lepr cre/cre ;Stat1 flox/flox (STAT1LepRb KO) mice null for Stat1 in LepRb neurons.

    Techniques: Mouse Assay, Expressing, Labeling

    Role of Y724 in full-length FGFR3. 293T cells transfected with full-length derivatives of FGFR3, with or without the Y724F mutation, were lysed and analyzed by immunoblotting. (A) Immunoblotting with antisera against FGFR3 shows equivalent expression levels of full-length FGFR3 derivatives. (B) Stat1 is phosphorylated in response to full-length constitutively active FGFR3 but not if the Y724F mutation is present. Lysates were examined by immunoblotting with anti-phospho-Stat1 (Y701) sera. Equal amounts of Stat1 were present in each sample.

    Journal: Molecular Biology of the Cell

    Article Title: Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation

    doi:

    Figure Lengend Snippet: Role of Y724 in full-length FGFR3. 293T cells transfected with full-length derivatives of FGFR3, with or without the Y724F mutation, were lysed and analyzed by immunoblotting. (A) Immunoblotting with antisera against FGFR3 shows equivalent expression levels of full-length FGFR3 derivatives. (B) Stat1 is phosphorylated in response to full-length constitutively active FGFR3 but not if the Y724F mutation is present. Lysates were examined by immunoblotting with anti-phospho-Stat1 (Y701) sera. Equal amounts of Stat1 were present in each sample.

    Article Snippet: Expression of either 4Y or the 3F-724Y Add-back construct led to phosphorylation of Stat1 and Stat3 proteins in 293T cells (Figure , A and C, lanes 3 and 5).

    Techniques: Transfection, Mutagenesis, Expressing

    Phosphorylation of Stat1 and Stat3. (A) Phosphorylation of Stat1 in cells expressing FGFR3 derivatives. (B) Immunoblotting of Stat1 protein indicated equivalent expression in each sample. (C) Phosphorylation of Stat3 in response to FGFR3 constructs. (D) Immunoblotting of Stat3 protein indicated equal expression in each lane. (E) Levels of the FGFR3 Add-back and Single F mutants were examined by immunoblotting of whole-cell lysates with FGFR3 antisera.

    Journal: Molecular Biology of the Cell

    Article Title: Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation

    doi:

    Figure Lengend Snippet: Phosphorylation of Stat1 and Stat3. (A) Phosphorylation of Stat1 in cells expressing FGFR3 derivatives. (B) Immunoblotting of Stat1 protein indicated equivalent expression in each sample. (C) Phosphorylation of Stat3 in response to FGFR3 constructs. (D) Immunoblotting of Stat3 protein indicated equal expression in each lane. (E) Levels of the FGFR3 Add-back and Single F mutants were examined by immunoblotting of whole-cell lysates with FGFR3 antisera.

    Article Snippet: Expression of either 4Y or the 3F-724Y Add-back construct led to phosphorylation of Stat1 and Stat3 proteins in 293T cells (Figure , A and C, lanes 3 and 5).

    Techniques: Expressing, Construct