Structured Review

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Expression of NFKB2 and <t>IRF1</t> predicts response to immunotherapy. (A) Various biomarkers for response to immunotherapy (y axis), including expression of NFKB2 + IRF1 expression, were compared using previously published genesets with TIDE online platform ( Jiang et al., 2018 ).
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1) Product Images from "Genome-wide profiling of druggable active tumor defense mechanisms to enhance cancer immunotherapy"

Article Title: Genome-wide profiling of druggable active tumor defense mechanisms to enhance cancer immunotherapy

Journal: bioRxiv

doi: 10.1101/843185

Expression of NFKB2 and IRF1 predicts response to immunotherapy. (A) Various biomarkers for response to immunotherapy (y axis), including expression of NFKB2 + IRF1 expression, were compared using previously published genesets with TIDE online platform ( Jiang et al., 2018 ).
Figure Legend Snippet: Expression of NFKB2 and IRF1 predicts response to immunotherapy. (A) Various biomarkers for response to immunotherapy (y axis), including expression of NFKB2 + IRF1 expression, were compared using previously published genesets with TIDE online platform ( Jiang et al., 2018 ).

Techniques Used: Expressing

2) Product Images from "ZBED2 is an antagonist of Interferon Regulatory Factor 1 and modulates cell identity in pancreatic cancer"

Article Title: ZBED2 is an antagonist of Interferon Regulatory Factor 1 and modulates cell identity in pancreatic cancer

Journal: bioRxiv

doi: 10.1101/868141

ChIP-seq analysis implicates ZBED2 as a sequence-specific repressor of ISG promoters. ( A ) Density plots showing FLAG-ZBED2 and H3K27ac enrichment surrounding a 2-kb interval centered on the summit of 2,451 high-confidence ZBED2 peaks in AsPC1 and SUIT2 cells, ranked by FLAG-ZBED2 peak intensity in AsPC1 cells. ( B ) Pie chart showing the distribution of high-confidence FLAG-ZBED2 peaks in AsPC1 cells. TTS, transcription termination site; TSS, transcription start site; UTR, untranslated region. ( C ) GSEA plot evaluating ZBED2 bound genes upon ZBED2 cDNA expression in HPAFII cells. Leading edge (indicated by the red line), Interferon Response genes are listed. ( D ) GAL4 fusion reporter assay testing full length ZBED2 and IRF1 transactivation activity normalized to Renilla luciferase internal control. Mean+SEM is shown. n=3. **p
Figure Legend Snippet: ChIP-seq analysis implicates ZBED2 as a sequence-specific repressor of ISG promoters. ( A ) Density plots showing FLAG-ZBED2 and H3K27ac enrichment surrounding a 2-kb interval centered on the summit of 2,451 high-confidence ZBED2 peaks in AsPC1 and SUIT2 cells, ranked by FLAG-ZBED2 peak intensity in AsPC1 cells. ( B ) Pie chart showing the distribution of high-confidence FLAG-ZBED2 peaks in AsPC1 cells. TTS, transcription termination site; TSS, transcription start site; UTR, untranslated region. ( C ) GSEA plot evaluating ZBED2 bound genes upon ZBED2 cDNA expression in HPAFII cells. Leading edge (indicated by the red line), Interferon Response genes are listed. ( D ) GAL4 fusion reporter assay testing full length ZBED2 and IRF1 transactivation activity normalized to Renilla luciferase internal control. Mean+SEM is shown. n=3. **p

Techniques Used: Chromatin Immunoprecipitation, Sequencing, Expressing, Reporter Assay, Activity Assay, Luciferase

Antagonistic regulation of ISG promoters by ZBED2 and IRF1. Related to Fig. 4. ( A ) Summary of CentriMo motif enrichment analysis for JASPAR motifs at ZBED2 binding sites. The top 10 transcription factor (TF) motifs ranked by E value are shown. The nucleotide sequence 200bp up- or downstream of the peak summit of the top 1000 ZBED2 peaks in AsPC1 cells was used for this analysis. ( B ) Expression of ZBED2 and IRF family TF genes in 15 human PDA cell lines. ( C ) ZBED2 expression versus IRF2 , IRF3 , IRF5 , IRF6 , IRF7 and IRF9 expression across 15 human PDA cell lines. ( D ) ZBED2 expression correlation with IRF family TF genes in 1,156 cancer cell lines from the CCLE database analyzed using CBioPortal. ( E and F ) Density plots showing IRF1 and FLAG-ZBED2 enrichment surrounding a 2-kb interval centered on the summit of all IRF1 peaks ( E ) or 140 random IRF1 peaks that do not intersect with FLAG-ZBED2 sites in AsPC1 cells ( F ), ranked by IRF1 peak intensity. ( G ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 in AsPC1 cells at the promoters of BTN3A3 and SAMD9 . ( H ) Gene ontology (GO) analysis with Metascape of genes annotated by HOMER to IRF1 only sites. Terms are ranked by their significance ( p value) and no terms reached the significant threshold (-log 10 p value > 12). ( I ) Pie chart showing the distribution of 140 IRF1 only peaks (left) or IRF1/ZBED2 peaks (right) in AsPC1 cells. TTS, transcription termination site; TSS, transcription start site; UTR, untranslated region. ( J and K ) GSEA plots evaluating protein coding genes annotated by HOMER to IRF1 only sites upon IRF1 ( J ) or ZBED2 ( K ) cDNA expression in AsPC1 cells. ( L and M ) GSEA plots evaluating the Interferon Response signature upon IRF1 cDNA expression in AsPC1 cells ( L ) or IRF1 knockout in PANC0403 cells ( M ). ( N ) Expression levels of protein coding genes annotated to IRF1 only sites following 12-hour treatment with 0.2ng/µl of IFN-β, IFN-γ or control. ***p
Figure Legend Snippet: Antagonistic regulation of ISG promoters by ZBED2 and IRF1. Related to Fig. 4. ( A ) Summary of CentriMo motif enrichment analysis for JASPAR motifs at ZBED2 binding sites. The top 10 transcription factor (TF) motifs ranked by E value are shown. The nucleotide sequence 200bp up- or downstream of the peak summit of the top 1000 ZBED2 peaks in AsPC1 cells was used for this analysis. ( B ) Expression of ZBED2 and IRF family TF genes in 15 human PDA cell lines. ( C ) ZBED2 expression versus IRF2 , IRF3 , IRF5 , IRF6 , IRF7 and IRF9 expression across 15 human PDA cell lines. ( D ) ZBED2 expression correlation with IRF family TF genes in 1,156 cancer cell lines from the CCLE database analyzed using CBioPortal. ( E and F ) Density plots showing IRF1 and FLAG-ZBED2 enrichment surrounding a 2-kb interval centered on the summit of all IRF1 peaks ( E ) or 140 random IRF1 peaks that do not intersect with FLAG-ZBED2 sites in AsPC1 cells ( F ), ranked by IRF1 peak intensity. ( G ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 in AsPC1 cells at the promoters of BTN3A3 and SAMD9 . ( H ) Gene ontology (GO) analysis with Metascape of genes annotated by HOMER to IRF1 only sites. Terms are ranked by their significance ( p value) and no terms reached the significant threshold (-log 10 p value > 12). ( I ) Pie chart showing the distribution of 140 IRF1 only peaks (left) or IRF1/ZBED2 peaks (right) in AsPC1 cells. TTS, transcription termination site; TSS, transcription start site; UTR, untranslated region. ( J and K ) GSEA plots evaluating protein coding genes annotated by HOMER to IRF1 only sites upon IRF1 ( J ) or ZBED2 ( K ) cDNA expression in AsPC1 cells. ( L and M ) GSEA plots evaluating the Interferon Response signature upon IRF1 cDNA expression in AsPC1 cells ( L ) or IRF1 knockout in PANC0403 cells ( M ). ( N ) Expression levels of protein coding genes annotated to IRF1 only sites following 12-hour treatment with 0.2ng/µl of IFN-β, IFN-γ or control. ***p

Techniques Used: Binding Assay, Sequencing, Expressing, Chromatin Immunoprecipitation, Knock-Out

Antagonistic regulation of ISG promoters by ZBED2 and IRF1. ( A ) CentriMo motif enrichment analysis for JASPAR motifs at ZBED2 binding sites. ( B ) ZBED2 expression versus the IRF1 expression across 15 human PDA cell lines. ( C ) Density plot showing IRF1 and FLAG-ZBED2 enrichment surrounding a 2-kb interval centered on the summit of 140 intersecting IRF1 and FLAG-ZBED2 sites in AsPC1 cells, ranked by IRF1 peak intensity. ( D ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 in AsPC1 cells at the promoters of CMPK2 and STAT2 . ( E ) Gene ontology (GO) analysis with Metascape of genes annotated by HOMER to IRF1/ZBED2 sites. Terms are ranked by their significance ( p value) and the most significant terms (-log 10 p value > 12) are shown. ( F ) RT-qPCR analysis of CMPK2 in AsPC1-empty and AsPC1-ZBED2 cells following IRF1 cDNA expression. Mean+SEM is shown. n=3. **p
Figure Legend Snippet: Antagonistic regulation of ISG promoters by ZBED2 and IRF1. ( A ) CentriMo motif enrichment analysis for JASPAR motifs at ZBED2 binding sites. ( B ) ZBED2 expression versus the IRF1 expression across 15 human PDA cell lines. ( C ) Density plot showing IRF1 and FLAG-ZBED2 enrichment surrounding a 2-kb interval centered on the summit of 140 intersecting IRF1 and FLAG-ZBED2 sites in AsPC1 cells, ranked by IRF1 peak intensity. ( D ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 in AsPC1 cells at the promoters of CMPK2 and STAT2 . ( E ) Gene ontology (GO) analysis with Metascape of genes annotated by HOMER to IRF1/ZBED2 sites. Terms are ranked by their significance ( p value) and the most significant terms (-log 10 p value > 12) are shown. ( F ) RT-qPCR analysis of CMPK2 in AsPC1-empty and AsPC1-ZBED2 cells following IRF1 cDNA expression. Mean+SEM is shown. n=3. **p

Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation, Quantitative RT-PCR

ZBED2 represses pancreatic progenitor lineage identity in PDA. Related to Fig. 6. ( A ) Summary of GSEA evaluating the Squamous PDA Identity signature upon ZBED2 cDNA expression in 15 PDA cell lines. ( B and C ) Expression changes at IRF1/ZBED2 bound genes in AsPC1 cells infected with ZBED2 cDNA ( B ) or IRF1 cDNA ( C ) versus those infected with an empty vector control. GATA6 and CMPK2 are labeled along with their rank with respect to downregulated ( B ) or upregulated ( C ) genes. ( D and E ) GATA6 ( D ) and IRF1 ( E ) expression in 15 human PDA cell lines. ( F and G ) Proportion of PDA patient samples from the indicated studies stratified as ZBED2 low or ZBED2 high classified based on their tumor differentiation status (grade). Statistical significance for the indicated comparisons was assessed using Fisher’s Exact Test, ns = not significant.
Figure Legend Snippet: ZBED2 represses pancreatic progenitor lineage identity in PDA. Related to Fig. 6. ( A ) Summary of GSEA evaluating the Squamous PDA Identity signature upon ZBED2 cDNA expression in 15 PDA cell lines. ( B and C ) Expression changes at IRF1/ZBED2 bound genes in AsPC1 cells infected with ZBED2 cDNA ( B ) or IRF1 cDNA ( C ) versus those infected with an empty vector control. GATA6 and CMPK2 are labeled along with their rank with respect to downregulated ( B ) or upregulated ( C ) genes. ( D and E ) GATA6 ( D ) and IRF1 ( E ) expression in 15 human PDA cell lines. ( F and G ) Proportion of PDA patient samples from the indicated studies stratified as ZBED2 low or ZBED2 high classified based on their tumor differentiation status (grade). Statistical significance for the indicated comparisons was assessed using Fisher’s Exact Test, ns = not significant.

Techniques Used: Expressing, Infection, Plasmid Preparation, Labeling

ZBED2 represses pancreatic progenitor lineage identity in PDA. ( A - C ) TF expression in Basal-like/Squamous and Classical/Progenitor subtypes of PDA. TFs are ranked by their mean log 2 fold change in Basal-like versus Classical ( A and C ) or Squamous versus Progenitor ( B ) patient samples from the indicated studies. ( D - F ) ZBED2 expression in PDA patient samples stratified according to molecular subtype. Each dot represents one patient sample. p value was calculated using Student’s t test. ( G ) Summary of GSEA evaluating the Progenitor PDA Identity signature upon ZBED2 cDNA expression in 15 PDA cell lines. ( H and I ) GSEA plots evaluating the Progenitor-PDA Identity signature following expression of ZBED2 ( H ) or IRF1 ( I ) in AsPC1 cells. ( J ) Overlap of Progenitor PDA Identity genes with protein coding genes associated with IRF1/ZBED2 sites. ( K ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 at the promoter of GATA6 in AsPC1 cells. ( L ) Overlap of leading edge (LE) genes associated with IRF1/ZBED2 sites that are repressed by ZBED2 or activated by IRF1 in AsPC1 cells. ( M ) Quantification of colony size in three-dimensional (3D) Matrigel colony formation assays. Means+SEM are shown. n=3. Representative images at day 7 are shown (right panel). ( N ) Quantification of scratch assays at the indicated time points post-seeding and representative images (right panel). Scale bar indicates 500µm. Means+SEM are shown. n=3. ***p
Figure Legend Snippet: ZBED2 represses pancreatic progenitor lineage identity in PDA. ( A - C ) TF expression in Basal-like/Squamous and Classical/Progenitor subtypes of PDA. TFs are ranked by their mean log 2 fold change in Basal-like versus Classical ( A and C ) or Squamous versus Progenitor ( B ) patient samples from the indicated studies. ( D - F ) ZBED2 expression in PDA patient samples stratified according to molecular subtype. Each dot represents one patient sample. p value was calculated using Student’s t test. ( G ) Summary of GSEA evaluating the Progenitor PDA Identity signature upon ZBED2 cDNA expression in 15 PDA cell lines. ( H and I ) GSEA plots evaluating the Progenitor-PDA Identity signature following expression of ZBED2 ( H ) or IRF1 ( I ) in AsPC1 cells. ( J ) Overlap of Progenitor PDA Identity genes with protein coding genes associated with IRF1/ZBED2 sites. ( K ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 at the promoter of GATA6 in AsPC1 cells. ( L ) Overlap of leading edge (LE) genes associated with IRF1/ZBED2 sites that are repressed by ZBED2 or activated by IRF1 in AsPC1 cells. ( M ) Quantification of colony size in three-dimensional (3D) Matrigel colony formation assays. Means+SEM are shown. n=3. Representative images at day 7 are shown (right panel). ( N ) Quantification of scratch assays at the indicated time points post-seeding and representative images (right panel). Scale bar indicates 500µm. Means+SEM are shown. n=3. ***p

Techniques Used: Expressing, Chromatin Immunoprecipitation

ZBED2 protects PDA cells from IRF1- and interferon- γ -induced growth arrest. ( A ) Luciferase-based quantification of cell viability of AsPC1 cells grown in Matrigel on day 7 post infection with IRF1 cDNA or the empty vector. Representative bright field images (right panel) are shown. Scale bar indicates 200µm. ( B ) Luciferase-based quantification of cell viability of AsPC1-empty and AsPC1-ZBED2 cells grown in Matrigel following co-expression of IRF1 cDNA or empty vector for 7 days. ( C ) Representative bright filed images from ( B ). Scale bar indicates 500µm. ( D ) Luciferase-based quantification of cell viability of KPC-derived FC1199 cells stably expressing ZBED2 or the empty vector grown in Matrigel following co-expression of IRF1 cDNA or empty vector for 7 days. ( E ) Representative bright filed images from ( D ). Scale bar indicates 200µm. ( F - H ) AsPC1 cells infected with sgRNAs targeting IRF1, IRF9 or a control sgRNA (sgNEG) were plated in Matrigel and grown for 7 days in the presence of 20ng/µl of IFN-γ, IFN-β or control. Representative western blots following overnight stimulation with 20ng/µl of IFN-γ ( F ), luciferase-based quantification ( G ) and representative bright field images on day 7 of the assay ( H ) are shown. Scale bar indicates 500µm. ( I and J ) AsPC1 cells ( I ) or the indicated mouse KPC cell lines ( J ) were infected with the ZBED2 cDNA or an empty vector and grown in Matrigel with the increasing concentrations of IFN-γ. Bar charts show luciferase-based quantification on day 7. Mean+SEM is shown. n=3. For ( A ), ( B ), ( D ) and ( G ), ***p
Figure Legend Snippet: ZBED2 protects PDA cells from IRF1- and interferon- γ -induced growth arrest. ( A ) Luciferase-based quantification of cell viability of AsPC1 cells grown in Matrigel on day 7 post infection with IRF1 cDNA or the empty vector. Representative bright field images (right panel) are shown. Scale bar indicates 200µm. ( B ) Luciferase-based quantification of cell viability of AsPC1-empty and AsPC1-ZBED2 cells grown in Matrigel following co-expression of IRF1 cDNA or empty vector for 7 days. ( C ) Representative bright filed images from ( B ). Scale bar indicates 500µm. ( D ) Luciferase-based quantification of cell viability of KPC-derived FC1199 cells stably expressing ZBED2 or the empty vector grown in Matrigel following co-expression of IRF1 cDNA or empty vector for 7 days. ( E ) Representative bright filed images from ( D ). Scale bar indicates 200µm. ( F - H ) AsPC1 cells infected with sgRNAs targeting IRF1, IRF9 or a control sgRNA (sgNEG) were plated in Matrigel and grown for 7 days in the presence of 20ng/µl of IFN-γ, IFN-β or control. Representative western blots following overnight stimulation with 20ng/µl of IFN-γ ( F ), luciferase-based quantification ( G ) and representative bright field images on day 7 of the assay ( H ) are shown. Scale bar indicates 500µm. ( I and J ) AsPC1 cells ( I ) or the indicated mouse KPC cell lines ( J ) were infected with the ZBED2 cDNA or an empty vector and grown in Matrigel with the increasing concentrations of IFN-γ. Bar charts show luciferase-based quantification on day 7. Mean+SEM is shown. n=3. For ( A ), ( B ), ( D ) and ( G ), ***p

Techniques Used: Luciferase, Infection, Plasmid Preparation, Expressing, Derivative Assay, Stable Transfection, Western Blot

ZBED2 protects PDA cells from IRF1- and interferon-γ-induced growth arrest. Related to Fig. 5. ( A ) Luciferase-based quantification of cell viability of SUIT2 cells grown in Matrigel on day 7 post infection with IRF1 cDNA or the empty vector. Representative bright field images (right panel) are shown. Scale bar indicates 200µm. ( B-D ) SUIT2 cells infected with sgRNAs targeting IRF1, IRF9 or a control sgRNA (sgNEG) were plated in Matrigel and grown for 7 days in the presence of 20ng/µl of IFN-γ, IFN-β or control. Representative western blots following overnight stimulation with 20ng/µl of IFN-γ ( B ), luciferase-based quantification ( C ) and representative bright field images on day 7 of the assay ( D ) are shown. Scale bar indicates 500µm. ( E ) FC1242 KPC cells were infected with the ZBED2 cDNA or an empty vector and grown in Matrigel with the increasing concentrations of IFN-γ. Bar chart shows luciferase-based quantification on day 7. Mean+SEM is shown. n=3. For ( A ) and ( C ), ***p
Figure Legend Snippet: ZBED2 protects PDA cells from IRF1- and interferon-γ-induced growth arrest. Related to Fig. 5. ( A ) Luciferase-based quantification of cell viability of SUIT2 cells grown in Matrigel on day 7 post infection with IRF1 cDNA or the empty vector. Representative bright field images (right panel) are shown. Scale bar indicates 200µm. ( B-D ) SUIT2 cells infected with sgRNAs targeting IRF1, IRF9 or a control sgRNA (sgNEG) were plated in Matrigel and grown for 7 days in the presence of 20ng/µl of IFN-γ, IFN-β or control. Representative western blots following overnight stimulation with 20ng/µl of IFN-γ ( B ), luciferase-based quantification ( C ) and representative bright field images on day 7 of the assay ( D ) are shown. Scale bar indicates 500µm. ( E ) FC1242 KPC cells were infected with the ZBED2 cDNA or an empty vector and grown in Matrigel with the increasing concentrations of IFN-γ. Bar chart shows luciferase-based quantification on day 7. Mean+SEM is shown. n=3. For ( A ) and ( C ), ***p

Techniques Used: Luciferase, Infection, Plasmid Preparation, Western Blot

3) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

4) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

5) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

6) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

7) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

8) Product Images from "MHC class I expression in intestinal cells is reduced by rotavirus infection and increased in bystander cells lacking rotavirus antigen"

Article Title: MHC class I expression in intestinal cells is reduced by rotavirus infection and increased in bystander cells lacking rotavirus antigen

Journal: Scientific Reports

doi: 10.1038/s41598-017-18464-x

Localization of activated STAT1 and IRF1 in rotavirus-infected and bystander cells. HT-29 cells were infected with RRV at a m.o.i. of 1 or mock-infected. After 7 h cells were fixed, permeabilised and stained with antibodies to rotavirus VP6 and phosphorylated STAT1 (pSTAT1; a , b ) or VP6 and IRF1 ( c , d ). Nuclei were stained with DAPI and representative images obtained by confocal microscopy. The VP6-positive (infected) cells in the merged images are indicated with white asterisks. Nuclear to cytoplasmic fluorescence ratios (Fn/c) of pSTAT1 ( b ) and IRF1 ( d ) were calculated for the uninfected and infected cell populations, analysing 7 to 14 randomly selected cells of each population type from 2 independent experiments. Images were obtained at ×1000 magnification. ***P
Figure Legend Snippet: Localization of activated STAT1 and IRF1 in rotavirus-infected and bystander cells. HT-29 cells were infected with RRV at a m.o.i. of 1 or mock-infected. After 7 h cells were fixed, permeabilised and stained with antibodies to rotavirus VP6 and phosphorylated STAT1 (pSTAT1; a , b ) or VP6 and IRF1 ( c , d ). Nuclei were stained with DAPI and representative images obtained by confocal microscopy. The VP6-positive (infected) cells in the merged images are indicated with white asterisks. Nuclear to cytoplasmic fluorescence ratios (Fn/c) of pSTAT1 ( b ) and IRF1 ( d ) were calculated for the uninfected and infected cell populations, analysing 7 to 14 randomly selected cells of each population type from 2 independent experiments. Images were obtained at ×1000 magnification. ***P

Techniques Used: Infection, Staining, Confocal Microscopy, Fluorescence

9) Product Images from "Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal"

Article Title: Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

Journal: Nature Communications

doi: 10.1038/ncomms12326

SUMOylation regulates TRIM21 expression through IRF1. ( a ) Representative IHC staining indicates higher TRIM21 level corresponding with lower Oct-1 in shSAE2 group tumour tissue compared with shCtrl group (scale bar, 100 μm). Tumour tissues were from LDA assay described in Fig. 2b . ( b ) Western blot (left) and quantification of SAE2, Oct-1, TRIM21 and ALDH levels in the same tumour tissues as a ; GAPDH, loading control. ‘1, 2 and 3' indicate tumour tissues from different mouse. ( c ) TRIM21 promoter activity was inhibited by overexpression of SAE2 and Ubc9 but increased by SENP1 overexpression as determined by luciferase reporter assay. HT29 cells were transfected with empty vector (Ctrl) or SAE2, UBC9 or SENP1 expression plasmid together with TRIM21 promoter luciferase reporter and Renilla plasmids. Dual-luciferase activity was measured after 48 h and normalized results were analysed with two-tailed Student's t -test. ( d ) TRIM21 mRNA level was suppressed by SAE2 or Ubc9 overexpression and enhanced with SENP1 overexpression in HT29 cells as determined by quantitative PCR (qPCR). ( e ) IRF1 SUMOylation site mutant K78R induced higher TRIM21 mRNA level than wild-type (WT) IRF1 as determined by qPCR. ( f ) TRIM21 protein level was suppressed on SAE2 or Ubc9 overexpression but enhanced with SENP1 overexpression as indicated by western blot; GAPDH, loading control. ( g ) Western blot showed overexpression of K78R mutant-induced higher TRIM21 protein level than WT IRF1 in shCtrl HT29 cells; GAPDH, loading control. * P
Figure Legend Snippet: SUMOylation regulates TRIM21 expression through IRF1. ( a ) Representative IHC staining indicates higher TRIM21 level corresponding with lower Oct-1 in shSAE2 group tumour tissue compared with shCtrl group (scale bar, 100 μm). Tumour tissues were from LDA assay described in Fig. 2b . ( b ) Western blot (left) and quantification of SAE2, Oct-1, TRIM21 and ALDH levels in the same tumour tissues as a ; GAPDH, loading control. ‘1, 2 and 3' indicate tumour tissues from different mouse. ( c ) TRIM21 promoter activity was inhibited by overexpression of SAE2 and Ubc9 but increased by SENP1 overexpression as determined by luciferase reporter assay. HT29 cells were transfected with empty vector (Ctrl) or SAE2, UBC9 or SENP1 expression plasmid together with TRIM21 promoter luciferase reporter and Renilla plasmids. Dual-luciferase activity was measured after 48 h and normalized results were analysed with two-tailed Student's t -test. ( d ) TRIM21 mRNA level was suppressed by SAE2 or Ubc9 overexpression and enhanced with SENP1 overexpression in HT29 cells as determined by quantitative PCR (qPCR). ( e ) IRF1 SUMOylation site mutant K78R induced higher TRIM21 mRNA level than wild-type (WT) IRF1 as determined by qPCR. ( f ) TRIM21 protein level was suppressed on SAE2 or Ubc9 overexpression but enhanced with SENP1 overexpression as indicated by western blot; GAPDH, loading control. ( g ) Western blot showed overexpression of K78R mutant-induced higher TRIM21 protein level than WT IRF1 in shCtrl HT29 cells; GAPDH, loading control. * P

Techniques Used: Expressing, Immunohistochemistry, Staining, Western Blot, Activity Assay, Over Expression, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Two Tailed Test, Real-time Polymerase Chain Reaction, Mutagenesis

A mechanism of how SUMOylation is involved in CSC function. ( a ) Overexpression of Oct-1 partially compensated for the reduction of CSC frequency by SAE2 knockdown in HT29 cells as determined by LDA. Stable cell lines were generated with lentivirus expressing pLenti CMV-hygro empty vector (EV) or pLenti CMV-Flag-Oct-1 in HT29 shCtrl and shSAE2 cells as shCtrl+EV, shSAE2+EV and shSAE2+Oct-1. ( b ) Representative western blot of the stable lines to confirm expression with Oct-1 and Flag-tag antibodies; GAPDH, loading control. ( c ) Overexpression of Oct-1 in SAE2 or Ubc9 knockdown cells restored ALDH + cells population in HT29 cells as measured by FACS analysis using the AldeFluor kit. HT29 cells were transfected with control non-targeting siRNA (SiCtrl), SAE2-targeting siRNA (SiSAE2) or Ubc9-targeting siRNA (SiUbc9) followed by Flag-Oct-1 plasmid or control empty vector transfection. After 3 days, cells were collected for FACS analysis using the AldeFluor kit. ( d ) Representative western blot of the samples from c ; quantification of the ALDH1A1 band intensity is shown on the bottom. ( e ) Knockdown of Ubc9 reduced CSC frequency, as shown by LDA using spheroid formation using HT29 stable cell lines expressing two different UBC9-targeting shRNA (shUBC9#1 and shUBC9#2). ( f ) Schematic diagram showing the mechanism of how SUMOylation regulates CSCs through Oct-1, TRIM21 and IRF1. * P
Figure Legend Snippet: A mechanism of how SUMOylation is involved in CSC function. ( a ) Overexpression of Oct-1 partially compensated for the reduction of CSC frequency by SAE2 knockdown in HT29 cells as determined by LDA. Stable cell lines were generated with lentivirus expressing pLenti CMV-hygro empty vector (EV) or pLenti CMV-Flag-Oct-1 in HT29 shCtrl and shSAE2 cells as shCtrl+EV, shSAE2+EV and shSAE2+Oct-1. ( b ) Representative western blot of the stable lines to confirm expression with Oct-1 and Flag-tag antibodies; GAPDH, loading control. ( c ) Overexpression of Oct-1 in SAE2 or Ubc9 knockdown cells restored ALDH + cells population in HT29 cells as measured by FACS analysis using the AldeFluor kit. HT29 cells were transfected with control non-targeting siRNA (SiCtrl), SAE2-targeting siRNA (SiSAE2) or Ubc9-targeting siRNA (SiUbc9) followed by Flag-Oct-1 plasmid or control empty vector transfection. After 3 days, cells were collected for FACS analysis using the AldeFluor kit. ( d ) Representative western blot of the samples from c ; quantification of the ALDH1A1 band intensity is shown on the bottom. ( e ) Knockdown of Ubc9 reduced CSC frequency, as shown by LDA using spheroid formation using HT29 stable cell lines expressing two different UBC9-targeting shRNA (shUBC9#1 and shUBC9#2). ( f ) Schematic diagram showing the mechanism of how SUMOylation regulates CSCs through Oct-1, TRIM21 and IRF1. * P

Techniques Used: Over Expression, Stable Transfection, Generated, Expressing, Plasmid Preparation, Western Blot, FLAG-tag, FACS, Transfection, shRNA

10) Product Images from "Respiratory virus-induced EGFR activation suppresses IRF1-dependent interferon ? and antiviral defense in airway epithelium"

Article Title: Respiratory virus-induced EGFR activation suppresses IRF1-dependent interferon ? and antiviral defense in airway epithelium

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20121401

EGFR activation suppresses IRF1 and IFN-λ. (A) BEAS-2b cells were transfected with IRF1 luciferase reporter (left), and after 24 h treated with serum-free medium alone (empty columns), 10 ng/ml EGF, IAV (striped columns), and IAV plus EGF for 3 h before luciferase activity was measured ( n = 3 independent experiments in duplicate; *, P
Figure Legend Snippet: EGFR activation suppresses IRF1 and IFN-λ. (A) BEAS-2b cells were transfected with IRF1 luciferase reporter (left), and after 24 h treated with serum-free medium alone (empty columns), 10 ng/ml EGF, IAV (striped columns), and IAV plus EGF for 3 h before luciferase activity was measured ( n = 3 independent experiments in duplicate; *, P

Techniques Used: Activation Assay, Transfection, Luciferase, Activity Assay

IRF1-dependent IFN-λ is required for EGFR inhibitor-induced suppression of viral infection. (A) BEAS-2b cells were treated with serum-free medium alone, or transfected with IRF1 or control (C) siRNA for 24 h and treated with serum-free medium alone (empty column), or IAV (striped column), RV1b (black column), and RV16. 24 h after viral infection secreted IFN-λ was measured by ELISA ( n = 6 independent experiments, mean ± SEM; *, P
Figure Legend Snippet: IRF1-dependent IFN-λ is required for EGFR inhibitor-induced suppression of viral infection. (A) BEAS-2b cells were treated with serum-free medium alone, or transfected with IRF1 or control (C) siRNA for 24 h and treated with serum-free medium alone (empty column), or IAV (striped column), RV1b (black column), and RV16. 24 h after viral infection secreted IFN-λ was measured by ELISA ( n = 6 independent experiments, mean ± SEM; *, P

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

11) Product Images from "Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells"

Article Title: Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-019-1531-3

Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P
Figure Legend Snippet: Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P

Techniques Used: Activation Assay, Migration, Indirect Immunoperoxidase Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

12) Product Images from "Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells"

Article Title: Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-019-1531-3

Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P
Figure Legend Snippet: Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P

Techniques Used: Activation Assay, Migration, Indirect Immunoperoxidase Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

13) Product Images from "Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells"

Article Title: Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-019-1531-3

Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P
Figure Legend Snippet: Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P

Techniques Used: Activation Assay, Migration, Indirect Immunoperoxidase Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

14) Product Images from "Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells"

Article Title: Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-019-1531-3

Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P
Figure Legend Snippet: Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P

Techniques Used: Activation Assay, Migration, Indirect Immunoperoxidase Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

15) Product Images from "miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma"

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S238975

miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P
Figure Legend Snippet: miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P

Techniques Used: Over Expression, Expressing

miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P
Figure Legend Snippet: miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P

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

IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P
Figure Legend Snippet: IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P

Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Inhibition

IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.
Figure Legend Snippet: IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.

Techniques Used: Expressing, Blocking Assay

16) Product Images from "miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma"

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S238975

miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P
Figure Legend Snippet: miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P

Techniques Used: Over Expression, Expressing

miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P
Figure Legend Snippet: miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P

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

IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P
Figure Legend Snippet: IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P

Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Inhibition

IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.
Figure Legend Snippet: IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.

Techniques Used: Expressing, Blocking Assay

17) Product Images from "miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma"

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S238975

miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P
Figure Legend Snippet: miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P

Techniques Used: Over Expression, Expressing

miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P
Figure Legend Snippet: miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P

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

IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P
Figure Legend Snippet: IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P

Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Inhibition

IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.
Figure Legend Snippet: IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.

Techniques Used: Expressing, Blocking Assay

18) Product Images from "Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer"

Article Title: Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer

Journal: JCI Insight

doi: 10.1172/jci.insight.136720

IRF1 functions in both the myeloid and epithelial cell to prevent colitis-associated colorectal tumorigenesis. ( A ) Representative images of colon tumors in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after azoxymethane (AOM) injection. ( B ) Number of colon tumors in WT ( n = 10), LysM Cre Irf1 fl/fl ( n = 10), Villin Cre Irf1 fl/fl ( n = 10), and Irf1 –/– ( n = 7) mice. ( C ) Percentage of tumors of various sizes in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after AOM injection. ( D ) Body weight change in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after AOM injection. Each symbol represents 1 individual mouse in B . * P
Figure Legend Snippet: IRF1 functions in both the myeloid and epithelial cell to prevent colitis-associated colorectal tumorigenesis. ( A ) Representative images of colon tumors in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after azoxymethane (AOM) injection. ( B ) Number of colon tumors in WT ( n = 10), LysM Cre Irf1 fl/fl ( n = 10), Villin Cre Irf1 fl/fl ( n = 10), and Irf1 –/– ( n = 7) mice. ( C ) Percentage of tumors of various sizes in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after AOM injection. ( D ) Body weight change in WT, LysM Cre Irf1 fl/fl , Villin Cre Irf1 fl/fl , and Irf1 –/– mice 80 days after AOM injection. Each symbol represents 1 individual mouse in B . * P

Techniques Used: Mouse Assay, Injection

IRF1 regulates PANoptosis. ( A ) Representative images of TUNEL staining of colons from azoxymethane/dextran sulfate sodium–treated (AOM/DSS-treated) WT and Irf1 –/– mice on days 0, 14, and 80. ( B ) Representative images of cleaved caspase-3 (CASP3) staining of colon tissues from DSS-treated WT and Irf1 –/– mice on days 0 and 14 after AOM injection. Scale bar: 100 μM. ( C ) Immunoblot analysis of the pro- and cleaved forms of CASP3 and caspase-7 (CASP7) in colons of WT and Irf1 –/– mice on days 0, 14, and 80 after AOM injection. Blots represent data from the same biological samples at the indicated time point run in parallel. ( D ) Immunoblot analysis of GSDMD and MLKL in colons of WT and Irf1 –/– mice 14 days after AOM injection. Blots represent data from the same biological samples run in parallel. ( E ) Cell death analysis of intestinal organoids derived from WT and Irf1 –/– mice after stimulation with TNF + zVAD. ** P
Figure Legend Snippet: IRF1 regulates PANoptosis. ( A ) Representative images of TUNEL staining of colons from azoxymethane/dextran sulfate sodium–treated (AOM/DSS-treated) WT and Irf1 –/– mice on days 0, 14, and 80. ( B ) Representative images of cleaved caspase-3 (CASP3) staining of colon tissues from DSS-treated WT and Irf1 –/– mice on days 0 and 14 after AOM injection. Scale bar: 100 μM. ( C ) Immunoblot analysis of the pro- and cleaved forms of CASP3 and caspase-7 (CASP7) in colons of WT and Irf1 –/– mice on days 0, 14, and 80 after AOM injection. Blots represent data from the same biological samples at the indicated time point run in parallel. ( D ) Immunoblot analysis of GSDMD and MLKL in colons of WT and Irf1 –/– mice 14 days after AOM injection. Blots represent data from the same biological samples run in parallel. ( E ) Cell death analysis of intestinal organoids derived from WT and Irf1 –/– mice after stimulation with TNF + zVAD. ** P

Techniques Used: TUNEL Assay, Staining, Mouse Assay, Injection, Derivative Assay

IRF1 prevents colitis-associated colorectal tumorigenesis. ( A ) Body weight change of WT ( n = 10) and Irf1 –/– ( n = 10) mice from 1 experiment (representative of 3 independent experiments). ( B ) Representative images of colon tumors in WT and Irf1 –/– mice 80 days after injection of azoxymethane (AOM). ( C ) Number of colon tumors in WT ( n = 14) and Irf1 –/– ( n = 12) mice. ( D ) Percentage of tumors of various sizes 80 days after AOM injection. ( E ) Representative H E staining of colon tumors. Scale bar: 200 μM. ( F ) Histological scores 80 days after injection of AOM. ( G ) Percentage of mice with dysplasia 80 days after AOM injection. ( H ) Percentage of mice with adenocarcinoma 80 days after AOM injection. Data are from 1 experiment (representative of 3 independent experiments). Each symbol represents 1 individual mouse ( C and F ). *** P
Figure Legend Snippet: IRF1 prevents colitis-associated colorectal tumorigenesis. ( A ) Body weight change of WT ( n = 10) and Irf1 –/– ( n = 10) mice from 1 experiment (representative of 3 independent experiments). ( B ) Representative images of colon tumors in WT and Irf1 –/– mice 80 days after injection of azoxymethane (AOM). ( C ) Number of colon tumors in WT ( n = 14) and Irf1 –/– ( n = 12) mice. ( D ) Percentage of tumors of various sizes 80 days after AOM injection. ( E ) Representative H E staining of colon tumors. Scale bar: 200 μM. ( F ) Histological scores 80 days after injection of AOM. ( G ) Percentage of mice with dysplasia 80 days after AOM injection. ( H ) Percentage of mice with adenocarcinoma 80 days after AOM injection. Data are from 1 experiment (representative of 3 independent experiments). Each symbol represents 1 individual mouse ( C and F ). *** P

Techniques Used: Mouse Assay, Injection, Staining

IRF1 prevents colorectal cancer in an Apc Min/+ model of tumorigenesis. ( A ) Representative images of colon tumors in 120-day-old littermate Apc Min/+ , Apc Min/+ Irf1 +/- , and Apc Min/+ Irf1 −/− mice. ( B ) Number of colon tumors in 120-day-old littermate Apc Min/+ ( n = 5), Apc Min/+ Irf1 +/– ( n = 5) , and Apc Min/+ Irf1 −/− ( n = 5) mice. ( C ) Percentage of tumors of various sizes in 120-day-old littermate Apc Min/+ , Apc Min/+ Irf1 +/– , and Apc Min/+ Irf1 −/− mice. Each symbol represents 1 individual mouse in B . ** P
Figure Legend Snippet: IRF1 prevents colorectal cancer in an Apc Min/+ model of tumorigenesis. ( A ) Representative images of colon tumors in 120-day-old littermate Apc Min/+ , Apc Min/+ Irf1 +/- , and Apc Min/+ Irf1 −/− mice. ( B ) Number of colon tumors in 120-day-old littermate Apc Min/+ ( n = 5), Apc Min/+ Irf1 +/– ( n = 5) , and Apc Min/+ Irf1 −/− ( n = 5) mice. ( C ) Percentage of tumors of various sizes in 120-day-old littermate Apc Min/+ , Apc Min/+ Irf1 +/– , and Apc Min/+ Irf1 −/− mice. Each symbol represents 1 individual mouse in B . ** P

Techniques Used: Mouse Assay

IRF1 does not regulate inflammation in the colon. ( A ) Body weight change of WT ( n = 10) and Irf1 –/– ( n = 10) mice from 1 experiment (representative of 3 independent experiments). ( B and C ) Representative images of colon ( B ) and length of colon ( C ) in WT ( n = 10) and Irf1 –/– ( n = 10) mice 14 days after azoxymethane (AOM) injection. ( D ) Histological scores. ( E ) Representative H E staining of colon. Scale bar: 500 μM. ( F ) Immunoblot analysis of IRF1, phosphorylated and total ERK1 and ERK2 (P-ERK1/2 and ERK1/2, respectively), phosphorylated and total IκBα (P-IκBα and IκBα, respectively), phosphorylated and total STAT3 (P-STAT3 and STAT3, respectively), and GAPDH (loading control) in colons of WT and Irf1 –/– mice. Blots represent data from the same biological samples run in parallel. ( G ) Levels of inflammatory cytokines in the colons of WT and Irf1 –/– mice at day 0, 14, and 80 after AOM injection. Each symbol represents 1 individual mouse ( C , D , and G ). * P
Figure Legend Snippet: IRF1 does not regulate inflammation in the colon. ( A ) Body weight change of WT ( n = 10) and Irf1 –/– ( n = 10) mice from 1 experiment (representative of 3 independent experiments). ( B and C ) Representative images of colon ( B ) and length of colon ( C ) in WT ( n = 10) and Irf1 –/– ( n = 10) mice 14 days after azoxymethane (AOM) injection. ( D ) Histological scores. ( E ) Representative H E staining of colon. Scale bar: 500 μM. ( F ) Immunoblot analysis of IRF1, phosphorylated and total ERK1 and ERK2 (P-ERK1/2 and ERK1/2, respectively), phosphorylated and total IκBα (P-IκBα and IκBα, respectively), phosphorylated and total STAT3 (P-STAT3 and STAT3, respectively), and GAPDH (loading control) in colons of WT and Irf1 –/– mice. Blots represent data from the same biological samples run in parallel. ( G ) Levels of inflammatory cytokines in the colons of WT and Irf1 –/– mice at day 0, 14, and 80 after AOM injection. Each symbol represents 1 individual mouse ( C , D , and G ). * P

Techniques Used: Mouse Assay, Injection, Staining

19) Product Images from "Co-Regulation of Immune Checkpoint PD-L1 with Interferon-Gamma Signaling is Associated with a Survival Benefit in Renal Cell Cancer"

Article Title: Co-Regulation of Immune Checkpoint PD-L1 with Interferon-Gamma Signaling is Associated with a Survival Benefit in Renal Cell Cancer

Journal: Targeted Oncology

doi: 10.1007/s11523-020-00728-8

Regulation of PD-L1 and components of the IFN-γ-signalling cascade in ccRCC cell lines (CaKi-1, A-498), pRCC cell line (CaKi-2) and Cal-54 RCC cell line. a Levels of mRNA (∆Ct) for PD-L1, PD-L2, CXCL10, JAK2, STAT1, IRF1, JAK1, IFN-γR1, IFN-γR2 in control cells (−con) and cells treated with IFN-γ (10 ng/ml) for 24 h (+IFN-γ) are shown. Transcripts that were not inducible by IFN-γ in CaKi-2 cells, in contrast to the other cell lines, are gray-shaded. Box plots indicate means with error bars corresponding to minimum and maximum values ( n = 3). b Western-blot analysis of control cells (con) or cells treated with IFN-γ (10 ng/ml) for 24 h with antibodies for PD-L1, p-JAK2, JAK2, p-JAK1, JAK1, IRF1, and cytoplasmic β-actin. The molecular weights are: PD-L1, ~ 50kd; PD-L2, ~ 50 kd; phosphate (P)-JAK2, 125 kd; JAK2, ~ 125 kd; phosphate (P)-JAK1, 130 kd; JAK1, ~ 130 kd; IRF1, ~ 48 kd; STAT1, ~ 90 kd; β-actin, ~ 43kd. c Schematic diagram of analyzed components of the IFN-γ-signaling cascade. nd below detection level, IFNG IFN-γ, Y tyrosine residue
Figure Legend Snippet: Regulation of PD-L1 and components of the IFN-γ-signalling cascade in ccRCC cell lines (CaKi-1, A-498), pRCC cell line (CaKi-2) and Cal-54 RCC cell line. a Levels of mRNA (∆Ct) for PD-L1, PD-L2, CXCL10, JAK2, STAT1, IRF1, JAK1, IFN-γR1, IFN-γR2 in control cells (−con) and cells treated with IFN-γ (10 ng/ml) for 24 h (+IFN-γ) are shown. Transcripts that were not inducible by IFN-γ in CaKi-2 cells, in contrast to the other cell lines, are gray-shaded. Box plots indicate means with error bars corresponding to minimum and maximum values ( n = 3). b Western-blot analysis of control cells (con) or cells treated with IFN-γ (10 ng/ml) for 24 h with antibodies for PD-L1, p-JAK2, JAK2, p-JAK1, JAK1, IRF1, and cytoplasmic β-actin. The molecular weights are: PD-L1, ~ 50kd; PD-L2, ~ 50 kd; phosphate (P)-JAK2, 125 kd; JAK2, ~ 125 kd; phosphate (P)-JAK1, 130 kd; JAK1, ~ 130 kd; IRF1, ~ 48 kd; STAT1, ~ 90 kd; β-actin, ~ 43kd. c Schematic diagram of analyzed components of the IFN-γ-signaling cascade. nd below detection level, IFNG IFN-γ, Y tyrosine residue

Techniques Used: Western Blot

20) Product Images from "miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma"

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S238975

miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P
Figure Legend Snippet: miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P

Techniques Used: Over Expression, Expressing

miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P
Figure Legend Snippet: miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P

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

IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P
Figure Legend Snippet: IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P

Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Inhibition

IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.
Figure Legend Snippet: IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.

Techniques Used: Expressing, Blocking Assay

21) Product Images from "miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma"

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S238975

miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P
Figure Legend Snippet: miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P

Techniques Used: Over Expression, Expressing

miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P
Figure Legend Snippet: miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P

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

IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P
Figure Legend Snippet: IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P

Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Inhibition

IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.
Figure Legend Snippet: IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.

Techniques Used: Expressing, Blocking Assay

22) Product Images from "miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma"

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S238975

miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P
Figure Legend Snippet: miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P

Techniques Used: Over Expression, Expressing

miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P
Figure Legend Snippet: miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P

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

IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P
Figure Legend Snippet: IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P

Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Inhibition

IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.
Figure Legend Snippet: IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.

Techniques Used: Expressing, Blocking Assay

23) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

24) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

25) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

26) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

27) Product Images from "ZBED2 is an antagonist of Interferon Regulatory Factor 1 and modulates cell identity in pancreatic cancer"

Article Title: ZBED2 is an antagonist of Interferon Regulatory Factor 1 and modulates cell identity in pancreatic cancer

Journal: bioRxiv

doi: 10.1101/868141

ChIP-seq analysis implicates ZBED2 as a sequence-specific repressor of ISG promoters. ( A ) Density plots showing FLAG-ZBED2 and H3K27ac enrichment surrounding a 2-kb interval centered on the summit of 2,451 high-confidence ZBED2 peaks in AsPC1 and SUIT2 cells, ranked by FLAG-ZBED2 peak intensity in AsPC1 cells. ( B ) Pie chart showing the distribution of high-confidence FLAG-ZBED2 peaks in AsPC1 cells. TTS, transcription termination site; TSS, transcription start site; UTR, untranslated region. ( C ) GSEA plot evaluating ZBED2 bound genes upon ZBED2 cDNA expression in HPAFII cells. Leading edge (indicated by the red line), Interferon Response genes are listed. ( D ) GAL4 fusion reporter assay testing full length ZBED2 and IRF1 transactivation activity normalized to Renilla luciferase internal control. Mean+SEM is shown. n=3. **p
Figure Legend Snippet: ChIP-seq analysis implicates ZBED2 as a sequence-specific repressor of ISG promoters. ( A ) Density plots showing FLAG-ZBED2 and H3K27ac enrichment surrounding a 2-kb interval centered on the summit of 2,451 high-confidence ZBED2 peaks in AsPC1 and SUIT2 cells, ranked by FLAG-ZBED2 peak intensity in AsPC1 cells. ( B ) Pie chart showing the distribution of high-confidence FLAG-ZBED2 peaks in AsPC1 cells. TTS, transcription termination site; TSS, transcription start site; UTR, untranslated region. ( C ) GSEA plot evaluating ZBED2 bound genes upon ZBED2 cDNA expression in HPAFII cells. Leading edge (indicated by the red line), Interferon Response genes are listed. ( D ) GAL4 fusion reporter assay testing full length ZBED2 and IRF1 transactivation activity normalized to Renilla luciferase internal control. Mean+SEM is shown. n=3. **p

Techniques Used: Chromatin Immunoprecipitation, Sequencing, Expressing, Reporter Assay, Activity Assay, Luciferase

Antagonistic regulation of ISG promoters by ZBED2 and IRF1. Related to Fig. 4. ( A ) Summary of CentriMo motif enrichment analysis for JASPAR motifs at ZBED2 binding sites. The top 10 transcription factor (TF) motifs ranked by E value are shown. The nucleotide sequence 200bp up- or downstream of the peak summit of the top 1000 ZBED2 peaks in AsPC1 cells was used for this analysis. ( B ) Expression of ZBED2 and IRF family TF genes in 15 human PDA cell lines. ( C ) ZBED2 expression versus IRF2 , IRF3 , IRF5 , IRF6 , IRF7 and IRF9 expression across 15 human PDA cell lines. ( D ) ZBED2 expression correlation with IRF family TF genes in 1,156 cancer cell lines from the CCLE database analyzed using CBioPortal. ( E and F ) Density plots showing IRF1 and FLAG-ZBED2 enrichment surrounding a 2-kb interval centered on the summit of all IRF1 peaks ( E ) or 140 random IRF1 peaks that do not intersect with FLAG-ZBED2 sites in AsPC1 cells ( F ), ranked by IRF1 peak intensity. ( G ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 in AsPC1 cells at the promoters of BTN3A3 and SAMD9 . ( H ) Gene ontology (GO) analysis with Metascape of genes annotated by HOMER to IRF1 only sites. Terms are ranked by their significance ( p value) and no terms reached the significant threshold (-log 10 p value > 12). ( I ) Pie chart showing the distribution of 140 IRF1 only peaks (left) or IRF1/ZBED2 peaks (right) in AsPC1 cells. TTS, transcription termination site; TSS, transcription start site; UTR, untranslated region. ( J and K ) GSEA plots evaluating protein coding genes annotated by HOMER to IRF1 only sites upon IRF1 ( J ) or ZBED2 ( K ) cDNA expression in AsPC1 cells. ( L and M ) GSEA plots evaluating the Interferon Response signature upon IRF1 cDNA expression in AsPC1 cells ( L ) or IRF1 knockout in PANC0403 cells ( M ). ( N ) Expression levels of protein coding genes annotated to IRF1 only sites following 12-hour treatment with 0.2ng/µl of IFN-β, IFN-γ or control. ***p
Figure Legend Snippet: Antagonistic regulation of ISG promoters by ZBED2 and IRF1. Related to Fig. 4. ( A ) Summary of CentriMo motif enrichment analysis for JASPAR motifs at ZBED2 binding sites. The top 10 transcription factor (TF) motifs ranked by E value are shown. The nucleotide sequence 200bp up- or downstream of the peak summit of the top 1000 ZBED2 peaks in AsPC1 cells was used for this analysis. ( B ) Expression of ZBED2 and IRF family TF genes in 15 human PDA cell lines. ( C ) ZBED2 expression versus IRF2 , IRF3 , IRF5 , IRF6 , IRF7 and IRF9 expression across 15 human PDA cell lines. ( D ) ZBED2 expression correlation with IRF family TF genes in 1,156 cancer cell lines from the CCLE database analyzed using CBioPortal. ( E and F ) Density plots showing IRF1 and FLAG-ZBED2 enrichment surrounding a 2-kb interval centered on the summit of all IRF1 peaks ( E ) or 140 random IRF1 peaks that do not intersect with FLAG-ZBED2 sites in AsPC1 cells ( F ), ranked by IRF1 peak intensity. ( G ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 in AsPC1 cells at the promoters of BTN3A3 and SAMD9 . ( H ) Gene ontology (GO) analysis with Metascape of genes annotated by HOMER to IRF1 only sites. Terms are ranked by their significance ( p value) and no terms reached the significant threshold (-log 10 p value > 12). ( I ) Pie chart showing the distribution of 140 IRF1 only peaks (left) or IRF1/ZBED2 peaks (right) in AsPC1 cells. TTS, transcription termination site; TSS, transcription start site; UTR, untranslated region. ( J and K ) GSEA plots evaluating protein coding genes annotated by HOMER to IRF1 only sites upon IRF1 ( J ) or ZBED2 ( K ) cDNA expression in AsPC1 cells. ( L and M ) GSEA plots evaluating the Interferon Response signature upon IRF1 cDNA expression in AsPC1 cells ( L ) or IRF1 knockout in PANC0403 cells ( M ). ( N ) Expression levels of protein coding genes annotated to IRF1 only sites following 12-hour treatment with 0.2ng/µl of IFN-β, IFN-γ or control. ***p

Techniques Used: Binding Assay, Sequencing, Expressing, Chromatin Immunoprecipitation, Knock-Out

Antagonistic regulation of ISG promoters by ZBED2 and IRF1. ( A ) CentriMo motif enrichment analysis for JASPAR motifs at ZBED2 binding sites. ( B ) ZBED2 expression versus the IRF1 expression across 15 human PDA cell lines. ( C ) Density plot showing IRF1 and FLAG-ZBED2 enrichment surrounding a 2-kb interval centered on the summit of 140 intersecting IRF1 and FLAG-ZBED2 sites in AsPC1 cells, ranked by IRF1 peak intensity. ( D ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 in AsPC1 cells at the promoters of CMPK2 and STAT2 . ( E ) Gene ontology (GO) analysis with Metascape of genes annotated by HOMER to IRF1/ZBED2 sites. Terms are ranked by their significance ( p value) and the most significant terms (-log 10 p value > 12) are shown. ( F ) RT-qPCR analysis of CMPK2 in AsPC1-empty and AsPC1-ZBED2 cells following IRF1 cDNA expression. Mean+SEM is shown. n=3. **p
Figure Legend Snippet: Antagonistic regulation of ISG promoters by ZBED2 and IRF1. ( A ) CentriMo motif enrichment analysis for JASPAR motifs at ZBED2 binding sites. ( B ) ZBED2 expression versus the IRF1 expression across 15 human PDA cell lines. ( C ) Density plot showing IRF1 and FLAG-ZBED2 enrichment surrounding a 2-kb interval centered on the summit of 140 intersecting IRF1 and FLAG-ZBED2 sites in AsPC1 cells, ranked by IRF1 peak intensity. ( D ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 in AsPC1 cells at the promoters of CMPK2 and STAT2 . ( E ) Gene ontology (GO) analysis with Metascape of genes annotated by HOMER to IRF1/ZBED2 sites. Terms are ranked by their significance ( p value) and the most significant terms (-log 10 p value > 12) are shown. ( F ) RT-qPCR analysis of CMPK2 in AsPC1-empty and AsPC1-ZBED2 cells following IRF1 cDNA expression. Mean+SEM is shown. n=3. **p

Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation, Quantitative RT-PCR

ZBED2 represses pancreatic progenitor lineage identity in PDA. Related to Fig. 6. ( A ) Summary of GSEA evaluating the Squamous PDA Identity signature upon ZBED2 cDNA expression in 15 PDA cell lines. ( B and C ) Expression changes at IRF1/ZBED2 bound genes in AsPC1 cells infected with ZBED2 cDNA ( B ) or IRF1 cDNA ( C ) versus those infected with an empty vector control. GATA6 and CMPK2 are labeled along with their rank with respect to downregulated ( B ) or upregulated ( C ) genes. ( D and E ) GATA6 ( D ) and IRF1 ( E ) expression in 15 human PDA cell lines. ( F and G ) Proportion of PDA patient samples from the indicated studies stratified as ZBED2 low or ZBED2 high classified based on their tumor differentiation status (grade). Statistical significance for the indicated comparisons was assessed using Fisher’s Exact Test, ns = not significant.
Figure Legend Snippet: ZBED2 represses pancreatic progenitor lineage identity in PDA. Related to Fig. 6. ( A ) Summary of GSEA evaluating the Squamous PDA Identity signature upon ZBED2 cDNA expression in 15 PDA cell lines. ( B and C ) Expression changes at IRF1/ZBED2 bound genes in AsPC1 cells infected with ZBED2 cDNA ( B ) or IRF1 cDNA ( C ) versus those infected with an empty vector control. GATA6 and CMPK2 are labeled along with their rank with respect to downregulated ( B ) or upregulated ( C ) genes. ( D and E ) GATA6 ( D ) and IRF1 ( E ) expression in 15 human PDA cell lines. ( F and G ) Proportion of PDA patient samples from the indicated studies stratified as ZBED2 low or ZBED2 high classified based on their tumor differentiation status (grade). Statistical significance for the indicated comparisons was assessed using Fisher’s Exact Test, ns = not significant.

Techniques Used: Expressing, Infection, Plasmid Preparation, Labeling

ZBED2 represses pancreatic progenitor lineage identity in PDA. ( A - C ) TF expression in Basal-like/Squamous and Classical/Progenitor subtypes of PDA. TFs are ranked by their mean log 2 fold change in Basal-like versus Classical ( A and C ) or Squamous versus Progenitor ( B ) patient samples from the indicated studies. ( D - F ) ZBED2 expression in PDA patient samples stratified according to molecular subtype. Each dot represents one patient sample. p value was calculated using Student’s t test. ( G ) Summary of GSEA evaluating the Progenitor PDA Identity signature upon ZBED2 cDNA expression in 15 PDA cell lines. ( H and I ) GSEA plots evaluating the Progenitor-PDA Identity signature following expression of ZBED2 ( H ) or IRF1 ( I ) in AsPC1 cells. ( J ) Overlap of Progenitor PDA Identity genes with protein coding genes associated with IRF1/ZBED2 sites. ( K ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 at the promoter of GATA6 in AsPC1 cells. ( L ) Overlap of leading edge (LE) genes associated with IRF1/ZBED2 sites that are repressed by ZBED2 or activated by IRF1 in AsPC1 cells. ( M ) Quantification of colony size in three-dimensional (3D) Matrigel colony formation assays. Means+SEM are shown. n=3. Representative images at day 7 are shown (right panel). ( N ) Quantification of scratch assays at the indicated time points post-seeding and representative images (right panel). Scale bar indicates 500µm. Means+SEM are shown. n=3. ***p
Figure Legend Snippet: ZBED2 represses pancreatic progenitor lineage identity in PDA. ( A - C ) TF expression in Basal-like/Squamous and Classical/Progenitor subtypes of PDA. TFs are ranked by their mean log 2 fold change in Basal-like versus Classical ( A and C ) or Squamous versus Progenitor ( B ) patient samples from the indicated studies. ( D - F ) ZBED2 expression in PDA patient samples stratified according to molecular subtype. Each dot represents one patient sample. p value was calculated using Student’s t test. ( G ) Summary of GSEA evaluating the Progenitor PDA Identity signature upon ZBED2 cDNA expression in 15 PDA cell lines. ( H and I ) GSEA plots evaluating the Progenitor-PDA Identity signature following expression of ZBED2 ( H ) or IRF1 ( I ) in AsPC1 cells. ( J ) Overlap of Progenitor PDA Identity genes with protein coding genes associated with IRF1/ZBED2 sites. ( K ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 at the promoter of GATA6 in AsPC1 cells. ( L ) Overlap of leading edge (LE) genes associated with IRF1/ZBED2 sites that are repressed by ZBED2 or activated by IRF1 in AsPC1 cells. ( M ) Quantification of colony size in three-dimensional (3D) Matrigel colony formation assays. Means+SEM are shown. n=3. Representative images at day 7 are shown (right panel). ( N ) Quantification of scratch assays at the indicated time points post-seeding and representative images (right panel). Scale bar indicates 500µm. Means+SEM are shown. n=3. ***p

Techniques Used: Expressing, Chromatin Immunoprecipitation

ZBED2 protects PDA cells from IRF1- and interferon- γ -induced growth arrest. ( A ) Luciferase-based quantification of cell viability of AsPC1 cells grown in Matrigel on day 7 post infection with IRF1 cDNA or the empty vector. Representative bright field images (right panel) are shown. Scale bar indicates 200µm. ( B ) Luciferase-based quantification of cell viability of AsPC1-empty and AsPC1-ZBED2 cells grown in Matrigel following co-expression of IRF1 cDNA or empty vector for 7 days. ( C ) Representative bright filed images from ( B ). Scale bar indicates 500µm. ( D ) Luciferase-based quantification of cell viability of KPC-derived FC1199 cells stably expressing ZBED2 or the empty vector grown in Matrigel following co-expression of IRF1 cDNA or empty vector for 7 days. ( E ) Representative bright filed images from ( D ). Scale bar indicates 200µm. ( F - H ) AsPC1 cells infected with sgRNAs targeting IRF1, IRF9 or a control sgRNA (sgNEG) were plated in Matrigel and grown for 7 days in the presence of 20ng/µl of IFN-γ, IFN-β or control. Representative western blots following overnight stimulation with 20ng/µl of IFN-γ ( F ), luciferase-based quantification ( G ) and representative bright field images on day 7 of the assay ( H ) are shown. Scale bar indicates 500µm. ( I and J ) AsPC1 cells ( I ) or the indicated mouse KPC cell lines ( J ) were infected with the ZBED2 cDNA or an empty vector and grown in Matrigel with the increasing concentrations of IFN-γ. Bar charts show luciferase-based quantification on day 7. Mean+SEM is shown. n=3. For ( A ), ( B ), ( D ) and ( G ), ***p
Figure Legend Snippet: ZBED2 protects PDA cells from IRF1- and interferon- γ -induced growth arrest. ( A ) Luciferase-based quantification of cell viability of AsPC1 cells grown in Matrigel on day 7 post infection with IRF1 cDNA or the empty vector. Representative bright field images (right panel) are shown. Scale bar indicates 200µm. ( B ) Luciferase-based quantification of cell viability of AsPC1-empty and AsPC1-ZBED2 cells grown in Matrigel following co-expression of IRF1 cDNA or empty vector for 7 days. ( C ) Representative bright filed images from ( B ). Scale bar indicates 500µm. ( D ) Luciferase-based quantification of cell viability of KPC-derived FC1199 cells stably expressing ZBED2 or the empty vector grown in Matrigel following co-expression of IRF1 cDNA or empty vector for 7 days. ( E ) Representative bright filed images from ( D ). Scale bar indicates 200µm. ( F - H ) AsPC1 cells infected with sgRNAs targeting IRF1, IRF9 or a control sgRNA (sgNEG) were plated in Matrigel and grown for 7 days in the presence of 20ng/µl of IFN-γ, IFN-β or control. Representative western blots following overnight stimulation with 20ng/µl of IFN-γ ( F ), luciferase-based quantification ( G ) and representative bright field images on day 7 of the assay ( H ) are shown. Scale bar indicates 500µm. ( I and J ) AsPC1 cells ( I ) or the indicated mouse KPC cell lines ( J ) were infected with the ZBED2 cDNA or an empty vector and grown in Matrigel with the increasing concentrations of IFN-γ. Bar charts show luciferase-based quantification on day 7. Mean+SEM is shown. n=3. For ( A ), ( B ), ( D ) and ( G ), ***p

Techniques Used: Luciferase, Infection, Plasmid Preparation, Expressing, Derivative Assay, Stable Transfection, Western Blot

ZBED2 protects PDA cells from IRF1- and interferon-γ-induced growth arrest. Related to Fig. 5. ( A ) Luciferase-based quantification of cell viability of SUIT2 cells grown in Matrigel on day 7 post infection with IRF1 cDNA or the empty vector. Representative bright field images (right panel) are shown. Scale bar indicates 200µm. ( B-D ) SUIT2 cells infected with sgRNAs targeting IRF1, IRF9 or a control sgRNA (sgNEG) were plated in Matrigel and grown for 7 days in the presence of 20ng/µl of IFN-γ, IFN-β or control. Representative western blots following overnight stimulation with 20ng/µl of IFN-γ ( B ), luciferase-based quantification ( C ) and representative bright field images on day 7 of the assay ( D ) are shown. Scale bar indicates 500µm. ( E ) FC1242 KPC cells were infected with the ZBED2 cDNA or an empty vector and grown in Matrigel with the increasing concentrations of IFN-γ. Bar chart shows luciferase-based quantification on day 7. Mean+SEM is shown. n=3. For ( A ) and ( C ), ***p
Figure Legend Snippet: ZBED2 protects PDA cells from IRF1- and interferon-γ-induced growth arrest. Related to Fig. 5. ( A ) Luciferase-based quantification of cell viability of SUIT2 cells grown in Matrigel on day 7 post infection with IRF1 cDNA or the empty vector. Representative bright field images (right panel) are shown. Scale bar indicates 200µm. ( B-D ) SUIT2 cells infected with sgRNAs targeting IRF1, IRF9 or a control sgRNA (sgNEG) were plated in Matrigel and grown for 7 days in the presence of 20ng/µl of IFN-γ, IFN-β or control. Representative western blots following overnight stimulation with 20ng/µl of IFN-γ ( B ), luciferase-based quantification ( C ) and representative bright field images on day 7 of the assay ( D ) are shown. Scale bar indicates 500µm. ( E ) FC1242 KPC cells were infected with the ZBED2 cDNA or an empty vector and grown in Matrigel with the increasing concentrations of IFN-γ. Bar chart shows luciferase-based quantification on day 7. Mean+SEM is shown. n=3. For ( A ) and ( C ), ***p

Techniques Used: Luciferase, Infection, Plasmid Preparation, Western Blot

28) Product Images from "The transcription factor IRF1 and guanylate-binding proteins target AIM2 inflammasome activation by Francisella infection"

Article Title: The transcription factor IRF1 and guanylate-binding proteins target AIM2 inflammasome activation by Francisella infection

Journal: Nature immunology

doi: 10.1038/ni.3118

GBPs target bacteria to mediate killing during F. novicida infection ( a ) Induction of IRF1, GBP2 and GBP5 expression in unprimed BMDMs infected with F. novicida (MOI 50). ( b ) Immunofluorescence staining of GBP5 in unprimed BMDMs infected with F. novicida for 16 h. Scale bar, 10 μm; insert scale bar, 1 μm. ( c ) The percentage of bacteria colocalized with GBP5 was quantified. n=3,575 bacteria in WT BMDMs and n=3,620 bacteria in Irf1 −/− BMDMs. ( d ) CFU recovered from WT, Irf1 −/− and Gbp chr3 -deleted BMDMs infected with F. novicida (MOI 100). ( e ) Quantification of the number of F. novicida bacteria per BMDMs using confocal microscopy. n=758 WT BMDMs and n=679 Gbp5 −/− BMDMs. ( f ) Quantification of the number of F. novicida bacteria per BMDMs using confocal microscopy. n=715 WT BMDMs, n=710 Irf1 −/− BMDMs, n=706 Aim2 −/− BMDMs. ( g ) Caspase-1 activation and IL-1β release in unprimed BMDMs infected with F. novicida (MOI 100) for 20 h. Data from one experiment representative of two ( d–f ) or from three ( a–c,g ) independent experiments. * P
Figure Legend Snippet: GBPs target bacteria to mediate killing during F. novicida infection ( a ) Induction of IRF1, GBP2 and GBP5 expression in unprimed BMDMs infected with F. novicida (MOI 50). ( b ) Immunofluorescence staining of GBP5 in unprimed BMDMs infected with F. novicida for 16 h. Scale bar, 10 μm; insert scale bar, 1 μm. ( c ) The percentage of bacteria colocalized with GBP5 was quantified. n=3,575 bacteria in WT BMDMs and n=3,620 bacteria in Irf1 −/− BMDMs. ( d ) CFU recovered from WT, Irf1 −/− and Gbp chr3 -deleted BMDMs infected with F. novicida (MOI 100). ( e ) Quantification of the number of F. novicida bacteria per BMDMs using confocal microscopy. n=758 WT BMDMs and n=679 Gbp5 −/− BMDMs. ( f ) Quantification of the number of F. novicida bacteria per BMDMs using confocal microscopy. n=715 WT BMDMs, n=710 Irf1 −/− BMDMs, n=706 Aim2 −/− BMDMs. ( g ) Caspase-1 activation and IL-1β release in unprimed BMDMs infected with F. novicida (MOI 100) for 20 h. Data from one experiment representative of two ( d–f ) or from three ( a–c,g ) independent experiments. * P

Techniques Used: Infection, Expressing, Immunofluorescence, Staining, Confocal Microscopy, Activation Assay

IRF1 is essential for AIM2 inflammasome activation by F. novicida infection ( a,b ) Caspase-1 activation, IL-1β and IL-18 release in unprimed BMDMs infected with F. novicida (MOI 100) for 20 h or transfected with poly(dA:dT) for 5 h. ( c,d ) Cell death in unprimed BMDMs infected with F. novicida for 20 h, transfected with poly(dA:dT) for 5 h or infected with mCMV (MOI 10) for 10 h. Arrowheads indicate dead cells. ( e,f ) Caspase-1 activation, IL-18 release and cell death in unprimed BMDMs transfected with pcDNA for 5 h. ( g,h ) Caspase-1 activation and IL-1β and IL-18 release in unprimed BMDMs infected with mCMV (MOI 10) for 10 h. ( i ) Caspase-1 activation in BMDMs infected with F. novicida with or without co-stimulation with recombinant mouse IFN-β (25, 250 and 500 U/ml). Graphs show mean and s.e.m. of two ( g,h ) or three ( a–f, i ) independent experiments. * P
Figure Legend Snippet: IRF1 is essential for AIM2 inflammasome activation by F. novicida infection ( a,b ) Caspase-1 activation, IL-1β and IL-18 release in unprimed BMDMs infected with F. novicida (MOI 100) for 20 h or transfected with poly(dA:dT) for 5 h. ( c,d ) Cell death in unprimed BMDMs infected with F. novicida for 20 h, transfected with poly(dA:dT) for 5 h or infected with mCMV (MOI 10) for 10 h. Arrowheads indicate dead cells. ( e,f ) Caspase-1 activation, IL-18 release and cell death in unprimed BMDMs transfected with pcDNA for 5 h. ( g,h ) Caspase-1 activation and IL-1β and IL-18 release in unprimed BMDMs infected with mCMV (MOI 10) for 10 h. ( i ) Caspase-1 activation in BMDMs infected with F. novicida with or without co-stimulation with recombinant mouse IFN-β (25, 250 and 500 U/ml). Graphs show mean and s.e.m. of two ( g,h ) or three ( a–f, i ) independent experiments. * P

Techniques Used: Activation Assay, Infection, Transfection, Recombinant

IRF1 is not required for canonical or non-canonical NLRP3 or NLRC4 inflammasome activation ( a ) Caspase-1 activation, ( b ) IL-1β and IL-18 release and cell death in LPS-primed BMDMs stimulated with ATP or nigericin or in unprimed BMDMs infected with Citrobacter rodentium (MOI 20) or ΔfliCΔfljB mutant S. Typhimurium (MOI 20) or wild-type S. Typhimurium ( S Tm) for 20 h. ( c ) Caspase-1 activation, ( d ) IL-1β and IL-18 release and cell death in unprimed BMDMs infected with log-phase grown S. Typhimurium (MOI 1) for 4 h. Graphs show mean and s.e.m. of three independent experiments. NS, not significant (two-tailed t -test).
Figure Legend Snippet: IRF1 is not required for canonical or non-canonical NLRP3 or NLRC4 inflammasome activation ( a ) Caspase-1 activation, ( b ) IL-1β and IL-18 release and cell death in LPS-primed BMDMs stimulated with ATP or nigericin or in unprimed BMDMs infected with Citrobacter rodentium (MOI 20) or ΔfliCΔfljB mutant S. Typhimurium (MOI 20) or wild-type S. Typhimurium ( S Tm) for 20 h. ( c ) Caspase-1 activation, ( d ) IL-1β and IL-18 release and cell death in unprimed BMDMs infected with log-phase grown S. Typhimurium (MOI 1) for 4 h. Graphs show mean and s.e.m. of three independent experiments. NS, not significant (two-tailed t -test).

Techniques Used: Activation Assay, Infection, Mutagenesis, Two Tailed Test

IRF1 controls expression of GBPs for AIM2 inflammasome activation ( a ) Heat map of microarray data showing relative expression of macrophage-mediated immunity genes found significantly enriched in unprimed Irf1 −/− , Ifnar1 −/− or Aim2 −/− BMDMs compared to unprimed WT BMDMs after 8 h of infection with F. novicida . ( b ) Gene expression of GBPs relative to β-actin in BMDMs infected with F. novicida for 8 h by real time qRT-PCR. ( c–f ) Caspase-1 activation, IL-1β and IL-18 release and cell death in unprimed WT, Gbp chr3 -deleted ( Gbp chr3- KO) or Aim2 −/− BMDMs infected with F. novicida (MOI 100, 20 h), log-phase grown S. Typhimurium (MOI 1, 4 h), or transfected with poly(dA:dT) or stimulated with LPS+ATP. Microarray analysis was performed using duplicate samples of each genotype ( a ). Graphs show mean and s.e.m. of three ( b–f ) independent experiments. * P
Figure Legend Snippet: IRF1 controls expression of GBPs for AIM2 inflammasome activation ( a ) Heat map of microarray data showing relative expression of macrophage-mediated immunity genes found significantly enriched in unprimed Irf1 −/− , Ifnar1 −/− or Aim2 −/− BMDMs compared to unprimed WT BMDMs after 8 h of infection with F. novicida . ( b ) Gene expression of GBPs relative to β-actin in BMDMs infected with F. novicida for 8 h by real time qRT-PCR. ( c–f ) Caspase-1 activation, IL-1β and IL-18 release and cell death in unprimed WT, Gbp chr3 -deleted ( Gbp chr3- KO) or Aim2 −/− BMDMs infected with F. novicida (MOI 100, 20 h), log-phase grown S. Typhimurium (MOI 1, 4 h), or transfected with poly(dA:dT) or stimulated with LPS+ATP. Microarray analysis was performed using duplicate samples of each genotype ( a ). Graphs show mean and s.e.m. of three ( b–f ) independent experiments. * P

Techniques Used: Expressing, Activation Assay, Microarray, Infection, Quantitative RT-PCR, Transfection

IRF1 provides host protection against F. novicida infection in vivo ( a ) % Survival of 8 week-old WT (n=28), Irf1 −/− (n=17), Aim2 −/− (n=13) and Casp1 x Casp11 double-deficient ( Casp1/11 −/− ) (n=5) mice subcutaneously infected with 7.5×10 4 CFU of F. novicida. ( b ) Body weight change of 8 week-old WT (n=16), Irf1 −/− (n=9), Aim2 −/− (n=9) and Casp1/11 −/− (n=5) subcutaneously infected with 7.5×10 4 CFU of F. novicida. ( c ) Bacterial burden in the liver and spleen of 8 week-old male WT (n=8) and Irf1 −/− (n=6) mice infected with 1×10 5 CFU of F. novicida . ( d ) Bacterial burden in the liver and spleen of 8-week old female WT (n=12) and Aim2 −/− (n=9) mice infected with 1×10 5 CFU of F. novicida . ( e ) Concentrations of IL-18 in the serum of WT (n=15), Irf1 −/− (n=11), Aim2 −/− (n=11), and Casp1/11 −/− (n=5) mice infected with 1×10 5 CFU of F. novicida for 24 h. ( f ) H E and ( g ) Gram staining of F. novicida -infected livers collected on day 3. ( h ) Myeloperoxidase (MPO) and ( i ) TUNEL staining of F. novicida -infected livers collected on day 3. Top panels, 10× magnification; bottom panels 40× magnification. The dashed circle indicates a granuloma. Arrowheads indicate bacterial colonies. Data are from one experiment representative of two independent experiments ( b ) or pooled from two independent experiments ( a,c–e ). Error bars indicate s.e.m. * P
Figure Legend Snippet: IRF1 provides host protection against F. novicida infection in vivo ( a ) % Survival of 8 week-old WT (n=28), Irf1 −/− (n=17), Aim2 −/− (n=13) and Casp1 x Casp11 double-deficient ( Casp1/11 −/− ) (n=5) mice subcutaneously infected with 7.5×10 4 CFU of F. novicida. ( b ) Body weight change of 8 week-old WT (n=16), Irf1 −/− (n=9), Aim2 −/− (n=9) and Casp1/11 −/− (n=5) subcutaneously infected with 7.5×10 4 CFU of F. novicida. ( c ) Bacterial burden in the liver and spleen of 8 week-old male WT (n=8) and Irf1 −/− (n=6) mice infected with 1×10 5 CFU of F. novicida . ( d ) Bacterial burden in the liver and spleen of 8-week old female WT (n=12) and Aim2 −/− (n=9) mice infected with 1×10 5 CFU of F. novicida . ( e ) Concentrations of IL-18 in the serum of WT (n=15), Irf1 −/− (n=11), Aim2 −/− (n=11), and Casp1/11 −/− (n=5) mice infected with 1×10 5 CFU of F. novicida for 24 h. ( f ) H E and ( g ) Gram staining of F. novicida -infected livers collected on day 3. ( h ) Myeloperoxidase (MPO) and ( i ) TUNEL staining of F. novicida -infected livers collected on day 3. Top panels, 10× magnification; bottom panels 40× magnification. The dashed circle indicates a granuloma. Arrowheads indicate bacterial colonies. Data are from one experiment representative of two independent experiments ( b ) or pooled from two independent experiments ( a,c–e ). Error bars indicate s.e.m. * P

Techniques Used: Infection, In Vivo, Mouse Assay, Staining, TUNEL Assay

F. novicida infection induces IRF1 expression in a manner that requires type I interferon signaling ( a ) Caspase-1 activation, IL-18 release and cell death in unprimed bone marrow-derived of the indicated strain macrophages (BMDMs) infected with F. novicida (MOI 100) for 20 h or transfected with poly(dA:dT) for 5 h. ( b ) IL-1β and IL-18 release and cell death in unprimed BMDMs infected with F. novicida for 20 h or transfected with poly(dA:dT) for 5 h. ( c ) Heat map of microarray analysis showing relative expression of interferon regulatory factors (IRFs) genes in Ifnar1 −/− BMDMs compared to wildtype (WT) BMDMs infected with F. novicida for 8 h. ( d ) Induction of IRF1 expression in unprimed BMDMs infected with F. novicida (MOI 50). Graphs show mean and s.e.m. of two ( b ) or three ( a,d ) independent experiments. Microarray analysis was performed using duplicate samples of each genotype ( c ). * P
Figure Legend Snippet: F. novicida infection induces IRF1 expression in a manner that requires type I interferon signaling ( a ) Caspase-1 activation, IL-18 release and cell death in unprimed bone marrow-derived of the indicated strain macrophages (BMDMs) infected with F. novicida (MOI 100) for 20 h or transfected with poly(dA:dT) for 5 h. ( b ) IL-1β and IL-18 release and cell death in unprimed BMDMs infected with F. novicida for 20 h or transfected with poly(dA:dT) for 5 h. ( c ) Heat map of microarray analysis showing relative expression of interferon regulatory factors (IRFs) genes in Ifnar1 −/− BMDMs compared to wildtype (WT) BMDMs infected with F. novicida for 8 h. ( d ) Induction of IRF1 expression in unprimed BMDMs infected with F. novicida (MOI 50). Graphs show mean and s.e.m. of two ( b ) or three ( a,d ) independent experiments. Microarray analysis was performed using duplicate samples of each genotype ( c ). * P

Techniques Used: Infection, Expressing, Activation Assay, Derivative Assay, Transfection, Microarray

29) Product Images from "Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells"

Article Title: Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-019-1531-3

Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P
Figure Legend Snippet: Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P

Techniques Used: Activation Assay, Migration, Indirect Immunoperoxidase Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

30) Product Images from "Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells"

Article Title: Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-019-1531-3

Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P
Figure Legend Snippet: Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P

Techniques Used: Activation Assay, Migration, Indirect Immunoperoxidase Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

31) Product Images from "Molecular Mechanisms for Synchronized Transcription of Three Complement C1q Subunit Genes in Dendritic Cells and Macrophages"

Article Title: Molecular Mechanisms for Synchronized Transcription of Three Complement C1q Subunit Genes in Dendritic Cells and Macrophages

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.286427

Roles of PU.1, IRF8, STAT1, and IRF1 in IFNγ stimulation of the C1qB promoter. RAW264.7 cells were transfected with the B273 promoter and also co-transfected with plasmids encoding shRNA for mouse IRF1 ( A ), STAT1 ( B ), IRF8 (IRF8–1, IRF8–2;
Figure Legend Snippet: Roles of PU.1, IRF8, STAT1, and IRF1 in IFNγ stimulation of the C1qB promoter. RAW264.7 cells were transfected with the B273 promoter and also co-transfected with plasmids encoding shRNA for mouse IRF1 ( A ), STAT1 ( B ), IRF8 (IRF8–1, IRF8–2;

Techniques Used: Transfection, shRNA

32) Product Images from "Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells"

Article Title: Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-019-1531-3

Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P
Figure Legend Snippet: Transcriptomic analysis of TFs in LPS-treated hMSCs. a Upstream regulator analysis predicted the activation state of TFs including IRF1, NF-κB, FOXO1, and NLRC5 in LPS-treated hMSCs. b The migration-related molecules were highly correlated with NF-κB and IRF1. These molecules are presented using the IPA molecule activity predictor. c Confirmation of the expression levels of the cell migration-related genes using quantitative real-time PCR (left). Gene expression was normalized to GAPDH transcript levels. ELISA results showing the release of CCL2 and CXCL10 upon TLR4 stimulation of hMSCs (right). The data represent three independent experiments. ** P

Techniques Used: Activation Assay, Migration, Indirect Immunoperoxidase Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

33) Product Images from "Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ–mediated host defenses"

Article Title: Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ–mediated host defenses

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20160340

IFN-γ–induced activated STAT1 DNA association is enhanced by T. gondii in a TgIST-dependent manner. (A–C) HFFs were infected for 24 h with Pru ku80 (WT) and Pru ku80 ΔTgIST ( ΔTgIST ) or left uninfected (ui) and stimulated 6 h with IFN-γ (+) or left unstimulated. Samples were analyzed by ChIP assay with antibodies to STAT1 Y701-P, STAT1 S727-P, and H3K4me3. IgG was used as negative control. Bound DNA corresponding to IRF1, CXCL10, and CIITA loci was quantified by qPCR-ChIP, and signals were normalized with the input DNA. Error bars represent SD ( n = 3). Data are from one representative of three independent experiments. (D) ChIP-qPCR was used as aforementioned to monitor enrichment of H3K9ac, H4panAc, and H3K27ac at IRF1, CXCL10, and CIITA promoters. Error bars represent SD ( n = 3). Data are from one representative of two independent experiments.
Figure Legend Snippet: IFN-γ–induced activated STAT1 DNA association is enhanced by T. gondii in a TgIST-dependent manner. (A–C) HFFs were infected for 24 h with Pru ku80 (WT) and Pru ku80 ΔTgIST ( ΔTgIST ) or left uninfected (ui) and stimulated 6 h with IFN-γ (+) or left unstimulated. Samples were analyzed by ChIP assay with antibodies to STAT1 Y701-P, STAT1 S727-P, and H3K4me3. IgG was used as negative control. Bound DNA corresponding to IRF1, CXCL10, and CIITA loci was quantified by qPCR-ChIP, and signals were normalized with the input DNA. Error bars represent SD ( n = 3). Data are from one representative of three independent experiments. (D) ChIP-qPCR was used as aforementioned to monitor enrichment of H3K9ac, H4panAc, and H3K27ac at IRF1, CXCL10, and CIITA promoters. Error bars represent SD ( n = 3). Data are from one representative of two independent experiments.

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

TgIST is required to inhibit IFN-γ–induced IRF1 expression. (A) HFFs were infected for 24 h with Pru ku80 TgIST-HAFlag or Pru ku80 ΔTgIST and stimulated 6 h with IFN-γ or left unstimulated (US). Cells were stained to detect nuclear localization of IRF1 (red) and TgIST (HA, green). (B) IFN-γ–induced IRF1 expression was monitored in mouse macrophages as described in A after infection with 76KGFP-WT and - ΔTgIST . GFP-expressing parasites (green) and IRF1 (red) were detected. (C) HFFs were infected for 24 h with ΔTgIST parasites transiently expressing a copy of P TgIST -TgIST-HAFlag whose expression was driven by its own promoter. After IFN-γ stimulation, TgIST (HA, green) and IRF1 expression (red) were detected. (D) IRF1 mRNA levels were determined by RT-qPCR in both HFFs and RAW264.7 infected and stimulated as described in A. β2-microglobulin was used for normalization. Data are displayed as fold difference relative to the uninfected cells. The mean of two experiments is shown; error bars represent SEM. This was performed three times with similar results. (E, left) Export of TgIST (HA, red) in U3A STAT1-deficient cells and the corresponding parental line 2fTGH. Cells were infected for 24 h with Pru ku80 TgIST-HAFlag and stimulated 6 h with IFN-γ or left unstimulated (US). (right) IFN-γ–induced IRF1 expression (magenta) was monitored in 2fTGH and U3A infected for 24 h with 76KGFP-WT and - ΔTgIST and stimulated for 6 h with IFN-γ. Immunofluorescence data are representative of at least three experiments. Yellow arrowheads indicate infected cells in which IRF1 expression was repressed.
Figure Legend Snippet: TgIST is required to inhibit IFN-γ–induced IRF1 expression. (A) HFFs were infected for 24 h with Pru ku80 TgIST-HAFlag or Pru ku80 ΔTgIST and stimulated 6 h with IFN-γ or left unstimulated (US). Cells were stained to detect nuclear localization of IRF1 (red) and TgIST (HA, green). (B) IFN-γ–induced IRF1 expression was monitored in mouse macrophages as described in A after infection with 76KGFP-WT and - ΔTgIST . GFP-expressing parasites (green) and IRF1 (red) were detected. (C) HFFs were infected for 24 h with ΔTgIST parasites transiently expressing a copy of P TgIST -TgIST-HAFlag whose expression was driven by its own promoter. After IFN-γ stimulation, TgIST (HA, green) and IRF1 expression (red) were detected. (D) IRF1 mRNA levels were determined by RT-qPCR in both HFFs and RAW264.7 infected and stimulated as described in A. β2-microglobulin was used for normalization. Data are displayed as fold difference relative to the uninfected cells. The mean of two experiments is shown; error bars represent SEM. This was performed three times with similar results. (E, left) Export of TgIST (HA, red) in U3A STAT1-deficient cells and the corresponding parental line 2fTGH. Cells were infected for 24 h with Pru ku80 TgIST-HAFlag and stimulated 6 h with IFN-γ or left unstimulated (US). (right) IFN-γ–induced IRF1 expression (magenta) was monitored in 2fTGH and U3A infected for 24 h with 76KGFP-WT and - ΔTgIST and stimulated for 6 h with IFN-γ. Immunofluorescence data are representative of at least three experiments. Yellow arrowheads indicate infected cells in which IRF1 expression was repressed.

Techniques Used: Expressing, Infection, Staining, Quantitative RT-PCR, Immunofluorescence

34) Product Images from "MiR-103 protects from recurrent spontaneous abortion via inhibiting STAT1 mediated M1 macrophage polarization"

Article Title: MiR-103 protects from recurrent spontaneous abortion via inhibiting STAT1 mediated M1 macrophage polarization

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.46144

miR-103 inhibits the STAT1/IRF1 signal pathway. RAW264.7 and PM cells were transfected with miR-103 mimics/NC or miR-103 inhibitor/INC, after 24h, the cells were stimulated with or without LPS/IFNγ for 24 h. ( A-B ) STAT1 and IRF1 mRNA expression were detected in RAW264.7 and PM cells transfected with miR-103 mimics or NC by qRT-PCR. ( C-D ) The protein levels of STAT1, p-STAT1 and IRF1 were measured in RAW264.7 and PM cells transfected with miR-103 mimics or NC by western blot. ( E-F ) STAT1 and IRF1 mRNA expression were detected in RAW264.7 and PM cells transfected with miR-103 inhibitor or INC by qRT-PCR. ( G-H ) STAT1, p-STAT1 and IRF1 protein levels were measured in RAW264.7 and PM cells transfected with miR-103 inhibitor or INC by western blot. ( I-J ) Expression of STAT1 and IRF1 were detected in RAW264.7 cells transfected with miR-103 mimics/NC or miR-103 inhibitor/INC by immunofluorescence. DAPI was used to stain the cell nucleus (Scale bar, 50 µm, 200×). Values were listed as the mean± SEM. * P
Figure Legend Snippet: miR-103 inhibits the STAT1/IRF1 signal pathway. RAW264.7 and PM cells were transfected with miR-103 mimics/NC or miR-103 inhibitor/INC, after 24h, the cells were stimulated with or without LPS/IFNγ for 24 h. ( A-B ) STAT1 and IRF1 mRNA expression were detected in RAW264.7 and PM cells transfected with miR-103 mimics or NC by qRT-PCR. ( C-D ) The protein levels of STAT1, p-STAT1 and IRF1 were measured in RAW264.7 and PM cells transfected with miR-103 mimics or NC by western blot. ( E-F ) STAT1 and IRF1 mRNA expression were detected in RAW264.7 and PM cells transfected with miR-103 inhibitor or INC by qRT-PCR. ( G-H ) STAT1, p-STAT1 and IRF1 protein levels were measured in RAW264.7 and PM cells transfected with miR-103 inhibitor or INC by western blot. ( I-J ) Expression of STAT1 and IRF1 were detected in RAW264.7 cells transfected with miR-103 mimics/NC or miR-103 inhibitor/INC by immunofluorescence. DAPI was used to stain the cell nucleus (Scale bar, 50 µm, 200×). Values were listed as the mean± SEM. * P

Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

miR-103 suppresses embryo resorption rate and M1 macrophages polarization in vivo . Mice were inspected every morning for vaginal plugs. The day when a plug became visible was designated as Day 0.5 of pregnancy. NP mice and RSA mice were administrated 10 nmol agomiR NC or agomiR-103 on Day 0.5, 3.5, 6.5, 9.5 via tail vein, and execute mice on Day 11.5 of pregnancy. ( A ) Treatment regime of agomiR-103 or agomiR NC and timeline for the measurement of parameters. ( B ) Relative expression of miR-103 was measured by qRT-PCR in the decidua of pregnant mice (n= 10). ( C-D ) Embryo resorption rate of three group mice, arrows indicate the embryo resorption (n= 10). ( E-F ) The mRNA level of STAT1, IRF1 and protein level of p-STAT1, STAT1 and IRF1 were detected in the decidua of pregnant mice (n= 10). ( G ) Dot plot represents labeling of F4/80 + CD80 + (M1) and F4/80 + MHCII + (M1) cell by flow cytometry in the decidua of pregnant mice (n= 10). ( H ) Relative expression of CCL2 , CXCL9 , CXCL10 , iNOS , IL6 , TNF-α was analysed by qRT-PCR in the decidua of pregnant mice (n= 10). Values were listed as the mean± SEM. * P
Figure Legend Snippet: miR-103 suppresses embryo resorption rate and M1 macrophages polarization in vivo . Mice were inspected every morning for vaginal plugs. The day when a plug became visible was designated as Day 0.5 of pregnancy. NP mice and RSA mice were administrated 10 nmol agomiR NC or agomiR-103 on Day 0.5, 3.5, 6.5, 9.5 via tail vein, and execute mice on Day 11.5 of pregnancy. ( A ) Treatment regime of agomiR-103 or agomiR NC and timeline for the measurement of parameters. ( B ) Relative expression of miR-103 was measured by qRT-PCR in the decidua of pregnant mice (n= 10). ( C-D ) Embryo resorption rate of three group mice, arrows indicate the embryo resorption (n= 10). ( E-F ) The mRNA level of STAT1, IRF1 and protein level of p-STAT1, STAT1 and IRF1 were detected in the decidua of pregnant mice (n= 10). ( G ) Dot plot represents labeling of F4/80 + CD80 + (M1) and F4/80 + MHCII + (M1) cell by flow cytometry in the decidua of pregnant mice (n= 10). ( H ) Relative expression of CCL2 , CXCL9 , CXCL10 , iNOS , IL6 , TNF-α was analysed by qRT-PCR in the decidua of pregnant mice (n= 10). Values were listed as the mean± SEM. * P

Techniques Used: In Vivo, Mouse Assay, Expressing, Quantitative RT-PCR, Labeling, Flow Cytometry

Overexpression of STAT1 can reverse the inhibitory effect of miR-103 on M1 polarization. RAW264.7 cells were co-transfected with miR-103 mimics, STAT1 plasmid or NC, vector for 24 h, followed by treating with LPS plus IFNγ for 24 h. ( A-B ) STAT1 and IRF1 mRNA levels were assessed by qRT-PCR. ( C ) p-STAT1, STAT1 and IRF1 protein levels were assessed by western blot. ( D ) mRNA expression level of M1 macrophages makers CXCL10 , IL6 , IL12b , iNOS were detected by qRT-PCR. Values were listed as the mean± SEM. * P
Figure Legend Snippet: Overexpression of STAT1 can reverse the inhibitory effect of miR-103 on M1 polarization. RAW264.7 cells were co-transfected with miR-103 mimics, STAT1 plasmid or NC, vector for 24 h, followed by treating with LPS plus IFNγ for 24 h. ( A-B ) STAT1 and IRF1 mRNA levels were assessed by qRT-PCR. ( C ) p-STAT1, STAT1 and IRF1 protein levels were assessed by western blot. ( D ) mRNA expression level of M1 macrophages makers CXCL10 , IL6 , IL12b , iNOS were detected by qRT-PCR. Values were listed as the mean± SEM. * P

Techniques Used: Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Expressing

35) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

36) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

37) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

38) Product Images from "iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice"

Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

Journal: Molecular Medicine

doi: 10.1186/s10020-020-00182-2

IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies
Figure Legend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

Techniques Used: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot
Figure Legend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

Techniques Used: Activity Assay, Translocation Assay, Western Blot

Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification
Figure Legend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

Techniques Used: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P
Figure Legend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003
Figure Legend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

Techniques Used: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results
Figure Legend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

Techniques Used: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase

39) Product Images from "miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma"

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S238975

miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P
Figure Legend Snippet: miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P

Techniques Used: Over Expression, Expressing

miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P
Figure Legend Snippet: miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P

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

IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P
Figure Legend Snippet: IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P

Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Inhibition

IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.
Figure Legend Snippet: IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.

Techniques Used: Expressing, Blocking Assay

40) Product Images from "miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma"

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S238975

miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P
Figure Legend Snippet: miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P

Techniques Used: Over Expression, Expressing

miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P
Figure Legend Snippet: miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P

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

IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P
Figure Legend Snippet: IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P

Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Inhibition

IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.
Figure Legend Snippet: IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.

Techniques Used: Expressing, Blocking Assay

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Incubation:

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Article Title: Co-Regulation of Immune Checkpoint PD-L1 with Interferon-Gamma Signaling is Associated with a Survival Benefit in Renal Cell Cancer
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Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma
Article Snippet: .. Membranes were incubated with primary antibody against IRF1 (#8478, Cell Signaling Technology) or β-actin (#3700, Cell Signaling Technology) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody. .. Proteins were visualized with the ECL reagent (Thermo Fisher).

Binding Assay:

Article Title: Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer
Article Snippet: .. Following electrophoretic transfer of proteins onto PVDF membranes (IPVH00010, MilliporeSigma), nonspecific binding was blocked by incubation with 5% skim milk, and then membranes were incubated with primary antibodies against CASP3 (9662, Cell Signaling Technology [CST]), cleaved CASP3 (9661, CST), CASP7 (9492, CST), cleaved CASP7 (9491, CST), CASP8 (AG-20T-0138-C100, AdipoGen), cleaved CASP8 (8592, CST), IRF1 (8478, CST), P-ERK (9101, CST), ERK (9102, CST), P-IκBα (9241, CST), IκBα (9242, CST), P-STAT3 Tyr705 (9131, CST), STAT3 (9139, CST), and GAPDH (5174, CST). .. Membranes were then washed and incubated with the appropriate HRP-conjugated secondary antibodies (111-035-047, anti-rabbit; 315-035-047, anti-mouse; 705-035-003, anti-goat; Jackson ImmunoResearch Laboratories).

other:

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Expressing:

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    Expression of NFKB2 and <t>IRF1</t> predicts response to immunotherapy. (A) Various biomarkers for response to immunotherapy (y axis), including expression of NFKB2 + IRF1 expression, were compared using previously published genesets with TIDE online platform ( Jiang et al., 2018 ).
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    Expression of NFKB2 and IRF1 predicts response to immunotherapy. (A) Various biomarkers for response to immunotherapy (y axis), including expression of NFKB2 + IRF1 expression, were compared using previously published genesets with TIDE online platform ( Jiang et al., 2018 ).

    Journal: bioRxiv

    Article Title: Genome-wide profiling of druggable active tumor defense mechanisms to enhance cancer immunotherapy

    doi: 10.1101/843185

    Figure Lengend Snippet: Expression of NFKB2 and IRF1 predicts response to immunotherapy. (A) Various biomarkers for response to immunotherapy (y axis), including expression of NFKB2 + IRF1 expression, were compared using previously published genesets with TIDE online platform ( Jiang et al., 2018 ).

    Article Snippet: This included the following antibodies: BIRC2 (108361, Abcam), BIRC3 (3130, Cell Signaling), HLA Class I (70328, Abcam), ICAM1 (4915, Cell Signaling), IRF1 (8478 Cell Signaling), phospho-JAK1 (3331, Cell Signaling), Lamin A/C (4777, Cell Signaling), phospho-NF-κB p65 (3033, Cell Signaling), NF-κB2 p100/p52 (4882, Cell Signaling), RelB (10544, Cell Signaling), XIAP (2042, Cell Signaling).

    Techniques: Expressing

    ChIP-seq analysis implicates ZBED2 as a sequence-specific repressor of ISG promoters. ( A ) Density plots showing FLAG-ZBED2 and H3K27ac enrichment surrounding a 2-kb interval centered on the summit of 2,451 high-confidence ZBED2 peaks in AsPC1 and SUIT2 cells, ranked by FLAG-ZBED2 peak intensity in AsPC1 cells. ( B ) Pie chart showing the distribution of high-confidence FLAG-ZBED2 peaks in AsPC1 cells. TTS, transcription termination site; TSS, transcription start site; UTR, untranslated region. ( C ) GSEA plot evaluating ZBED2 bound genes upon ZBED2 cDNA expression in HPAFII cells. Leading edge (indicated by the red line), Interferon Response genes are listed. ( D ) GAL4 fusion reporter assay testing full length ZBED2 and IRF1 transactivation activity normalized to Renilla luciferase internal control. Mean+SEM is shown. n=3. **p

    Journal: bioRxiv

    Article Title: ZBED2 is an antagonist of Interferon Regulatory Factor 1 and modulates cell identity in pancreatic cancer

    doi: 10.1101/868141

    Figure Lengend Snippet: ChIP-seq analysis implicates ZBED2 as a sequence-specific repressor of ISG promoters. ( A ) Density plots showing FLAG-ZBED2 and H3K27ac enrichment surrounding a 2-kb interval centered on the summit of 2,451 high-confidence ZBED2 peaks in AsPC1 and SUIT2 cells, ranked by FLAG-ZBED2 peak intensity in AsPC1 cells. ( B ) Pie chart showing the distribution of high-confidence FLAG-ZBED2 peaks in AsPC1 cells. TTS, transcription termination site; TSS, transcription start site; UTR, untranslated region. ( C ) GSEA plot evaluating ZBED2 bound genes upon ZBED2 cDNA expression in HPAFII cells. Leading edge (indicated by the red line), Interferon Response genes are listed. ( D ) GAL4 fusion reporter assay testing full length ZBED2 and IRF1 transactivation activity normalized to Renilla luciferase internal control. Mean+SEM is shown. n=3. **p

    Article Snippet: Peaks that may be recognized by both ZBED2 and IRF1 were defined as those intervals in high confidence ZBED2 and IRF1 peaks that overlap by at least 1bp.

    Techniques: Chromatin Immunoprecipitation, Sequencing, Expressing, Reporter Assay, Activity Assay, Luciferase

    Antagonistic regulation of ISG promoters by ZBED2 and IRF1. Related to Fig. 4. ( A ) Summary of CentriMo motif enrichment analysis for JASPAR motifs at ZBED2 binding sites. The top 10 transcription factor (TF) motifs ranked by E value are shown. The nucleotide sequence 200bp up- or downstream of the peak summit of the top 1000 ZBED2 peaks in AsPC1 cells was used for this analysis. ( B ) Expression of ZBED2 and IRF family TF genes in 15 human PDA cell lines. ( C ) ZBED2 expression versus IRF2 , IRF3 , IRF5 , IRF6 , IRF7 and IRF9 expression across 15 human PDA cell lines. ( D ) ZBED2 expression correlation with IRF family TF genes in 1,156 cancer cell lines from the CCLE database analyzed using CBioPortal. ( E and F ) Density plots showing IRF1 and FLAG-ZBED2 enrichment surrounding a 2-kb interval centered on the summit of all IRF1 peaks ( E ) or 140 random IRF1 peaks that do not intersect with FLAG-ZBED2 sites in AsPC1 cells ( F ), ranked by IRF1 peak intensity. ( G ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 in AsPC1 cells at the promoters of BTN3A3 and SAMD9 . ( H ) Gene ontology (GO) analysis with Metascape of genes annotated by HOMER to IRF1 only sites. Terms are ranked by their significance ( p value) and no terms reached the significant threshold (-log 10 p value > 12). ( I ) Pie chart showing the distribution of 140 IRF1 only peaks (left) or IRF1/ZBED2 peaks (right) in AsPC1 cells. TTS, transcription termination site; TSS, transcription start site; UTR, untranslated region. ( J and K ) GSEA plots evaluating protein coding genes annotated by HOMER to IRF1 only sites upon IRF1 ( J ) or ZBED2 ( K ) cDNA expression in AsPC1 cells. ( L and M ) GSEA plots evaluating the Interferon Response signature upon IRF1 cDNA expression in AsPC1 cells ( L ) or IRF1 knockout in PANC0403 cells ( M ). ( N ) Expression levels of protein coding genes annotated to IRF1 only sites following 12-hour treatment with 0.2ng/µl of IFN-β, IFN-γ or control. ***p

    Journal: bioRxiv

    Article Title: ZBED2 is an antagonist of Interferon Regulatory Factor 1 and modulates cell identity in pancreatic cancer

    doi: 10.1101/868141

    Figure Lengend Snippet: Antagonistic regulation of ISG promoters by ZBED2 and IRF1. Related to Fig. 4. ( A ) Summary of CentriMo motif enrichment analysis for JASPAR motifs at ZBED2 binding sites. The top 10 transcription factor (TF) motifs ranked by E value are shown. The nucleotide sequence 200bp up- or downstream of the peak summit of the top 1000 ZBED2 peaks in AsPC1 cells was used for this analysis. ( B ) Expression of ZBED2 and IRF family TF genes in 15 human PDA cell lines. ( C ) ZBED2 expression versus IRF2 , IRF3 , IRF5 , IRF6 , IRF7 and IRF9 expression across 15 human PDA cell lines. ( D ) ZBED2 expression correlation with IRF family TF genes in 1,156 cancer cell lines from the CCLE database analyzed using CBioPortal. ( E and F ) Density plots showing IRF1 and FLAG-ZBED2 enrichment surrounding a 2-kb interval centered on the summit of all IRF1 peaks ( E ) or 140 random IRF1 peaks that do not intersect with FLAG-ZBED2 sites in AsPC1 cells ( F ), ranked by IRF1 peak intensity. ( G ) ChIP-seq profiles of IRF1 and FLAG-ZBED2 in AsPC1 cells at the promoters of BTN3A3 and SAMD9 . ( H ) Gene ontology (GO) analysis with Metascape of genes annotated by HOMER to IRF1 only sites. Terms are ranked by their significance ( p value) and no terms reached the significant threshold (-log 10 p value > 12). ( I ) Pie chart showing the distribution of 140 IRF1 only peaks (left) or IRF1/ZBED2 peaks (right) in AsPC1 cells. TTS, transcription termination site; TSS, transcription start site; UTR, untranslated region. ( J and K ) GSEA plots evaluating protein coding genes annotated by HOMER to IRF1 only sites upon IRF1 ( J ) or ZBED2 ( K ) cDNA expression in AsPC1 cells. ( L and M ) GSEA plots evaluating the Interferon Response signature upon IRF1 cDNA expression in AsPC1 cells ( L ) or IRF1 knockout in PANC0403 cells ( M ). ( N ) Expression levels of protein coding genes annotated to IRF1 only sites following 12-hour treatment with 0.2ng/µl of IFN-β, IFN-γ or control. ***p

    Article Snippet: Peaks that may be recognized by both ZBED2 and IRF1 were defined as those intervals in high confidence ZBED2 and IRF1 peaks that overlap by at least 1bp.

    Techniques: Binding Assay, Sequencing, Expressing, Chromatin Immunoprecipitation, Knock-Out

    ZBED2 represses pancreatic progenitor lineage identity in PDA. Related to Fig. 6. ( A ) Summary of GSEA evaluating the Squamous PDA Identity signature upon ZBED2 cDNA expression in 15 PDA cell lines. ( B and C ) Expression changes at IRF1/ZBED2 bound genes in AsPC1 cells infected with ZBED2 cDNA ( B ) or IRF1 cDNA ( C ) versus those infected with an empty vector control. GATA6 and CMPK2 are labeled along with their rank with respect to downregulated ( B ) or upregulated ( C ) genes. ( D and E ) GATA6 ( D ) and IRF1 ( E ) expression in 15 human PDA cell lines. ( F and G ) Proportion of PDA patient samples from the indicated studies stratified as ZBED2 low or ZBED2 high classified based on their tumor differentiation status (grade). Statistical significance for the indicated comparisons was assessed using Fisher’s Exact Test, ns = not significant.

    Journal: bioRxiv

    Article Title: ZBED2 is an antagonist of Interferon Regulatory Factor 1 and modulates cell identity in pancreatic cancer

    doi: 10.1101/868141

    Figure Lengend Snippet: ZBED2 represses pancreatic progenitor lineage identity in PDA. Related to Fig. 6. ( A ) Summary of GSEA evaluating the Squamous PDA Identity signature upon ZBED2 cDNA expression in 15 PDA cell lines. ( B and C ) Expression changes at IRF1/ZBED2 bound genes in AsPC1 cells infected with ZBED2 cDNA ( B ) or IRF1 cDNA ( C ) versus those infected with an empty vector control. GATA6 and CMPK2 are labeled along with their rank with respect to downregulated ( B ) or upregulated ( C ) genes. ( D and E ) GATA6 ( D ) and IRF1 ( E ) expression in 15 human PDA cell lines. ( F and G ) Proportion of PDA patient samples from the indicated studies stratified as ZBED2 low or ZBED2 high classified based on their tumor differentiation status (grade). Statistical significance for the indicated comparisons was assessed using Fisher’s Exact Test, ns = not significant.

    Article Snippet: Peaks that may be recognized by both ZBED2 and IRF1 were defined as those intervals in high confidence ZBED2 and IRF1 peaks that overlap by at least 1bp.

    Techniques: Expressing, Infection, Plasmid Preparation, Labeling

    ZBED2 protects PDA cells from IRF1- and interferon- γ -induced growth arrest. ( A ) Luciferase-based quantification of cell viability of AsPC1 cells grown in Matrigel on day 7 post infection with IRF1 cDNA or the empty vector. Representative bright field images (right panel) are shown. Scale bar indicates 200µm. ( B ) Luciferase-based quantification of cell viability of AsPC1-empty and AsPC1-ZBED2 cells grown in Matrigel following co-expression of IRF1 cDNA or empty vector for 7 days. ( C ) Representative bright filed images from ( B ). Scale bar indicates 500µm. ( D ) Luciferase-based quantification of cell viability of KPC-derived FC1199 cells stably expressing ZBED2 or the empty vector grown in Matrigel following co-expression of IRF1 cDNA or empty vector for 7 days. ( E ) Representative bright filed images from ( D ). Scale bar indicates 200µm. ( F - H ) AsPC1 cells infected with sgRNAs targeting IRF1, IRF9 or a control sgRNA (sgNEG) were plated in Matrigel and grown for 7 days in the presence of 20ng/µl of IFN-γ, IFN-β or control. Representative western blots following overnight stimulation with 20ng/µl of IFN-γ ( F ), luciferase-based quantification ( G ) and representative bright field images on day 7 of the assay ( H ) are shown. Scale bar indicates 500µm. ( I and J ) AsPC1 cells ( I ) or the indicated mouse KPC cell lines ( J ) were infected with the ZBED2 cDNA or an empty vector and grown in Matrigel with the increasing concentrations of IFN-γ. Bar charts show luciferase-based quantification on day 7. Mean+SEM is shown. n=3. For ( A ), ( B ), ( D ) and ( G ), ***p

    Journal: bioRxiv

    Article Title: ZBED2 is an antagonist of Interferon Regulatory Factor 1 and modulates cell identity in pancreatic cancer

    doi: 10.1101/868141

    Figure Lengend Snippet: ZBED2 protects PDA cells from IRF1- and interferon- γ -induced growth arrest. ( A ) Luciferase-based quantification of cell viability of AsPC1 cells grown in Matrigel on day 7 post infection with IRF1 cDNA or the empty vector. Representative bright field images (right panel) are shown. Scale bar indicates 200µm. ( B ) Luciferase-based quantification of cell viability of AsPC1-empty and AsPC1-ZBED2 cells grown in Matrigel following co-expression of IRF1 cDNA or empty vector for 7 days. ( C ) Representative bright filed images from ( B ). Scale bar indicates 500µm. ( D ) Luciferase-based quantification of cell viability of KPC-derived FC1199 cells stably expressing ZBED2 or the empty vector grown in Matrigel following co-expression of IRF1 cDNA or empty vector for 7 days. ( E ) Representative bright filed images from ( D ). Scale bar indicates 200µm. ( F - H ) AsPC1 cells infected with sgRNAs targeting IRF1, IRF9 or a control sgRNA (sgNEG) were plated in Matrigel and grown for 7 days in the presence of 20ng/µl of IFN-γ, IFN-β or control. Representative western blots following overnight stimulation with 20ng/µl of IFN-γ ( F ), luciferase-based quantification ( G ) and representative bright field images on day 7 of the assay ( H ) are shown. Scale bar indicates 500µm. ( I and J ) AsPC1 cells ( I ) or the indicated mouse KPC cell lines ( J ) were infected with the ZBED2 cDNA or an empty vector and grown in Matrigel with the increasing concentrations of IFN-γ. Bar charts show luciferase-based quantification on day 7. Mean+SEM is shown. n=3. For ( A ), ( B ), ( D ) and ( G ), ***p

    Article Snippet: Peaks that may be recognized by both ZBED2 and IRF1 were defined as those intervals in high confidence ZBED2 and IRF1 peaks that overlap by at least 1bp.

    Techniques: Luciferase, Infection, Plasmid Preparation, Expressing, Derivative Assay, Stable Transfection, Western Blot

    IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

    Journal: Molecular Medicine

    Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

    doi: 10.1186/s10020-020-00182-2

    Figure Lengend Snippet: IRF-1 synergistically targets PUMA gene expression with p53. a HCT116TP53 +/+ and HCT116TP53 −/− cells were treated with IFNγ (24 h). Immunofluorescence staining was performed with the indicated antibodies. Representative images are shown, red: IRF1; green: PUMA. b 293 T cells were transfected with pCMV6-xl 5 -hIRF1 (3 μg) or pCMV-xl 5 -TP53 (1 μg); and co-transfected with pCMV6-xl 5 -hIRF1 (3 μg) and pCMV-xl 5 -TP53 (1 μg) for 24 h. Total proteins were analyzed by Western blot with the indicated antibodies

    Article Snippet: A co-transfection experiment was conducted to overexpress IRF1 and p53 in 293 T cells, and observe for synergistic effects on the induction of PUMA.

    Techniques: Expressing, Immunofluorescence, Staining, Transfection, Western Blot

    iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

    Journal: Molecular Medicine

    Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

    doi: 10.1186/s10020-020-00182-2

    Figure Lengend Snippet: iNOS/NO was required for HDAC2 activity which up-regulated IRF1 nuclear translocation. a Mouse (left) and human (right) hepatocytes were treated with GSNO (1 μM) or SNAP (500 μM) for 3 h, respectively. Nuclear expressions of IRF1, HDAC2 and H3AcK9 were analyzed by Western blot; lamin A/C was loading controls. b Mouse hepatic I/R were performed 1 h ischemia and a various times of reperfusion as indicated. The nuclear expressions of HDAC2 and H3AcK9 in liver tissues were measured by Western blot. c 293 T cells were treated with GSNO (1 μM) and romidepsin (5 μM) for 3 h. The expressions of IRF1, HDAC2 and H3AcK9 (nuclear extracts), and PUMA and p21 (whole cell extracts) were analyzed by Western blot

    Article Snippet: A co-transfection experiment was conducted to overexpress IRF1 and p53 in 293 T cells, and observe for synergistic effects on the induction of PUMA.

    Techniques: Activity Assay, Translocation Assay, Western Blot

    Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

    Journal: Molecular Medicine

    Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

    doi: 10.1186/s10020-020-00182-2

    Figure Lengend Snippet: Schematic of the proposed model of iNOS/NO-mediated IRF1 activation in response to hepatic I/R in mice. Ischemia and reperfusion induces iNOS/NO, which activates IRF1 transcriptional activities. This process requires iNOS/NO induced HDAC2 activation to catalyze deacetylation of histone H3. On the other hand, iNOS gene deficiency decreases IRF1 and HDAC2 activities. The activated IRF1 as a transcription factor is translocated into the nucleus, where it regulates transcription of the target genes associated with cell death and cell cycle repression such as, iNOS, PUMA and p21. A positive feedback loop between IRF1 and iNOS may lead to IRF1 continuatively activated. Inhibition of HDAC2 by its inhibitor leads to an increase in histone H3 acetylation, and a decrease in IRF1 nuclear translocation and its target gene expressions (see Results and Discussion). I/R induced IRF1 activation requires iNOS/NO, which recruits HDAC2 as a co-activator to mediate chromatin modification

    Article Snippet: A co-transfection experiment was conducted to overexpress IRF1 and p53 in 293 T cells, and observe for synergistic effects on the induction of PUMA.

    Techniques: Activation Assay, Mouse Assay, Inhibition, Translocation Assay, Modification

    iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

    Journal: Molecular Medicine

    Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

    doi: 10.1186/s10020-020-00182-2

    Figure Lengend Snippet: iNOS is required for IRF-1 translocation to the nucleus and liver injury in I/R mice. a iNOS +/+ and iNOS −/− mice were used to generate I/R with 1 h ischemia and reperfusion as indicated. The nuclear and cytosolic proteins from the livers were analyzed by Western blot. b Livers from iNOS +/+ and iNOS −/− I/R mice (6 h reperfusion) were subjected to immunofluorescence staining. Representative images are shown in the comparison of IRF1 expressions between the iNOS +/+ and iNOS −/− mice. IRF1 is stained with FITC (green), and nucleus is stained with Hoechst dye (bis-benzimide) and is shown as blue color (upper). Moreover, to confirm the translocation of IRF1 to nucleus we used staining for IRF1 with Cy3 (red), and counterstaining for nucleus with SYTOX (green). Merging of the images shows the translocation to the nucleus of IRF1 as yellow color (lower). c ALT was detected in I/R iNOS +/+ vs. iNOS −/− mice at the indicated time points. I/R more likely induced ALT releases with a time-course dependent manner in iNOS +/+ mice compared with that in iNOS −/− mice, P

    Article Snippet: A co-transfection experiment was conducted to overexpress IRF1 and p53 in 293 T cells, and observe for synergistic effects on the induction of PUMA.

    Techniques: Translocation Assay, Mouse Assay, Western Blot, Immunofluorescence, Staining

    iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

    Journal: Molecular Medicine

    Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

    doi: 10.1186/s10020-020-00182-2

    Figure Lengend Snippet: iNOS inhibition reversed the iNOS/NO induced signaling. a Human hepatocytes were treated with L-NIL (100 μM, 24 h), and IRF1 (nuclear extracts) and PUMA (whole cell extracts) were analyzed by Western blot. b Similar to ( a ), but representative images of immunofluorescence staining are shown, red: IRF1; green: F-actin; blue: nucleus. c Mouse hepatocytes were transfected with IRF1-luciferase reporter for 24 h, and followed the treatments as indicated for 9 h. Luciferase reporter assay was performed. L-NIL decreased IFNγ-induced IRF1 transcriptional response, *P = 0.03. The data shown are representative of three experiments with similar results. d Warm I/R mice ( n = 4) were used for the study of iNOS inhibition reducing liver injury. Ischemia was performed for 1 h, and then reperfusion with the treatment of BYK191023 (60 mg/kg, 6 h). TUNEL staining of apoptotic cells with green color on the liver tissues (left) and ALT concentrations (right) were reduced by BYK191023 vs. the controls, ** P = 0.003

    Article Snippet: A co-transfection experiment was conducted to overexpress IRF1 and p53 in 293 T cells, and observe for synergistic effects on the induction of PUMA.

    Techniques: Inhibition, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Reporter Assay, Mouse Assay, TUNEL Assay

    iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

    Journal: Molecular Medicine

    Article Title: iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

    doi: 10.1186/s10020-020-00182-2

    Figure Lengend Snippet: iNOS regulation of IRF1 transcriptional activity. a 293 T cells were transfected with plasmids pcDNA3 or pcDNA3-hiNOS for 24 h, and treated with L-NIL (100 μM, 24 h). The iNOS/NO-induced nuclear IRF1 was analyzed by Western blot (upper). Similar as (upper), but the NO production was detected (lower). b Similar to ( a ) upper, but the iNOS/NO induced-IRF1 was evaluated by immunofluorescence staining, green: iNOS or IRF1. c Human hepatocytes were infected by AdhiNOS or Adlacz for 24 h. Representative images of immunofluorescence staining are shown for IRF1 expression, green: iNOS; red: IRF1. d 293 T cells were transfected with pT109-IRF1 (0.5 μg) and iNOS expression plasmid, pT109-IRF1-iNOS with different concentrations of iNOS. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3A vector. Transcriptional activities of IRF1 were analyzed by luciferase assay (RLA: relative luciferase activity), cells transfected with pT109-IRF1-iNOS vs. pT109-IRF1, * P = 0.0003. The data shown are representative of three experiments with similar results

    Article Snippet: A co-transfection experiment was conducted to overexpress IRF1 and p53 in 293 T cells, and observe for synergistic effects on the induction of PUMA.

    Techniques: Activity Assay, Transfection, Western Blot, Immunofluorescence, Staining, Infection, Expressing, Plasmid Preparation, Luciferase