Structured Review

Santa Cruz Biotechnology anti stat1
Other TFs or signaling proteins are present in MAM but not in mitochondria. Western blot analysis of the Percoll density centrifugation result showed that TFs or signaling proteins including <t>STAT1,</t> MAPKs, AMPK, AKT, mTOR, and RELA could only be found in the MAM fractions but not in pure mitochondria. Mito.C., crude mitochondrial fraction; Mito.P., pure mitochondrial fraction; MAM.H, heavy MAM; MAM.L, light MAM.
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1) Product Images from "STAT3 localizes in mitochondria-associated ER membranes instead of in mitochondria"

Article Title: STAT3 localizes in mitochondria-associated ER membranes instead of in mitochondria

Journal: bioRxiv

doi: 10.1101/2019.12.18.880567

Other TFs or signaling proteins are present in MAM but not in mitochondria. Western blot analysis of the Percoll density centrifugation result showed that TFs or signaling proteins including STAT1, MAPKs, AMPK, AKT, mTOR, and RELA could only be found in the MAM fractions but not in pure mitochondria. Mito.C., crude mitochondrial fraction; Mito.P., pure mitochondrial fraction; MAM.H, heavy MAM; MAM.L, light MAM.
Figure Legend Snippet: Other TFs or signaling proteins are present in MAM but not in mitochondria. Western blot analysis of the Percoll density centrifugation result showed that TFs or signaling proteins including STAT1, MAPKs, AMPK, AKT, mTOR, and RELA could only be found in the MAM fractions but not in pure mitochondria. Mito.C., crude mitochondrial fraction; Mito.P., pure mitochondrial fraction; MAM.H, heavy MAM; MAM.L, light MAM.

Techniques Used: Western Blot, Centrifugation

2) Product Images from "ROS via BTK-p300-STAT1-PPARγ signaling activation mediates cholesterol crystals-induced CD36 expression and foam cell formation"

Article Title: ROS via BTK-p300-STAT1-PPARγ signaling activation mediates cholesterol crystals-induced CD36 expression and foam cell formation

Journal: Redox Biology

doi: 10.1016/j.redox.2016.12.005

Interactions between STAT1, PPARγ and p300 are required for CC-induced STAT1 binding to CD36 promoter. A. Nuclear extracts of control and 2 h of CC (40 μg/ml)-treated cells were analyzed by EMSA and supershift EMSA for the presence of STAT1, PPARγ and p300 in the STAT-DNA complexes using STAT binding site at −107 nt in the CD36 promoter as a biotin-labeled probe. B. Quiescent cells were treated with vehicle or CC for the indicated time periods and subjected to ChIP and re-ChIP assays of CD36 promoter using anti-STAT1, anti-PPARγ or anti-p300 antibodies with the indicated sequential order. C and D. Cells were transfected with vector, PFS1YF, K410R/K413R, p300WT or p300ΔHAT, quiesced, treated with and without CC for 2 h and subjected to ChIP and re-ChIP assays for STAT1, PPARγ and p300 binding to CD36 promoter as described in panel B. E. Quiescent cells were treated with vehicle or CC in the presence and absence of GW9662 (5 μM) for 2 h and subjected to ChIP and re-ChIP assays for STAT1, PPARγ and p300 binding to CD36 promoter as described in panel B. F and G. Cells were transfected with the indicated ASOs, quiesced, treated with and without CC for 2 h and subjected to ChIP and re-ChIP assays for STAT1, PPARγ and p300 binding to CD36 promoter as described in panel B. IgG was used as a negative control.
Figure Legend Snippet: Interactions between STAT1, PPARγ and p300 are required for CC-induced STAT1 binding to CD36 promoter. A. Nuclear extracts of control and 2 h of CC (40 μg/ml)-treated cells were analyzed by EMSA and supershift EMSA for the presence of STAT1, PPARγ and p300 in the STAT-DNA complexes using STAT binding site at −107 nt in the CD36 promoter as a biotin-labeled probe. B. Quiescent cells were treated with vehicle or CC for the indicated time periods and subjected to ChIP and re-ChIP assays of CD36 promoter using anti-STAT1, anti-PPARγ or anti-p300 antibodies with the indicated sequential order. C and D. Cells were transfected with vector, PFS1YF, K410R/K413R, p300WT or p300ΔHAT, quiesced, treated with and without CC for 2 h and subjected to ChIP and re-ChIP assays for STAT1, PPARγ and p300 binding to CD36 promoter as described in panel B. E. Quiescent cells were treated with vehicle or CC in the presence and absence of GW9662 (5 μM) for 2 h and subjected to ChIP and re-ChIP assays for STAT1, PPARγ and p300 binding to CD36 promoter as described in panel B. F and G. Cells were transfected with the indicated ASOs, quiesced, treated with and without CC for 2 h and subjected to ChIP and re-ChIP assays for STAT1, PPARγ and p300 binding to CD36 promoter as described in panel B. IgG was used as a negative control.

Techniques Used: Binding Assay, Labeling, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Negative Control

STAT1, PPARγ and p300 mediate CC-induced CD36 promoter activity. A. Cells were transfected with vector, pGL3-hCD36 or pGL3-hCD36m (STAT-binding site at −107 nt was mutated), growth-arrested, treated with and without CC (40 μg/ml) for 6 h and the luciferase activity was measured. B, C and E. After transfection with vector or pGL3-hCD36, cells were quiesced, treated with vehicle or CC in the presence and absence of Apocyanin (100 μM), DPI (10 μM), Allopurinol (100 μM), PCI32765 (10 μM) or GW9662 (5 μM) for 6 h and the luciferase activity was measured. D and F. Cells were co-transfected with vector or pGL3-hCD36 in combination with pcDNA3.1, PFS1YF, K410R/K413R, p300WT or p300ΔHAT, quiesced, treated with vehicle or CC for 6 h and the luciferase activity was measured. *p
Figure Legend Snippet: STAT1, PPARγ and p300 mediate CC-induced CD36 promoter activity. A. Cells were transfected with vector, pGL3-hCD36 or pGL3-hCD36m (STAT-binding site at −107 nt was mutated), growth-arrested, treated with and without CC (40 μg/ml) for 6 h and the luciferase activity was measured. B, C and E. After transfection with vector or pGL3-hCD36, cells were quiesced, treated with vehicle or CC in the presence and absence of Apocyanin (100 μM), DPI (10 μM), Allopurinol (100 μM), PCI32765 (10 μM) or GW9662 (5 μM) for 6 h and the luciferase activity was measured. D and F. Cells were co-transfected with vector or pGL3-hCD36 in combination with pcDNA3.1, PFS1YF, K410R/K413R, p300WT or p300ΔHAT, quiesced, treated with vehicle or CC for 6 h and the luciferase activity was measured. *p

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

CC-induced BTK activation depends on NADPH and xanthine oxidases-mediated ROS production. A. Quiescent cells were treated with vehicle or CC (40 μg/ml) for the indicated time periods or for 30 min in the presence and absence of Apocyanin (100 μM), DPI (10 μM) or Allopurinol (100 μM) and ROS production was measured using CM-H2DCFDA and Amplex Red. B. Cells were transfected with control, p47Phox or xanthine oxidase ASOs, quiesced, treated with vehicle or CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in Fig. 1 , panel C. The CD36 blot was reprobed for p47Phox, xanthine oxidase and β-tubulin levels to show the effects of the ASOs on their target and off target molecules levels or normalization. C. Cells that were transfected with control, p47Phox or xanthine oxidase ASOs and quiesced were treated with vehicle or CC for 1 h and analyzed for BTK and p300 tyrosine phosphorylation, p300 association with STAT1, STAT1 acetylation and its interaction with PPARγ as described in Fig. 4 , panels B, A and D, respectively, and the blots were reprobed for BTK, p300 or STAT1 levels for normalization. An equal amount of protein from control and each treatment was also assayed for p300 acetyltransferase activity. *p
Figure Legend Snippet: CC-induced BTK activation depends on NADPH and xanthine oxidases-mediated ROS production. A. Quiescent cells were treated with vehicle or CC (40 μg/ml) for the indicated time periods or for 30 min in the presence and absence of Apocyanin (100 μM), DPI (10 μM) or Allopurinol (100 μM) and ROS production was measured using CM-H2DCFDA and Amplex Red. B. Cells were transfected with control, p47Phox or xanthine oxidase ASOs, quiesced, treated with vehicle or CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in Fig. 1 , panel C. The CD36 blot was reprobed for p47Phox, xanthine oxidase and β-tubulin levels to show the effects of the ASOs on their target and off target molecules levels or normalization. C. Cells that were transfected with control, p47Phox or xanthine oxidase ASOs and quiesced were treated with vehicle or CC for 1 h and analyzed for BTK and p300 tyrosine phosphorylation, p300 association with STAT1, STAT1 acetylation and its interaction with PPARγ as described in Fig. 4 , panels B, A and D, respectively, and the blots were reprobed for BTK, p300 or STAT1 levels for normalization. An equal amount of protein from control and each treatment was also assayed for p300 acetyltransferase activity. *p

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

CC-induced CD36 expression, oxLDL uptake and foam cell formation require STAT1 acetylation. A. Quiescent cells were treated with vehicle or CC (40 µg/ml) for the indicated time periods and either protein extracts were prepared or RNA was isolated. The protein extracts and RNA were analyzed by Western blotting and RT-PCR for the indicated scavenger receptors expression and normalized to β-tubulin protein and β-actin mRNA levels, respectively. B. Cells were treated with vehicle or CC (40 µg/ml) for 6 h and tested for their cytotoxicity and proliferation by LDH release and MTT assays, respectively. C. Upper panel: Cells were transfected with the indicated ASO, quiesced, treated with vehicle or CC for 4 h and analyzed by Western blotting for CD36 levels and the blot was reprobed for β-tubulin to show the effects of the ASO on its target and off target molecules levels. Middle and bottom panels: All the conditions were the same as in the upper panel except that cells were subjected to CC-induced oxLDL uptake (middle panel) or foam cell formation (bottom panel) assays. D. Equal amounts of protein from control and various time periods of CC-treated cells were analyzed by Western blotting for pSTAT1, pSTAT2, pSTAT3, pSTAT4, pSTAT5 and pSTAT6 levels and normalized to their total levels. E. Upper panel: Cells were transfected with vector or PFS1YF, quiesced, treated with and without CC for 1 h or 4 h, cell extracts were prepared and analyzed by Western blotting for pSTAT1 (1 h samples) and CD36 levels (4 h samples) and the blots were reprobed for STAT1 over expression and β-tubulin normalization. Middle and bottom panels: All the conditions were the same as in the upper panel except that after quiescence cells were subjected to CC-induced oxLDL uptake (middle panel) and foam cell formation (bottom panel) assays. F. Cells were transfected with control or STAT1 ASO, quiesced, treated with and without CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in panel C. The CD36 blot was reprobed for STAT1 and β-tubulin to show the effect of the ASO on its target and off target molecules levels. G. Upper panel: Equal amounts of proteins from control and the indicated time periods of CC-treated cells were immunoprecipitated with anti-STAT1 antibodies or IgG and the immunocomplexes were analyzed by Western blotting using anti-acetyl lysine (Ac-Lys) antibodies followed by normalization to STAT1. Lower panels: Cells were transfected with vector, K410R/K413R or PFS1YF, quiesced, treated with and without CC for 1 h and analyzed for STAT1 acetylation and phosphorylation as described in the upper panel and panel C, respectively and the blots were reprobed for STAT1 levels. H. Cells were transfected with vector or K410R/K413R, quiesced, treated with and without CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in panel C. The bar graphs represent Mean±S.D. values of three experiments. *p
Figure Legend Snippet: CC-induced CD36 expression, oxLDL uptake and foam cell formation require STAT1 acetylation. A. Quiescent cells were treated with vehicle or CC (40 µg/ml) for the indicated time periods and either protein extracts were prepared or RNA was isolated. The protein extracts and RNA were analyzed by Western blotting and RT-PCR for the indicated scavenger receptors expression and normalized to β-tubulin protein and β-actin mRNA levels, respectively. B. Cells were treated with vehicle or CC (40 µg/ml) for 6 h and tested for their cytotoxicity and proliferation by LDH release and MTT assays, respectively. C. Upper panel: Cells were transfected with the indicated ASO, quiesced, treated with vehicle or CC for 4 h and analyzed by Western blotting for CD36 levels and the blot was reprobed for β-tubulin to show the effects of the ASO on its target and off target molecules levels. Middle and bottom panels: All the conditions were the same as in the upper panel except that cells were subjected to CC-induced oxLDL uptake (middle panel) or foam cell formation (bottom panel) assays. D. Equal amounts of protein from control and various time periods of CC-treated cells were analyzed by Western blotting for pSTAT1, pSTAT2, pSTAT3, pSTAT4, pSTAT5 and pSTAT6 levels and normalized to their total levels. E. Upper panel: Cells were transfected with vector or PFS1YF, quiesced, treated with and without CC for 1 h or 4 h, cell extracts were prepared and analyzed by Western blotting for pSTAT1 (1 h samples) and CD36 levels (4 h samples) and the blots were reprobed for STAT1 over expression and β-tubulin normalization. Middle and bottom panels: All the conditions were the same as in the upper panel except that after quiescence cells were subjected to CC-induced oxLDL uptake (middle panel) and foam cell formation (bottom panel) assays. F. Cells were transfected with control or STAT1 ASO, quiesced, treated with and without CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in panel C. The CD36 blot was reprobed for STAT1 and β-tubulin to show the effect of the ASO on its target and off target molecules levels. G. Upper panel: Equal amounts of proteins from control and the indicated time periods of CC-treated cells were immunoprecipitated with anti-STAT1 antibodies or IgG and the immunocomplexes were analyzed by Western blotting using anti-acetyl lysine (Ac-Lys) antibodies followed by normalization to STAT1. Lower panels: Cells were transfected with vector, K410R/K413R or PFS1YF, quiesced, treated with and without CC for 1 h and analyzed for STAT1 acetylation and phosphorylation as described in the upper panel and panel C, respectively and the blots were reprobed for STAT1 levels. H. Cells were transfected with vector or K410R/K413R, quiesced, treated with and without CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in panel C. The bar graphs represent Mean±S.D. values of three experiments. *p

Techniques Used: Expressing, Isolation, Western Blot, Reverse Transcription Polymerase Chain Reaction, MTT Assay, Transfection, Allele-specific Oligonucleotide, Plasmid Preparation, Over Expression, Immunoprecipitation

CC-induced STAT1 acetylation requires p300 acetyltransferase activity. A. Equal amounts of protein from control and the indicated time periods of CC (40 μg/ml)-treated cells were immunoprecipitated with anti-p300 antibodies or IgG and the immunocomplexes were analyzed by Western blotting for STAT1, STAT3, or STAT5 levels and normalized for p300. B. Quiescent cells were transfected with control or p300 ASO, quiesced, treated with and without CC for 1 h, cell extracts were prepared and equal amounts proteins from each condition were immunoprecipitated with anti-STAT1 antibodies and the immunocomplexes were analyzed by Western blotting for STAT1 acetylation and its association with PPARγ as described in Fig. 1 , panel F and Fig. 2 , panel C, respectively. C. All the conditions were same as in panel B except that cells were treated with vehicle or CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in Fig. 1 , panel C. The blot was reprobed for p300 and β-tubulin to show the effect of the ASO on its target and off target molecules levels. D and E. Cells were transfected with p300WT or p300ΔHAT, quiesced, treated with and without CC and analyzed for STAT1 acetylation, its association with PPARγ, CD36 expression, oxLDL uptake or foam cell formation as described above in panels B and C, respectively. The blots were reprobed for STAT1 or β-tubulin for normalization. F and G. Cells were transfected with control or CBP ASO, quiesced, treated with and without CC and analyzed for STAT1 acetylation and its association with PPARγ, CD36 expression and oxLDL uptake as described above in panels B and C, respectively. The blots were reprobed for CBP, STAT1 or β-tubulin to show the effect of the ASO on its target and off target molecules levels. The bar graphs represent Mean±S.D. values of three experiments. *p
Figure Legend Snippet: CC-induced STAT1 acetylation requires p300 acetyltransferase activity. A. Equal amounts of protein from control and the indicated time periods of CC (40 μg/ml)-treated cells were immunoprecipitated with anti-p300 antibodies or IgG and the immunocomplexes were analyzed by Western blotting for STAT1, STAT3, or STAT5 levels and normalized for p300. B. Quiescent cells were transfected with control or p300 ASO, quiesced, treated with and without CC for 1 h, cell extracts were prepared and equal amounts proteins from each condition were immunoprecipitated with anti-STAT1 antibodies and the immunocomplexes were analyzed by Western blotting for STAT1 acetylation and its association with PPARγ as described in Fig. 1 , panel F and Fig. 2 , panel C, respectively. C. All the conditions were same as in panel B except that cells were treated with vehicle or CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in Fig. 1 , panel C. The blot was reprobed for p300 and β-tubulin to show the effect of the ASO on its target and off target molecules levels. D and E. Cells were transfected with p300WT or p300ΔHAT, quiesced, treated with and without CC and analyzed for STAT1 acetylation, its association with PPARγ, CD36 expression, oxLDL uptake or foam cell formation as described above in panels B and C, respectively. The blots were reprobed for STAT1 or β-tubulin for normalization. F and G. Cells were transfected with control or CBP ASO, quiesced, treated with and without CC and analyzed for STAT1 acetylation and its association with PPARγ, CD36 expression and oxLDL uptake as described above in panels B and C, respectively. The blots were reprobed for CBP, STAT1 or β-tubulin to show the effect of the ASO on its target and off target molecules levels. The bar graphs represent Mean±S.D. values of three experiments. *p

Techniques Used: Activity Assay, Immunoprecipitation, Western Blot, Transfection, Allele-specific Oligonucleotide, Expressing

CC-induced CD36 expression, oxLDL uptake and foam cell formation require STAT1 interaction with PPARγ. A. Quiescent cells were treated with vehicle or CC (40 μg/ml) in the presence and absence of GW9662 (5 μM) and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in Fig. 1 , panel C. B. Upper panel: Quiescent cells were treated with and without CC in the presence and absence of GW9662 for 1 h and analyzed for STAT1 acetylation as described in Fig. 1 , panel F. Middle and bottom panels: Equal amounts of protein from control and the indicated time periods of CC-treated cells were immunoprecipitated with anti-STAT1 or anti-PPARγ antibodies or IgG and the immunocomplexes were analyzed by Western blotting for the indicated proteins using their specific antibodies and normalized for STAT1 or PPARγ. C. Cells were transfected with vector, PFS1YF or K410R/K413R, quiesced, treated with and without CC for 1 h and equal amounts of protein from control and each treatment were immunoprecipitated with anti-STAT1 antibodies and the immunocomplexes were analyzed by Western blotting for PPARγ. The blots were reprobed for STAT1 over expression. The bar graphs represent Mean±S.D. of three experiments *p
Figure Legend Snippet: CC-induced CD36 expression, oxLDL uptake and foam cell formation require STAT1 interaction with PPARγ. A. Quiescent cells were treated with vehicle or CC (40 μg/ml) in the presence and absence of GW9662 (5 μM) and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in Fig. 1 , panel C. B. Upper panel: Quiescent cells were treated with and without CC in the presence and absence of GW9662 for 1 h and analyzed for STAT1 acetylation as described in Fig. 1 , panel F. Middle and bottom panels: Equal amounts of protein from control and the indicated time periods of CC-treated cells were immunoprecipitated with anti-STAT1 or anti-PPARγ antibodies or IgG and the immunocomplexes were analyzed by Western blotting for the indicated proteins using their specific antibodies and normalized for STAT1 or PPARγ. C. Cells were transfected with vector, PFS1YF or K410R/K413R, quiesced, treated with and without CC for 1 h and equal amounts of protein from control and each treatment were immunoprecipitated with anti-STAT1 antibodies and the immunocomplexes were analyzed by Western blotting for PPARγ. The blots were reprobed for STAT1 over expression. The bar graphs represent Mean±S.D. of three experiments *p

Techniques Used: Expressing, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Over Expression

CC induces p300 activation in BTK-dependent manner. A. Equal amounts of protein from control and the indicated time periods of CC (40 μg/ml)-treated cells were immunoprecipitated with anti-p300 antibodies or IgG and the immunocomplexes were analyzed by Western blotting with anti-PY20 or anti-phosphoserine/threonine antibodies followed by normalization for p300. B. All the conditions were same as in panel A except that the cell extracts were analyzed by Western blotting for pBTK, pPyk2, pSrc and pSyk levels using their phospho-specific antibodies and normalized for their total levels. C. Cells were transfected with the indicated ASO, quiesced, treated with and without CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in Fig. 1 , panel C. The blot was reprobed for BTK, Pyk2 and β-tubulin levels to show the effects of the ASOs on their target and off target molecules levels. D. Cells were transfected with control or BTK ASO, quiesced, treated with and without CC for 1 h and cell extracts were prepared. Equal amount of protein from control and each treatment were immunoprecipitated with anti-p300 or anti-STAT1 antibodies and the immunocomplexes were analyzed by Western blotting for anti-PY20, anti-STAT1, anti-Ac-Lys or anti-PPARγ antibodies and normalized for p300 or STAT1. An equal amount of protein from control and each treatment was also assayed for p300 acetyltransferase activity. *p
Figure Legend Snippet: CC induces p300 activation in BTK-dependent manner. A. Equal amounts of protein from control and the indicated time periods of CC (40 μg/ml)-treated cells were immunoprecipitated with anti-p300 antibodies or IgG and the immunocomplexes were analyzed by Western blotting with anti-PY20 or anti-phosphoserine/threonine antibodies followed by normalization for p300. B. All the conditions were same as in panel A except that the cell extracts were analyzed by Western blotting for pBTK, pPyk2, pSrc and pSyk levels using their phospho-specific antibodies and normalized for their total levels. C. Cells were transfected with the indicated ASO, quiesced, treated with and without CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in Fig. 1 , panel C. The blot was reprobed for BTK, Pyk2 and β-tubulin levels to show the effects of the ASOs on their target and off target molecules levels. D. Cells were transfected with control or BTK ASO, quiesced, treated with and without CC for 1 h and cell extracts were prepared. Equal amount of protein from control and each treatment were immunoprecipitated with anti-p300 or anti-STAT1 antibodies and the immunocomplexes were analyzed by Western blotting for anti-PY20, anti-STAT1, anti-Ac-Lys or anti-PPARγ antibodies and normalized for p300 or STAT1. An equal amount of protein from control and each treatment was also assayed for p300 acetyltransferase activity. *p

Techniques Used: Activation Assay, Immunoprecipitation, Western Blot, Transfection, Allele-specific Oligonucleotide, Expressing, Activity Assay

CC induces ROS production, BTK activation, p300-STAT1-PPARγ interactions, CD36 expression and foam cell formation in mouse primary peritoneal macrophages. A–C. Mouse primary peritoneal macrophages were isolated from WT mice, quiesced overnight, treated with and without CC (40 μg/ml) for the indicated time periods and either ROS production was measured or cell extracts were prepared and analyzed for BTK and p300 tyrosine phosphorylation, p300 association with STAT1, STAT1 acetylation and its association with PPARγ and CD36 expression as described in Figure legends 5A, 4B, 4A, 3A, 1F, 2B and IA, respectively. D and E. Mouse primary peritoneal macrophages after overnight growth arrest were treated with and without CC (40 μg/ml) for 6 h and analyzed for oxLDL uptake and foam cell formation as described in Figure legend 1 C. *p
Figure Legend Snippet: CC induces ROS production, BTK activation, p300-STAT1-PPARγ interactions, CD36 expression and foam cell formation in mouse primary peritoneal macrophages. A–C. Mouse primary peritoneal macrophages were isolated from WT mice, quiesced overnight, treated with and without CC (40 μg/ml) for the indicated time periods and either ROS production was measured or cell extracts were prepared and analyzed for BTK and p300 tyrosine phosphorylation, p300 association with STAT1, STAT1 acetylation and its association with PPARγ and CD36 expression as described in Figure legends 5A, 4B, 4A, 3A, 1F, 2B and IA, respectively. D and E. Mouse primary peritoneal macrophages after overnight growth arrest were treated with and without CC (40 μg/ml) for 6 h and analyzed for oxLDL uptake and foam cell formation as described in Figure legend 1 C. *p

Techniques Used: Activation Assay, Expressing, Isolation, Mouse Assay, IA

3) Product Images from "Alkylation of cysteine 468 in Stat3 defines a novel site for therapeutic development"

Article Title: Alkylation of cysteine 468 in Stat3 defines a novel site for therapeutic development

Journal: ACS chemical biology

doi: 10.1021/cb100253e

Effect of C48 on Stat3- and Stat1-mediated transcriptional activity A) Effect of C48 on Oncostatin M (OSM)-induced, Stat3-mediated expression of luciferase as a measure of transcription activity. Serum-starved HeLa-Stat3-Luc cells were pre-incubated with C48 1 h prior to stimulation with OSM. Luminescence was measured 8 h post stimulation. B) Effect of C48 on IFNγ-induced, Stat1-mediated expression of luciferase as a measure of transcription activity. Serum-starved HeLa-Stat1-Luc cells were pre-incubated with C48 1 h prior to stimulation with IFNγ. Luminescence was measured 8 h post stimulation. One representative result is shown (n=3, in triplicate).
Figure Legend Snippet: Effect of C48 on Stat3- and Stat1-mediated transcriptional activity A) Effect of C48 on Oncostatin M (OSM)-induced, Stat3-mediated expression of luciferase as a measure of transcription activity. Serum-starved HeLa-Stat3-Luc cells were pre-incubated with C48 1 h prior to stimulation with OSM. Luminescence was measured 8 h post stimulation. B) Effect of C48 on IFNγ-induced, Stat1-mediated expression of luciferase as a measure of transcription activity. Serum-starved HeLa-Stat1-Luc cells were pre-incubated with C48 1 h prior to stimulation with IFNγ. Luminescence was measured 8 h post stimulation. One representative result is shown (n=3, in triplicate).

Techniques Used: Activity Assay, Expressing, Luciferase, Incubation

Structure of Stat3 inhibitor compounds and effect of C48 on STAT DNA-binding activity A) Structure of the initial lead Stat3 inhibitor compound (C36) and structures of Stat3 inhibitor compounds identified through a similarity search (C29, C30 and C48). B) Dose-response effect of compound C48 on the DNA-binding activity of Stat1 and Stat3 homodimers, as well as Stat1/3 heterodimers in vitro , as assessed by EMSA. C48 and nuclear extract (NE) that contained activated STAT proteins were pre-incubated for 30 min prior to addition of DNA for 30 min. SS, Super-shifted STAT proteins. C) Mode of action of C48 on STAT DNA-binding. As a control, NE was incubated with DNA for 30 minutes (lanes 1-2), C48 and NE that contained activated STAT proteins were pre-incubated prior to addition of DNA (lanes 3-6), NE and DNA were pre-incubated prior to addition of C48 (lanes 7-10), or C48 and DNA were pre-incubated prior to addition of NE (lanes 11-14).
Figure Legend Snippet: Structure of Stat3 inhibitor compounds and effect of C48 on STAT DNA-binding activity A) Structure of the initial lead Stat3 inhibitor compound (C36) and structures of Stat3 inhibitor compounds identified through a similarity search (C29, C30 and C48). B) Dose-response effect of compound C48 on the DNA-binding activity of Stat1 and Stat3 homodimers, as well as Stat1/3 heterodimers in vitro , as assessed by EMSA. C48 and nuclear extract (NE) that contained activated STAT proteins were pre-incubated for 30 min prior to addition of DNA for 30 min. SS, Super-shifted STAT proteins. C) Mode of action of C48 on STAT DNA-binding. As a control, NE was incubated with DNA for 30 minutes (lanes 1-2), C48 and NE that contained activated STAT proteins were pre-incubated prior to addition of DNA (lanes 3-6), NE and DNA were pre-incubated prior to addition of C48 (lanes 7-10), or C48 and DNA were pre-incubated prior to addition of NE (lanes 11-14).

Techniques Used: Binding Assay, Activity Assay, In Vitro, Incubation

4) Product Images from "Activation of Oas1a gene expression by type I IFN requires both STAT1 and STAT2 while only STAT2 is required for Oas1b activation"

Article Title: Activation of Oas1a gene expression by type I IFN requires both STAT1 and STAT2 while only STAT2 is required for Oas1b activation

Journal: Virology

doi: 10.1016/j.virol.2011.11.025

Binding of STAT1 and STAT2 to the Oas1a and Oas1b promoters in vitro and in vivo (A) DIG-labeled Oas1b and Oas1a DNA probes and nuclear extracts from untreated or murine IFN beta (1000 U/ml) treated C3H/RV MEFs were used for EMSA. Anti-STAT1, anti-STAT2 or a nonspecific IgG antibody was added to the nuclear extract prior to addition of the probe. DNA-protein complexes were resolved on 6% native gels and the labeled probe was detected with anti-DIG antibody. The results shown are representative of at least two independent experiments. ChIP analysis of the binding of STAT1 or STAT2 to the (B) Oas1a or (C) Oas1b promoter in control or 30 min IFN beta treated C3H/He cells. The amounts of precipitated Oas1a and Oas1b promoter DNA were quantified by real-time qPCR using promoter-specific primers and fluorogenic TaqMan FAM/MGB probes. Nonspecific IgG antibody was used as a negative control and averaged values are shown. The bars represent standard deviation (SD) (n=3).
Figure Legend Snippet: Binding of STAT1 and STAT2 to the Oas1a and Oas1b promoters in vitro and in vivo (A) DIG-labeled Oas1b and Oas1a DNA probes and nuclear extracts from untreated or murine IFN beta (1000 U/ml) treated C3H/RV MEFs were used for EMSA. Anti-STAT1, anti-STAT2 or a nonspecific IgG antibody was added to the nuclear extract prior to addition of the probe. DNA-protein complexes were resolved on 6% native gels and the labeled probe was detected with anti-DIG antibody. The results shown are representative of at least two independent experiments. ChIP analysis of the binding of STAT1 or STAT2 to the (B) Oas1a or (C) Oas1b promoter in control or 30 min IFN beta treated C3H/He cells. The amounts of precipitated Oas1a and Oas1b promoter DNA were quantified by real-time qPCR using promoter-specific primers and fluorogenic TaqMan FAM/MGB probes. Nonspecific IgG antibody was used as a negative control and averaged values are shown. The bars represent standard deviation (SD) (n=3).

Techniques Used: Binding Assay, In Vitro, In Vivo, Labeling, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Standard Deviation

5) Product Images from "Global changes in STAT target selection and transcription regulation upon interferon treatments"

Article Title: Global changes in STAT target selection and transcription regulation upon interferon treatments

Journal: Genes & Development

doi: 10.1101/gad.1371305

Locations of all the ChIP–chip and expression results across chromosome 22q based on Ensembl gene annotations. The gray center track represents the regions of chromosome 22 represented on the microarray, from the centromere ( top left ) to the telomere ( bottom right ). Black bars within the gray track represent the locations of ChIP–chip results from the IFN-γ STAT1 ( top third), IFN-α–STAT1 ( middle ), and IFN-α–STAT2 ( bottom third) assays. Open diamonds and black triangles signify STAT-binding sites identified by two or three different ChIP–chip assays, respectively. Expression results from up-regulated (red), down-regulated (green), and unaffected (yellow) genes are located above (sense) and below .
Figure Legend Snippet: Locations of all the ChIP–chip and expression results across chromosome 22q based on Ensembl gene annotations. The gray center track represents the regions of chromosome 22 represented on the microarray, from the centromere ( top left ) to the telomere ( bottom right ). Black bars within the gray track represent the locations of ChIP–chip results from the IFN-γ STAT1 ( top third), IFN-α–STAT1 ( middle ), and IFN-α–STAT2 ( bottom third) assays. Open diamonds and black triangles signify STAT-binding sites identified by two or three different ChIP–chip assays, respectively. Expression results from up-regulated (red), down-regulated (green), and unaffected (yellow) genes are located above (sense) and below .

Techniques Used: Chromatin Immunoprecipitation, Expressing, Microarray, Binding Assay

IFN stimulations activate the STATs and induce their binding to correct DNA target sequences. ( A ) Western blotting of STAT1 and STAT2 immunoprecipitations from nuclear extracts detected the increase in nuclear localization and phosphorylation of STAT1 and STAT2. ( B ) PCR analysis of STAT1 and STAT2 ChIP DNA confirmed the IFN-induced enriched presence of the STAT-binding sites in the IRF1 and OAS1 promoters. Positive and negative controls used genomic DNA and no template, respectively.
Figure Legend Snippet: IFN stimulations activate the STATs and induce their binding to correct DNA target sequences. ( A ) Western blotting of STAT1 and STAT2 immunoprecipitations from nuclear extracts detected the increase in nuclear localization and phosphorylation of STAT1 and STAT2. ( B ) PCR analysis of STAT1 and STAT2 ChIP DNA confirmed the IFN-induced enriched presence of the STAT-binding sites in the IRF1 and OAS1 promoters. Positive and negative controls used genomic DNA and no template, respectively.

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

Schematic of IFN-induced STAT1-binding site selection. ( A ) IFN-γ and IFN-α both induce STAT1 homodimers, which bind DNA (Gene A); however, we also observed IFN-α-induced, STAT2-independent binding of STAT1 to sites not occupied by IFN-γ-induced STAT1 (Gene C). ( B ) Changes in binding site accessibility and/or the presence of a cooperating cofactor may account for the binding of STAT1 to new sites upon IFN-α treatment. Also, the binding of STAT1 to alternate dimerization partners (such as STAT2) reduces the relative quantity of STAT1 homodimers; thus, a loss of STAT1 binding to some IFN-γ-induced sites occurs.
Figure Legend Snippet: Schematic of IFN-induced STAT1-binding site selection. ( A ) IFN-γ and IFN-α both induce STAT1 homodimers, which bind DNA (Gene A); however, we also observed IFN-α-induced, STAT2-independent binding of STAT1 to sites not occupied by IFN-γ-induced STAT1 (Gene C). ( B ) Changes in binding site accessibility and/or the presence of a cooperating cofactor may account for the binding of STAT1 to new sites upon IFN-α treatment. Also, the binding of STAT1 to alternate dimerization partners (such as STAT2) reduces the relative quantity of STAT1 homodimers; thus, a loss of STAT1 binding to some IFN-γ-induced sites occurs.

Techniques Used: Binding Assay, Selection

6) Product Images from "Induction of IFN-? and the Innate Antiviral Response in Myeloid Cells Occurs through an IPS-1-Dependent Signal That Does Not Require IRF-3 and IRF-7"

Article Title: Induction of IFN-? and the Innate Antiviral Response in Myeloid Cells Occurs through an IPS-1-Dependent Signal That Does Not Require IRF-3 and IRF-7

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000607

A model for detection of WNV and IFN-α/β gene activation in MEF. (1). The host through recognition of an as yet undefined viral RNA PAMP in the cytoplasm detects WNV. RIG-I acts as the primary PRR sensor for WNV during the early stages of infection. RIG-I activation promotes association with IPS-1, which leads to recruitment of TRAF3 and TBK1, and phosphorylation of IRF-3. NF-κB and ATF-2/c-Jun and the small amounts of constitutively expressed IRF-7 may also be activated via this IPS-1-dependent pathway. IRF-3, IRF-7 NF-κB, ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and promote transcription. Secretion of IFN-β by infected cells during this early phase results in autocrine and paracrine type I IFN signaling through binding of the IFN-αβR. (2). Activation of IFN-αβR results in phosphorylation of JAK1 and Tyk2, which activate STAT1 and STAT2 leading to formation of the heterotrimer ISGF3 (STAT1, STAT2 and IRF-9). Nuclear translocation and promoter binding of ISGF3 upregulates hundreds of different ISG, including IRF-7. (3). During a later phase of infection, detection of WNV in MEF also relies on MDA5 and PKR. Recruitment of TRAF3 and TRAF6 activates IRF-3 and IRF-7. NF-κB and ATF-2/c-Jun are also activated via an as yet undefined mechanism. Subsequently, IRF-3, IRF-7, NF-κB, and ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and induce optimal transcription. Induction of IFN-α genes occurs through TRAF6 and the transcriptional activation of IRF-7.
Figure Legend Snippet: A model for detection of WNV and IFN-α/β gene activation in MEF. (1). The host through recognition of an as yet undefined viral RNA PAMP in the cytoplasm detects WNV. RIG-I acts as the primary PRR sensor for WNV during the early stages of infection. RIG-I activation promotes association with IPS-1, which leads to recruitment of TRAF3 and TBK1, and phosphorylation of IRF-3. NF-κB and ATF-2/c-Jun and the small amounts of constitutively expressed IRF-7 may also be activated via this IPS-1-dependent pathway. IRF-3, IRF-7 NF-κB, ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and promote transcription. Secretion of IFN-β by infected cells during this early phase results in autocrine and paracrine type I IFN signaling through binding of the IFN-αβR. (2). Activation of IFN-αβR results in phosphorylation of JAK1 and Tyk2, which activate STAT1 and STAT2 leading to formation of the heterotrimer ISGF3 (STAT1, STAT2 and IRF-9). Nuclear translocation and promoter binding of ISGF3 upregulates hundreds of different ISG, including IRF-7. (3). During a later phase of infection, detection of WNV in MEF also relies on MDA5 and PKR. Recruitment of TRAF3 and TRAF6 activates IRF-3 and IRF-7. NF-κB and ATF-2/c-Jun are also activated via an as yet undefined mechanism. Subsequently, IRF-3, IRF-7, NF-κB, and ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and induce optimal transcription. Induction of IFN-α genes occurs through TRAF6 and the transcriptional activation of IRF-7.

Techniques Used: Activation Assay, Infection, Binding Assay, Translocation Assay

IRF-3 and IRF-7 partially modulate the IFN-β response and ISG expression in primary Mφ. A. Mφ generated from wild type, IFN-αβR −/− and DKO mice were infected at an MOI of 0.01 and virus production was evaluated at the indicated times post infection by plaque assay. Values are an average of quadruplicate samples generated from at least three independent experiments. B. Whole cell lysates were generated at the indicated times from wild type and DKO Mφ that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, PKR, STAT1, RIG-I, MDA5 and tubulin were examined by immunoblot analysis. C and D. The induction of (C) IFN-α and (D) IFN-β mRNA in WNV-infected Mφ was analyzed by qRT-PCR as described in Figure 3 . Asterisks indicate values that are statistically significant (***, P
Figure Legend Snippet: IRF-3 and IRF-7 partially modulate the IFN-β response and ISG expression in primary Mφ. A. Mφ generated from wild type, IFN-αβR −/− and DKO mice were infected at an MOI of 0.01 and virus production was evaluated at the indicated times post infection by plaque assay. Values are an average of quadruplicate samples generated from at least three independent experiments. B. Whole cell lysates were generated at the indicated times from wild type and DKO Mφ that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, PKR, STAT1, RIG-I, MDA5 and tubulin were examined by immunoblot analysis. C and D. The induction of (C) IFN-α and (D) IFN-β mRNA in WNV-infected Mφ was analyzed by qRT-PCR as described in Figure 3 . Asterisks indicate values that are statistically significant (***, P

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

7) Product Images from "Identification of Residues of SARS-CoV nsp1 That Differentially Affect Inhibition of Gene Expression and Antiviral Signaling"

Article Title: Identification of Residues of SARS-CoV nsp1 That Differentially Affect Inhibition of Gene Expression and Antiviral Signaling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0062416

Graphical representation of experimental design. (A) SARS-CoV nsp1 was cloned into a plasmid driven by the CMV enhancer with a N-terminal triple FLAG tag. Surface residues that could be important for function were identified and mutated. (B) SARS-CoV nsp1-wt and mutants were cotransfected into 293T cells with reporter plasmids. Plasmid expressing eGFP was used to visually inspect for high transfection efficiency. (C) Standard assays were performed: luciferase and β-galactosidase assays were used as a proxy to measure level of nsp1 inhibition of gene expression; CAT gene under the control of three ISRE copies was used as a proxy for nsp1 inhibition of interferon- and virus-dependent signaling; immunoblots were run to directly measure nsp1 inhibition of STAT1 phosphorylation and IRF3 dimerization, and nsp1 levels.
Figure Legend Snippet: Graphical representation of experimental design. (A) SARS-CoV nsp1 was cloned into a plasmid driven by the CMV enhancer with a N-terminal triple FLAG tag. Surface residues that could be important for function were identified and mutated. (B) SARS-CoV nsp1-wt and mutants were cotransfected into 293T cells with reporter plasmids. Plasmid expressing eGFP was used to visually inspect for high transfection efficiency. (C) Standard assays were performed: luciferase and β-galactosidase assays were used as a proxy to measure level of nsp1 inhibition of gene expression; CAT gene under the control of three ISRE copies was used as a proxy for nsp1 inhibition of interferon- and virus-dependent signaling; immunoblots were run to directly measure nsp1 inhibition of STAT1 phosphorylation and IRF3 dimerization, and nsp1 levels.

Techniques Used: Clone Assay, Plasmid Preparation, FLAG-tag, Expressing, Transfection, Luciferase, Inhibition, Western Blot

SARS-CoV nsp1 mutants show altered inhibition of signaling molecules. To confirm previous results, selected nsp1 mutants were tested for ability to directly inhibit signaling molecules. Cell extracts were separated by SDS-PAGE, and probed for levels of active phosphorylated STAT1 (P-STAT1) and for levels of total STAT1 (STAT1) (A). Cell extracts were separated by native-PAGE (B), and probed with anti-IRF3 antibody to detect both monomeric (inactive) and dimeric (active) forms. Immunoblots were quantitated and the relative activity of signaling molecules was calculated. Immunoblots confirm previous results showing that nsp1-m16 had indeed lost its ability to strongly inhibit both the phosphorylation of STAT1 and the dimerization of IRF3 after cells had been stimulated with either IFNα or SeV. Error bars are standard error; P-values are result of a t-Test and significance for all mutants is shown in (C).
Figure Legend Snippet: SARS-CoV nsp1 mutants show altered inhibition of signaling molecules. To confirm previous results, selected nsp1 mutants were tested for ability to directly inhibit signaling molecules. Cell extracts were separated by SDS-PAGE, and probed for levels of active phosphorylated STAT1 (P-STAT1) and for levels of total STAT1 (STAT1) (A). Cell extracts were separated by native-PAGE (B), and probed with anti-IRF3 antibody to detect both monomeric (inactive) and dimeric (active) forms. Immunoblots were quantitated and the relative activity of signaling molecules was calculated. Immunoblots confirm previous results showing that nsp1-m16 had indeed lost its ability to strongly inhibit both the phosphorylation of STAT1 and the dimerization of IRF3 after cells had been stimulated with either IFNα or SeV. Error bars are standard error; P-values are result of a t-Test and significance for all mutants is shown in (C).

Techniques Used: Inhibition, SDS Page, Clear Native PAGE, Western Blot, Activity Assay

8) Product Images from "Alterations in the properties of the cell membrane due to glycosphingolipid accumulation in a model of Gaucher disease"

Article Title: Alterations in the properties of the cell membrane due to glycosphingolipid accumulation in a model of Gaucher disease

Journal: Scientific Reports

doi: 10.1038/s41598-017-18405-8

Effect of the Gaucher phenotype on STAT phosphorylation. Control THP-1-derived macrophages and those exhibiting the Gaucher phenotype were serum-starved overnight followed by a 30-min stimulation with 100 ng/ml IFN-γ. Cells were analyzed by microscopy ( A–C ) or flow cytometry after trypsinization ( D ). The level of tyrosine phosphorylated STAT1 was calculated in the whole cell ( A ), in the nucleus ( B ) or in the cytoplasm ( C ) using quantitative image analysis, or it was determined from the mean of flow cytometric histograms ( D ). The fluorescence intensity values were normalized to the unstimulated control. The error bars represent the standard error of the mean of four independent experiments in the case of microscopy and three independent experiments in the case of flow cytometric results. Control and Gaucher-type cells were compared by two-way ANOVA followed by Tukey’s HSD test (*p
Figure Legend Snippet: Effect of the Gaucher phenotype on STAT phosphorylation. Control THP-1-derived macrophages and those exhibiting the Gaucher phenotype were serum-starved overnight followed by a 30-min stimulation with 100 ng/ml IFN-γ. Cells were analyzed by microscopy ( A–C ) or flow cytometry after trypsinization ( D ). The level of tyrosine phosphorylated STAT1 was calculated in the whole cell ( A ), in the nucleus ( B ) or in the cytoplasm ( C ) using quantitative image analysis, or it was determined from the mean of flow cytometric histograms ( D ). The fluorescence intensity values were normalized to the unstimulated control. The error bars represent the standard error of the mean of four independent experiments in the case of microscopy and three independent experiments in the case of flow cytometric results. Control and Gaucher-type cells were compared by two-way ANOVA followed by Tukey’s HSD test (*p

Techniques Used: Derivative Assay, Microscopy, Flow Cytometry, Cytometry, Fluorescence

9) Product Images from "The Type III Effectors NleE and NleB from Enteropathogenic E. coli and OspZ from Shigella Block Nuclear Translocation of NF-?B p65"

Article Title: The Type III Effectors NleE and NleB from Enteropathogenic E. coli and OspZ from Shigella Block Nuclear Translocation of NF-?B p65

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000898

Effect of NleE on nuclear translocation of STAT1 and STAT2. A . Representative immunofluorescence fields and quantification of nuclear exclusion using antibodies to STAT1 ( A ) or STAT2 ( B ) (red) of HeLa cells transfected with pEGFP-C2 (GFP only) or pGFP-NleE (green), stimulated with IFNα for 30 min and stained for nucleic acid with DAPI (blue). Results are expressed as the percentage of GFP-positive cells that exclude STAT1 or STAT2 and are the mean ± SEM of three independent experiments performed in duplicate. At least 100 GFP-positive cells were counted per test. C . Fold increase in STAT1/2 dependent luciferase activity in HeLa cells transfected with pEGFP-C2 (GFP), pGFP-NleE or pGFP-OspZ. Results are the mean ± SEM of 3 independent experiments performed in triplicate. Differences between GFP and GFP-NleE or GFP-OspZ were not significant ( P > 0.05, one way ANOVA).
Figure Legend Snippet: Effect of NleE on nuclear translocation of STAT1 and STAT2. A . Representative immunofluorescence fields and quantification of nuclear exclusion using antibodies to STAT1 ( A ) or STAT2 ( B ) (red) of HeLa cells transfected with pEGFP-C2 (GFP only) or pGFP-NleE (green), stimulated with IFNα for 30 min and stained for nucleic acid with DAPI (blue). Results are expressed as the percentage of GFP-positive cells that exclude STAT1 or STAT2 and are the mean ± SEM of three independent experiments performed in duplicate. At least 100 GFP-positive cells were counted per test. C . Fold increase in STAT1/2 dependent luciferase activity in HeLa cells transfected with pEGFP-C2 (GFP), pGFP-NleE or pGFP-OspZ. Results are the mean ± SEM of 3 independent experiments performed in triplicate. Differences between GFP and GFP-NleE or GFP-OspZ were not significant ( P > 0.05, one way ANOVA).

Techniques Used: Translocation Assay, Immunofluorescence, Transfection, Staining, Luciferase, Activity Assay

10) Product Images from "Interaction between MUC1 and STAT1 drives IFITM1 overexpression in aromatase inhibitor-resistant breast cancer cells and mediates estrogen-induced apoptosis"

Article Title: Interaction between MUC1 and STAT1 drives IFITM1 overexpression in aromatase inhibitor-resistant breast cancer cells and mediates estrogen-induced apoptosis

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-18-0916

E 2 treatment reduces tumor size and reduces MUC1, P-STAT1, and IFITM1 levels in AI-resistant cells in vivo . ( a ) 3 million MCF-7 and MCF-7:5C cells were bilaterally injected into the 4 th mammary fat pad of ovariectomized female NSG mice with and without E 2 capsule implants respectively. 35 days post-injection, mice were randomized into two groups with half of the E 2 capsule implants removed from MCF-7 injected mice and half of the MCF-7:5C injected mice received estrogen capsule implants. Tumors were measured using digital calipers and measurements were used to calculate tumor volume (mm 3 ) over time. ( b ) At the end of the experiment tumors were excised and final tumor volume was measured. In the MCF-7 injected mice, 12 tumors were collected from the −E 2 group and 11 tumors were collected from the +E 2 group. In the MCF-7:5C injected mice, 14 tumors were collected from the −E 2 group and 16 tumors were collected from the +E 2 group. ( c ) Cell death was analyzed by TUNEL staining and quantified by Image J software. ( d ) Hematoxylin and Eosin (H+E), IgG (negative staining control) and MUC1, P-STAT1, and IFITM1 staining in NSG mice from untreated and E 2 treated mice. *p
Figure Legend Snippet: E 2 treatment reduces tumor size and reduces MUC1, P-STAT1, and IFITM1 levels in AI-resistant cells in vivo . ( a ) 3 million MCF-7 and MCF-7:5C cells were bilaterally injected into the 4 th mammary fat pad of ovariectomized female NSG mice with and without E 2 capsule implants respectively. 35 days post-injection, mice were randomized into two groups with half of the E 2 capsule implants removed from MCF-7 injected mice and half of the MCF-7:5C injected mice received estrogen capsule implants. Tumors were measured using digital calipers and measurements were used to calculate tumor volume (mm 3 ) over time. ( b ) At the end of the experiment tumors were excised and final tumor volume was measured. In the MCF-7 injected mice, 12 tumors were collected from the −E 2 group and 11 tumors were collected from the +E 2 group. In the MCF-7:5C injected mice, 14 tumors were collected from the −E 2 group and 16 tumors were collected from the +E 2 group. ( c ) Cell death was analyzed by TUNEL staining and quantified by Image J software. ( d ) Hematoxylin and Eosin (H+E), IgG (negative staining control) and MUC1, P-STAT1, and IFITM1 staining in NSG mice from untreated and E 2 treated mice. *p

Techniques Used: In Vivo, Injection, Mouse Assay, TUNEL Assay, Staining, Software, Negative Staining

Rux treatment reduces tumor size and reduces MUC1, P-STAT1, and IFITM1 levels in AI-resistant cells in vivo . ( a ) Whole cell lysates from MCF-7 and MCF-7:5C cells treated with 10 μmol Rux for 24 hours were immunoblotted for P-STAT1, STAT1 and IFITM1 protein expression. ( b ) 3 million MCF-7 or MCF-7:5C cells were injected into the 4 th mammary fat pad of female NSG mice. After 32 days of tumor growth, mice were randomized to treatment groups and half were given 50 mg/kg Rux by oral gavage every other day. At the end of the experiment tumors were excised and tumor volumes were determined by digital caliper measurement. In the MCF-7 injected mice, 8 tumors were collected from the -Rux group and 7 tumors were collected from the +Rux group. In the MCF-7:5C injected mice, 5 tumors were collected from the -Rux group and 6 tumors were collected from the +Rux group. ( c ) Tumors were subjected to TUNEL staining to quantify apoptotic cells. The intensity of TUNEL (red) staining was quantified using ten separate images with Image J Software ( bottom panel ). ( d ) Tumor expression of P-STAT1, STAT1 and IFITM1 after 21 days of treatment was determined by immunoblot. ( e ) Tumors were fixed, embedded in paraffin and sectioned onto glass slides. H+E staining revealed tumor architecture and MUC1, IFITM1, and P-STAT1 expression was determined by immunohistochemistry. *p
Figure Legend Snippet: Rux treatment reduces tumor size and reduces MUC1, P-STAT1, and IFITM1 levels in AI-resistant cells in vivo . ( a ) Whole cell lysates from MCF-7 and MCF-7:5C cells treated with 10 μmol Rux for 24 hours were immunoblotted for P-STAT1, STAT1 and IFITM1 protein expression. ( b ) 3 million MCF-7 or MCF-7:5C cells were injected into the 4 th mammary fat pad of female NSG mice. After 32 days of tumor growth, mice were randomized to treatment groups and half were given 50 mg/kg Rux by oral gavage every other day. At the end of the experiment tumors were excised and tumor volumes were determined by digital caliper measurement. In the MCF-7 injected mice, 8 tumors were collected from the -Rux group and 7 tumors were collected from the +Rux group. In the MCF-7:5C injected mice, 5 tumors were collected from the -Rux group and 6 tumors were collected from the +Rux group. ( c ) Tumors were subjected to TUNEL staining to quantify apoptotic cells. The intensity of TUNEL (red) staining was quantified using ten separate images with Image J Software ( bottom panel ). ( d ) Tumor expression of P-STAT1, STAT1 and IFITM1 after 21 days of treatment was determined by immunoblot. ( e ) Tumors were fixed, embedded in paraffin and sectioned onto glass slides. H+E staining revealed tumor architecture and MUC1, IFITM1, and P-STAT1 expression was determined by immunohistochemistry. *p

Techniques Used: In Vivo, Expressing, Injection, Mouse Assay, TUNEL Assay, Staining, Software, Immunohistochemistry

MUC1 stabilizes JAK/STAT signaling which is necessary for IFITM1 expression. ( a ) T-47D, MCF-7, and MCF-7:5C cells were transiently transfected with siCon or siMUC1 for 72 hours and immunoblotted for MUC1, P-STAT2, STAT2, P-STAT1, STAT1, and IFITM1 expression. ( b ) T-47D, MCF-7, and MCF-7:5C cells were treated for 48 hours with Ruxolitinib (Rux) and immunoblotted for MUC1, P-STAT2, STAT2, P-STAT1, STAT1, and IFITM1 expression. ( c ) T-47D, MCF-7, and MCF-7:5C cells were treated for 24 hours with E 2 and immunoblotted for MUC1, P-STAT2, STAT2, P-STAT1, STAT1, and IFITM1 expression. ( d ) T-47D and MCF-7:5C cells were transiently transfected with control or MUC1 siRNA or treated with Rux. After 24 hours, mRNA expression of IFITM1 was determined by RT-PCR. ( e ) Whole cell lysates from T-47D and MCF-7:5C cells were treated with vehicle (Con) or 10 μmol Rux for 8 hours were immunoprecipitated with anti-MUC1 antibody or rabbit IgG and samples immunoblotted for MUC1, P-STAT2, P-STAT1 and ERα protein interaction. ( f ) Fixed T-47D and MCF-7:5C cell lysates were subjected to chromatin immunoprecipitation (ChIP) with antibodies against STAT1, STAT2, MUC1 or species-specific IgG control. As indicated, fixed whole cell lysates from T-47D and MCF-7:5C cells treated with 10 μmol Rux for 24 hours. qPCR was performed on the isolated DNA using primers designed to amplify the ISRE regulatory regions. Recruitment of the indicated proteins to the ISRE site was compared to input and IgG DNA and displayed as mean ± SD of technical triplicates in two independent experiments. Fold change between control and treatment is displayed above the indicated protein. ***p
Figure Legend Snippet: MUC1 stabilizes JAK/STAT signaling which is necessary for IFITM1 expression. ( a ) T-47D, MCF-7, and MCF-7:5C cells were transiently transfected with siCon or siMUC1 for 72 hours and immunoblotted for MUC1, P-STAT2, STAT2, P-STAT1, STAT1, and IFITM1 expression. ( b ) T-47D, MCF-7, and MCF-7:5C cells were treated for 48 hours with Ruxolitinib (Rux) and immunoblotted for MUC1, P-STAT2, STAT2, P-STAT1, STAT1, and IFITM1 expression. ( c ) T-47D, MCF-7, and MCF-7:5C cells were treated for 24 hours with E 2 and immunoblotted for MUC1, P-STAT2, STAT2, P-STAT1, STAT1, and IFITM1 expression. ( d ) T-47D and MCF-7:5C cells were transiently transfected with control or MUC1 siRNA or treated with Rux. After 24 hours, mRNA expression of IFITM1 was determined by RT-PCR. ( e ) Whole cell lysates from T-47D and MCF-7:5C cells were treated with vehicle (Con) or 10 μmol Rux for 8 hours were immunoprecipitated with anti-MUC1 antibody or rabbit IgG and samples immunoblotted for MUC1, P-STAT2, P-STAT1 and ERα protein interaction. ( f ) Fixed T-47D and MCF-7:5C cell lysates were subjected to chromatin immunoprecipitation (ChIP) with antibodies against STAT1, STAT2, MUC1 or species-specific IgG control. As indicated, fixed whole cell lysates from T-47D and MCF-7:5C cells treated with 10 μmol Rux for 24 hours. qPCR was performed on the isolated DNA using primers designed to amplify the ISRE regulatory regions. Recruitment of the indicated proteins to the ISRE site was compared to input and IgG DNA and displayed as mean ± SD of technical triplicates in two independent experiments. Fold change between control and treatment is displayed above the indicated protein. ***p

Techniques Used: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation

11) Product Images from "Stat1 Acetylation Inhibits Inducible Nitric Oxide Synthase Expression in Interferon-γ Treated RAW264.7 Murine Macrophages"

Article Title: Stat1 Acetylation Inhibits Inducible Nitric Oxide Synthase Expression in Interferon-γ Treated RAW264.7 Murine Macrophages

Journal:

doi: 10.1016/j.surg.2007.02.016

Immunoprecipitation of Ac-Stat1 from IFN-γ stimulated macrophages
Figure Legend Snippet: Immunoprecipitation of Ac-Stat1 from IFN-γ stimulated macrophages

Techniques Used: Immunoprecipitation

Stat1 and NF-κB binding to the iNOS promoter
Figure Legend Snippet: Stat1 and NF-κB binding to the iNOS promoter

Techniques Used: Binding Assay

12) Product Images from "STAT1 Is a Master Regulator of Pancreatic ?-Cell Apoptosis and Islet Inflammation *"

Article Title: STAT1 Is a Master Regulator of Pancreatic ?-Cell Apoptosis and Islet Inflammation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.162131

Schematic representation of selected cytokine-dependent genes differentially regulated by the transcription factors STAT1 ( left ), IRF-1 ( right ), or both STAT1 and IRF-1 ( center ). Of note, some genes ( e.g. chemokines) are regulated, at least in part, in opposite directions by STAT1 and IRF-1.
Figure Legend Snippet: Schematic representation of selected cytokine-dependent genes differentially regulated by the transcription factors STAT1 ( left ), IRF-1 ( right ), or both STAT1 and IRF-1 ( center ). Of note, some genes ( e.g. chemokines) are regulated, at least in part, in opposite directions by STAT1 and IRF-1.

Techniques Used:

13) Product Images from "IRF9 is a key factor for eliciting the antiproliferative activity of IFN-?"

Article Title: IRF9 is a key factor for eliciting the antiproliferative activity of IFN-?

Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

doi: 10.1097/CJI.0b013e3181ad4092

IRF9-RNAi inhibited the antiproliferative activity of IFN-α2c, but Stat1-RNAi did not. A (IFN-α2c treatment) and B (IFN-γ treatment), Twenty nM of RNAi-transfected OVCAR3 cells were treated with or without IFNs for 3 days. Absorbance
Figure Legend Snippet: IRF9-RNAi inhibited the antiproliferative activity of IFN-α2c, but Stat1-RNAi did not. A (IFN-α2c treatment) and B (IFN-γ treatment), Twenty nM of RNAi-transfected OVCAR3 cells were treated with or without IFNs for 3 days. Absorbance

Techniques Used: Activity Assay, Transfection

IRF9-RNAi inhibited transcription of TRAIL, IFIT1, and IFIT3 in response to IFN-α2c, but Stat1-RNAi did not. Twenty nM of RNAi-transfected OVCAR3 cells were treated with or without 10 ng/ml of IFN-α2c for 24 h, then transcription of these
Figure Legend Snippet: IRF9-RNAi inhibited transcription of TRAIL, IFIT1, and IFIT3 in response to IFN-α2c, but Stat1-RNAi did not. Twenty nM of RNAi-transfected OVCAR3 cells were treated with or without 10 ng/ml of IFN-α2c for 24 h, then transcription of these

Techniques Used: Transfection

14) Product Images from "IFN-β Plays Both Pro- and Anti-inflammatory Roles in the Rat Cardiac Fibroblast Through Differential STAT Protein Activation"

Article Title: IFN-β Plays Both Pro- and Anti-inflammatory Roles in the Rat Cardiac Fibroblast Through Differential STAT Protein Activation

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.01368

Effects of IFN-β and STAT proteins on LPS-induced cytokine secretion: (A–E) CF were transfected with scramble or 200 ng of si-STAT1, si-STAT2, and si-STAT3 for 8 h, serum-deprived of for 24 h, pre-treated with IFN-β for 1 h, and stimulated with LPS for 24 h. (A) IL-6, (B) TNF-β, (C) IL-10, (D) MCP-1 and (E) IP-10 secretion to the culture medium was determined by LUMINEX assay. Error bars indicate the SD for three independent experiments. ∗∗∗ p
Figure Legend Snippet: Effects of IFN-β and STAT proteins on LPS-induced cytokine secretion: (A–E) CF were transfected with scramble or 200 ng of si-STAT1, si-STAT2, and si-STAT3 for 8 h, serum-deprived of for 24 h, pre-treated with IFN-β for 1 h, and stimulated with LPS for 24 h. (A) IL-6, (B) TNF-β, (C) IL-10, (D) MCP-1 and (E) IP-10 secretion to the culture medium was determined by LUMINEX assay. Error bars indicate the SD for three independent experiments. ∗∗∗ p

Techniques Used: Transfection, Luminex

Proinflammatory and anti-inflammatory effects of IFN-β in CF: (A) IFN-β triggers JAK/STATsignaling pathway activation (STAT1, STAT3, and STAT3 proteins). (1) IFN-β induces a pro-inflammatory response in the CF through STAT1 protein activation (green arrows), characterized by increased expression of the chemokines IP-10 and MCP-1, as well as (2) increased neutrophil migration, favored by chemokines released from CF.
Figure Legend Snippet: Proinflammatory and anti-inflammatory effects of IFN-β in CF: (A) IFN-β triggers JAK/STATsignaling pathway activation (STAT1, STAT3, and STAT3 proteins). (1) IFN-β induces a pro-inflammatory response in the CF through STAT1 protein activation (green arrows), characterized by increased expression of the chemokines IP-10 and MCP-1, as well as (2) increased neutrophil migration, favored by chemokines released from CF.

Techniques Used: Activation Assay, Expressing, Migration

Anti-inflammatory effect of IFN-β in CF: (A) IFN-β induces the activation of its JAK/STAT transduction pathway (proteins Stat1, Stat3, Stat3); and directly through Stat3 induces the secretion of IL-10 (1). (B) The LPS is a pro-inflammatory stimulus that favors the secretion of cytokines (2), chemokines (3), expression of adhesion molecules (4), which finally favors the recruitment of neutrophils by the FC (5 and 6) . (7) IFN-β through the activation of Stat2 and Stat3 proteins (red and blue STOP lines), inhibits the pro-inflammatory effects of LPS.
Figure Legend Snippet: Anti-inflammatory effect of IFN-β in CF: (A) IFN-β induces the activation of its JAK/STAT transduction pathway (proteins Stat1, Stat3, Stat3); and directly through Stat3 induces the secretion of IL-10 (1). (B) The LPS is a pro-inflammatory stimulus that favors the secretion of cytokines (2), chemokines (3), expression of adhesion molecules (4), which finally favors the recruitment of neutrophils by the FC (5 and 6) . (7) IFN-β through the activation of Stat2 and Stat3 proteins (red and blue STOP lines), inhibits the pro-inflammatory effects of LPS.

Techniques Used: Activation Assay, Transduction, Expressing

Effects of IFN-β and STAT proteins on LPS-induced cytokine secretion: (A–E) CF were transfected with scramble or 200 ng of si-STAT1, si-STAT2, and si-STAT3 for 8 h, serum-deprived of for 24 h, pre-treated with IFN-β for 1 h, and stimulated with LPS for 24 h. (A) IL-6, (B) TNF-β, (C) IL-10, (D) MCP-1 and (E) IP-10 secretion to the culture medium was determined by LUMINEX assay. Error bars indicate the SD for three independent experiments. ∗∗∗ p
Figure Legend Snippet: Effects of IFN-β and STAT proteins on LPS-induced cytokine secretion: (A–E) CF were transfected with scramble or 200 ng of si-STAT1, si-STAT2, and si-STAT3 for 8 h, serum-deprived of for 24 h, pre-treated with IFN-β for 1 h, and stimulated with LPS for 24 h. (A) IL-6, (B) TNF-β, (C) IL-10, (D) MCP-1 and (E) IP-10 secretion to the culture medium was determined by LUMINEX assay. Error bars indicate the SD for three independent experiments. ∗∗∗ p

Techniques Used: Transfection, Luminex

IFN-β decreases LPS-induced ICAM-1/VCAM-1 expression through STAT3. (A–B) CF were transfected with scramble or 200 ng of si-STAT1, si-STAT2, and si-STAT3 for 8 h, serum-deprived for 24 h, pretreated with IFN-β (500 U/ml) for 1 h, and stimulated with LPS (1 μg/ml) for 8 h. (A) The upper panel shows a representative Western blot image, indicating expression levels of ICAM-1 and GAPDH (used as charge control). The graphical analysis is presented at the bottom of each panel. Error bars indicate the SD for three independent experiments. ∗∗∗ p
Figure Legend Snippet: IFN-β decreases LPS-induced ICAM-1/VCAM-1 expression through STAT3. (A–B) CF were transfected with scramble or 200 ng of si-STAT1, si-STAT2, and si-STAT3 for 8 h, serum-deprived for 24 h, pretreated with IFN-β (500 U/ml) for 1 h, and stimulated with LPS (1 μg/ml) for 8 h. (A) The upper panel shows a representative Western blot image, indicating expression levels of ICAM-1 and GAPDH (used as charge control). The graphical analysis is presented at the bottom of each panel. Error bars indicate the SD for three independent experiments. ∗∗∗ p

Techniques Used: Expressing, Transfection, Western Blot

Neutrophil migration induced by conditioned culture media derived from IFN-β- and LPS-treated CF: assays were performed in transwell chambers. Neutrophils charged with fluorescent calcein were seeded in the upper chamber and allowed to migrate at 37°C for 3 h to the lower chamber containing conditioned culture medium derived from CF that were transfected with scramble or 200 ng of STAT1 siRNA, STAT2, and STAT3 for 8 h, serum-deprived for 24 h, pretreated with IFN-β (500 U/ml) for 1 h, and stimulated with LPS (1 μg/ml) for 8 h. Fluorescent neutrophils that migrated to the lower compartment were measured using a fluorescence spectrometer, and the chemotactic activity was expressed as the percentage of fluorescent cells in the lower chamber as compared to the fluorescence of the total neutrophils added. Error bars indicate the SD for three independent experiments. ∗∗∗ p
Figure Legend Snippet: Neutrophil migration induced by conditioned culture media derived from IFN-β- and LPS-treated CF: assays were performed in transwell chambers. Neutrophils charged with fluorescent calcein were seeded in the upper chamber and allowed to migrate at 37°C for 3 h to the lower chamber containing conditioned culture medium derived from CF that were transfected with scramble or 200 ng of STAT1 siRNA, STAT2, and STAT3 for 8 h, serum-deprived for 24 h, pretreated with IFN-β (500 U/ml) for 1 h, and stimulated with LPS (1 μg/ml) for 8 h. Fluorescent neutrophils that migrated to the lower compartment were measured using a fluorescence spectrometer, and the chemotactic activity was expressed as the percentage of fluorescent cells in the lower chamber as compared to the fluorescence of the total neutrophils added. Error bars indicate the SD for three independent experiments. ∗∗∗ p

Techniques Used: Migration, Derivative Assay, Transfection, Fluorescence, Activity Assay

IFN-β activates the JAK/STAT signaling pathway in CF. (A–C) The upper panelshows a Western blot image, indicating the INF-β-induced p-STAT1, p-STAT2, and p-STAT3 expression levels; STAT1, STAT2, and STAT3 were used as loading controls. The graphical analysis is presented at the bottom of each panel. Error bars indicate theSD for four independent experiments. ∗∗∗ p
Figure Legend Snippet: IFN-β activates the JAK/STAT signaling pathway in CF. (A–C) The upper panelshows a Western blot image, indicating the INF-β-induced p-STAT1, p-STAT2, and p-STAT3 expression levels; STAT1, STAT2, and STAT3 were used as loading controls. The graphical analysis is presented at the bottom of each panel. Error bars indicate theSD for four independent experiments. ∗∗∗ p

Techniques Used: Western Blot, Expressing

15) Product Images from "Prostaglandin E2 and SOCS1 have a role in intestinal immune tolerance"

Article Title: Prostaglandin E2 and SOCS1 have a role in intestinal immune tolerance

Journal: Nature Communications

doi: 10.1038/ncomms1181

IFNγ antagonizes cAMP but not Treg-derived IL-10 in Socs1 −/− BMDCs. ( a ) Socs1 +/+ (5×10 5 cells per ml; black bars) and Socs1 −/− BMDCs (5×10 5 cells per ml; grey bars) were stimulated with LPS+IFNγ (10 ng ml −1 each) for 12 h in the presence or absence of graded concentrations of membrane-permeable cAMP analogue, 8-Br-cAMP. The levels of IL-12p70 and TNFα in the culture supernatant were measured by ELISA. ( b ) Socs1 +/+ (5×10 5 cells per ml; left half of each panel) and Socs1 −/− BMDCs (5×10 5 cells per ml; right half of each panel) were stimulated with LPS (10 ng ml −1 ) for 12 h with (white bars) or without (black bars) 100 μM of 8-Br-cAMP with graded concentrations of murine recombinant IFNγ. The levels of IL-12p70 and TNFα in the culture supernatant were measured by ELISA. IFNγ dose-dependently antagonized cAMP-mediated immunosuppression, which was enhanced by SOCS1 deficiency. ( c ) Stat1 +/+ (5×10 5 cells per ml; white bars) and Stat1 −/− (5×10 5 cells per ml; grey bars) BMDCs were stimulated with LPS (10 ng ml −1 ) for 24 h in the absence or presence of PGE2 and graded concentrations of murine recombinant IFNγ. The level of TNFα in the culture supernatant was measured by ELISA. ( d ) Socs1 +/+ and Socs1 −/− BMDCs (5×10 5 cells per ml) were co-cultured with 2.5×10 5 cells per ml of CD4 + CD25 high regulatory T cells in the presence of soluble anti-CD3 Ab (1 μg ml −1 ). Cells were stimulated with 10 ng ml −1 of LPS+IFNγ (10 ng ml −1 , each) for 12 h with or without anti-IL-10 neutralizing Ab (10 μg ml −1 ) in the presence or absence of membrane-permeable cAMP. The levels of IL-12p70 and TNFα in the culture supernatant were measured by ELISA. Data are representative of two ( b – d ) to three ( a ) independent experiments. Error bars represent +s.d. Cells were stimulated in triplicate wells for each condition and s.d. was calculated from the values determined by ELISA. ** P
Figure Legend Snippet: IFNγ antagonizes cAMP but not Treg-derived IL-10 in Socs1 −/− BMDCs. ( a ) Socs1 +/+ (5×10 5 cells per ml; black bars) and Socs1 −/− BMDCs (5×10 5 cells per ml; grey bars) were stimulated with LPS+IFNγ (10 ng ml −1 each) for 12 h in the presence or absence of graded concentrations of membrane-permeable cAMP analogue, 8-Br-cAMP. The levels of IL-12p70 and TNFα in the culture supernatant were measured by ELISA. ( b ) Socs1 +/+ (5×10 5 cells per ml; left half of each panel) and Socs1 −/− BMDCs (5×10 5 cells per ml; right half of each panel) were stimulated with LPS (10 ng ml −1 ) for 12 h with (white bars) or without (black bars) 100 μM of 8-Br-cAMP with graded concentrations of murine recombinant IFNγ. The levels of IL-12p70 and TNFα in the culture supernatant were measured by ELISA. IFNγ dose-dependently antagonized cAMP-mediated immunosuppression, which was enhanced by SOCS1 deficiency. ( c ) Stat1 +/+ (5×10 5 cells per ml; white bars) and Stat1 −/− (5×10 5 cells per ml; grey bars) BMDCs were stimulated with LPS (10 ng ml −1 ) for 24 h in the absence or presence of PGE2 and graded concentrations of murine recombinant IFNγ. The level of TNFα in the culture supernatant was measured by ELISA. ( d ) Socs1 +/+ and Socs1 −/− BMDCs (5×10 5 cells per ml) were co-cultured with 2.5×10 5 cells per ml of CD4 + CD25 high regulatory T cells in the presence of soluble anti-CD3 Ab (1 μg ml −1 ). Cells were stimulated with 10 ng ml −1 of LPS+IFNγ (10 ng ml −1 , each) for 12 h with or without anti-IL-10 neutralizing Ab (10 μg ml −1 ) in the presence or absence of membrane-permeable cAMP. The levels of IL-12p70 and TNFα in the culture supernatant were measured by ELISA. Data are representative of two ( b – d ) to three ( a ) independent experiments. Error bars represent +s.d. Cells were stimulated in triplicate wells for each condition and s.d. was calculated from the values determined by ELISA. ** P

Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Cell Culture

16) Product Images from "Inhibition of Alpha Interferon Signaling by Hepatitis B Virus ▿"

Article Title: Inhibition of Alpha Interferon Signaling by Hepatitis B Virus ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01292-06

(A) STAT1 methylation (asterisk), as detected by an immunoprecipitation ( ip ) with antibodies to monomethyl- and dimethylarginine and Western blotting with STAT1 antibodies, is impaired in cells expressing viral proteins (lane 2) compared to that in Huh7 cells (lane 1). (B) Binding of PIAS1 to STAT1, as detected by immunoprecipitation ( ip ) with STAT1 antibodies and Western blotting with PIAS1 antibodies in Huh7.93 cells, is enhanced (lane 2) compared to that in Huh7 cells (lane 1). Densitometric analysis of the PIAS1 signals is shown in the lower panel. The values are the integrated densities measured with NIH Image software and expressed as arbitrary units. (C) Reduced IFN-α target gene induction in the presence of HBV proteins. Huh7 and Huh7.93 cells were stimulated with human IFN-α (1,000 U/ml) for 6 h. The amount of the interferon target gene IP10 was measured with real-time RT-PCR in three independent samples (each sample was measured in duplicate). The induction of IP10 mRNA was calculated as severalfold increase of the mRNA amounts in IFN-α-treated samples versus that in untreated samples. Shown are the mean values and the standard errors of the means (error bars). The P value was obtained using the analysis of variance test. The transcriptional induction of IP10 is inhibited in Huh7.93 cells (right bar) compared to that in the parental cell line Huh7 (left bar).
Figure Legend Snippet: (A) STAT1 methylation (asterisk), as detected by an immunoprecipitation ( ip ) with antibodies to monomethyl- and dimethylarginine and Western blotting with STAT1 antibodies, is impaired in cells expressing viral proteins (lane 2) compared to that in Huh7 cells (lane 1). (B) Binding of PIAS1 to STAT1, as detected by immunoprecipitation ( ip ) with STAT1 antibodies and Western blotting with PIAS1 antibodies in Huh7.93 cells, is enhanced (lane 2) compared to that in Huh7 cells (lane 1). Densitometric analysis of the PIAS1 signals is shown in the lower panel. The values are the integrated densities measured with NIH Image software and expressed as arbitrary units. (C) Reduced IFN-α target gene induction in the presence of HBV proteins. Huh7 and Huh7.93 cells were stimulated with human IFN-α (1,000 U/ml) for 6 h. The amount of the interferon target gene IP10 was measured with real-time RT-PCR in three independent samples (each sample was measured in duplicate). The induction of IP10 mRNA was calculated as severalfold increase of the mRNA amounts in IFN-α-treated samples versus that in untreated samples. Shown are the mean values and the standard errors of the means (error bars). The P value was obtained using the analysis of variance test. The transcriptional induction of IP10 is inhibited in Huh7.93 cells (right bar) compared to that in the parental cell line Huh7 (left bar).

Techniques Used: Methylation, Immunoprecipitation, Western Blot, Expressing, Binding Assay, Software, Quantitative RT-PCR

(A) IFN-α-induced binding of activated STAT1 is impaired in Huh7.93 cells compared to that in the parental cell lines Huh7 and H7TA-61. Shown are EMSA results using the SIE-m67 oligonucleotide probe. Cells were left untreated (−) (lanes 1, 3, 6, and 8) or they were treated (+) for 20 min with 1,000 U/ml human IFN-α (hIFN-α) (lanes 2, 4, 5, 7, and 9). In lane 5, the nuclear extract of Huh7 (hIFN-α treated) was incubated with anti-STAT1 antibody prior to the binding reaction to perform a supershift (asterisk). (B) Huh7.93 cells were cultured in medium with (+) or without (−) Dox as indicated. Cells were then stimulated for 20 min with 1,000 U/ml hIFN-α. Nuclear extracts were analyzed with EMSA with SIE-m67. No further decrease in signal intensity was observed in derepressed cells. (C) Cytoplasmatic extracts were used to perform Western blot analysis. There is no difference in the phosphorylation of STAT1 on tyrosine 701 between the control cells and the HBV-expressing cells (upper part). The membrane was reblotted for STAT 1 as a loading control (lower part). +, with; −, without.
Figure Legend Snippet: (A) IFN-α-induced binding of activated STAT1 is impaired in Huh7.93 cells compared to that in the parental cell lines Huh7 and H7TA-61. Shown are EMSA results using the SIE-m67 oligonucleotide probe. Cells were left untreated (−) (lanes 1, 3, 6, and 8) or they were treated (+) for 20 min with 1,000 U/ml human IFN-α (hIFN-α) (lanes 2, 4, 5, 7, and 9). In lane 5, the nuclear extract of Huh7 (hIFN-α treated) was incubated with anti-STAT1 antibody prior to the binding reaction to perform a supershift (asterisk). (B) Huh7.93 cells were cultured in medium with (+) or without (−) Dox as indicated. Cells were then stimulated for 20 min with 1,000 U/ml hIFN-α. Nuclear extracts were analyzed with EMSA with SIE-m67. No further decrease in signal intensity was observed in derepressed cells. (C) Cytoplasmatic extracts were used to perform Western blot analysis. There is no difference in the phosphorylation of STAT1 on tyrosine 701 between the control cells and the HBV-expressing cells (upper part). The membrane was reblotted for STAT 1 as a loading control (lower part). +, with; −, without.

Techniques Used: Binding Assay, Incubation, Cell Culture, Western Blot, Expressing

17) Product Images from "IFNα enhances the production of IL-6 by human neutrophils activated via TLR8"

Article Title: IFNα enhances the production of IL-6 by human neutrophils activated via TLR8

Journal: Scientific Reports

doi: 10.1038/srep19674

Effect of IFNα on Pol II, STAT1 and C/EBPβ recruitment to the IL-6 promoter in R848-treated neutrophils. (a) Neutrophils (5 × 10 6 /ml), isolated from the peripheral blood of healthy donors, were cultured with or without 5 μM R848, 1000 U/ml IFNα or IFNα plus R848 for 6, 12 and 20 h to evaluate IL-6 primary transcript (PT) expression by RT-qPCR (n = 3–8). Asterisks indicate a significant increase: **p
Figure Legend Snippet: Effect of IFNα on Pol II, STAT1 and C/EBPβ recruitment to the IL-6 promoter in R848-treated neutrophils. (a) Neutrophils (5 × 10 6 /ml), isolated from the peripheral blood of healthy donors, were cultured with or without 5 μM R848, 1000 U/ml IFNα or IFNα plus R848 for 6, 12 and 20 h to evaluate IL-6 primary transcript (PT) expression by RT-qPCR (n = 3–8). Asterisks indicate a significant increase: **p

Techniques Used: Isolation, Cell Culture, Expressing, Quantitative RT-PCR

18) Product Images from "Double-Stranded RNA Induces Biphasic STAT1 Phosphorylation by both Type I Interferon (IFN)-Dependent and Type I IFN-Independent Pathways"

Article Title: Double-Stranded RNA Induces Biphasic STAT1 Phosphorylation by both Type I Interferon (IFN)-Dependent and Type I IFN-Independent Pathways

Journal: Journal of Virology

doi: 10.1128/JVI.01881-12

Influence of a neutralizing IFN receptor antibody on STAT1 phosphorylation in response to poly(I:C). (A) Following pretreatment with the anti-IFNAR neutralizing antibody (2.5 μg/well) for 1 h, A549 cells were transfected with poly(I:C) (200 ng)
Figure Legend Snippet: Influence of a neutralizing IFN receptor antibody on STAT1 phosphorylation in response to poly(I:C). (A) Following pretreatment with the anti-IFNAR neutralizing antibody (2.5 μg/well) for 1 h, A549 cells were transfected with poly(I:C) (200 ng)

Techniques Used: Transfection

Effect of MAVS on RIG-I-CARD-induced STAT1 phosphorylation. Either stably MAVS-silenced 293-flp cells (MAVS) or a negative control, lacZ -silenced cells (Lac), were transfected with RIG-I-CARD (CARD) or an empty vector (mock) for the indicated periods.
Figure Legend Snippet: Effect of MAVS on RIG-I-CARD-induced STAT1 phosphorylation. Either stably MAVS-silenced 293-flp cells (MAVS) or a negative control, lacZ -silenced cells (Lac), were transfected with RIG-I-CARD (CARD) or an empty vector (mock) for the indicated periods.

Techniques Used: Stable Transfection, Negative Control, Transfection, Plasmid Preparation

Mechanism of dsRNA-mediated STAT1 phosphorylation. (A) Our results indicate that the initial phosphorylation of STAT1 is RIG-I-MAVS dependent. MDA-5 is not involved in the initial dsRNA-induced STAT1 phosphorylation. dsRNA-induced type I IFN is essential
Figure Legend Snippet: Mechanism of dsRNA-mediated STAT1 phosphorylation. (A) Our results indicate that the initial phosphorylation of STAT1 is RIG-I-MAVS dependent. MDA-5 is not involved in the initial dsRNA-induced STAT1 phosphorylation. dsRNA-induced type I IFN is essential

Techniques Used: Multiple Displacement Amplification

Involvement of RIG-I, MDA-5, and MAVS in STAT1 phosphorylation in response to poly(I:C). A549 cells were transfected with control siRNA or gene-specific siRNAs against RIG-I, MDA-5, or MAVS for 48 h. After the incubation, the cells were transfected with
Figure Legend Snippet: Involvement of RIG-I, MDA-5, and MAVS in STAT1 phosphorylation in response to poly(I:C). A549 cells were transfected with control siRNA or gene-specific siRNAs against RIG-I, MDA-5, or MAVS for 48 h. After the incubation, the cells were transfected with

Techniques Used: Multiple Displacement Amplification, Transfection, Incubation

Influence of the IFN receptor on ISG induction. (A) U5A cells were transfected with poly(I:C) (200 ng) for 8 h or treated with IFN-γ (5 ng/ml) for 30 min, followed by fixation with 4% paraformaldehyde. STAT1 (green) and DAPI (blue; nuclei) stains
Figure Legend Snippet: Influence of the IFN receptor on ISG induction. (A) U5A cells were transfected with poly(I:C) (200 ng) for 8 h or treated with IFN-γ (5 ng/ml) for 30 min, followed by fixation with 4% paraformaldehyde. STAT1 (green) and DAPI (blue; nuclei) stains

Techniques Used: Transfection

Kinetics of IFN-β production and STAT1 phosphorylation in response to poly(I:C) transfection. (A) A549 cells were transfected with poly(I:C) (200 ng), and the levels of IFN-β mRNA were determined by quantitative real-time PCR. Glyceraldehyde-3-phosphate
Figure Legend Snippet: Kinetics of IFN-β production and STAT1 phosphorylation in response to poly(I:C) transfection. (A) A549 cells were transfected with poly(I:C) (200 ng), and the levels of IFN-β mRNA were determined by quantitative real-time PCR. Glyceraldehyde-3-phosphate

Techniques Used: Transfection, Real-time Polymerase Chain Reaction

Effect of RIG-CARD on STAT1 phosphorylation in U5A cells. (A) The cells were transfected with either an empty control vector (mock) or a vector encoding the CARD of RIG-I for 24 h. The levels of pSTAT1, STAT1, and FLAG-RIG-CARD were analyzed by immunoblotting.
Figure Legend Snippet: Effect of RIG-CARD on STAT1 phosphorylation in U5A cells. (A) The cells were transfected with either an empty control vector (mock) or a vector encoding the CARD of RIG-I for 24 h. The levels of pSTAT1, STAT1, and FLAG-RIG-CARD were analyzed by immunoblotting.

Techniques Used: Transfection, Plasmid Preparation

dsRNA can induce STAT1 phosphorylation in IFNAR-deficient cells. (A) Characterization of U5A cells. A549, 2fTGH, and U5A cells were treated with IFN-α2b (α2b; 200 pg/ml) or IFN-γ (γ; 5 ng/ml) for 30 min or left untreated
Figure Legend Snippet: dsRNA can induce STAT1 phosphorylation in IFNAR-deficient cells. (A) Characterization of U5A cells. A549, 2fTGH, and U5A cells were treated with IFN-α2b (α2b; 200 pg/ml) or IFN-γ (γ; 5 ng/ml) for 30 min or left untreated

Techniques Used:

Time course of STAT1 phosphorylation following the transfection of A549 cells with vectors expressing RIG-I constructs. (A) A549 cells were transfected with FLC-RIG-I, RIG-I-CARD, or an empty control vector for up to 8 h. (B) A549 cells were transfected
Figure Legend Snippet: Time course of STAT1 phosphorylation following the transfection of A549 cells with vectors expressing RIG-I constructs. (A) A549 cells were transfected with FLC-RIG-I, RIG-I-CARD, or an empty control vector for up to 8 h. (B) A549 cells were transfected

Techniques Used: Transfection, Expressing, Construct, Plasmid Preparation

Effect of an anti-IFNAR neutralizing antibody on RIG-I-CARD-induced STAT1 phosphorylation. Either stably MAVS-silenced 293-flp cells (M) or lacZ -silenced cells (L) were transfected with FLAG-RIG-I-CARD or an empty vector (mock) for the indicated periods
Figure Legend Snippet: Effect of an anti-IFNAR neutralizing antibody on RIG-I-CARD-induced STAT1 phosphorylation. Either stably MAVS-silenced 293-flp cells (M) or lacZ -silenced cells (L) were transfected with FLAG-RIG-I-CARD or an empty vector (mock) for the indicated periods

Techniques Used: Stable Transfection, Transfection, Plasmid Preparation

19) Product Images from "High Resistance of Human Parainfluenza Type 2 Virus Protein-Expressing Cells to the Antiviral and Anti-Cell Proliferative Activities of Alpha/Beta Interferons: Cysteine-Rich V-Specific Domain Is Required for High Resistance to the Interferons"

Article Title: High Resistance of Human Parainfluenza Type 2 Virus Protein-Expressing Cells to the Antiviral and Anti-Cell Proliferative Activities of Alpha/Beta Interferons: Cysteine-Rich V-Specific Domain Is Required for High Resistance to the Interferons

Journal: Journal of Virology

doi: 10.1128/JVI.75.19.9165-9176.2001

Effects of proteasome inhibitors on the expression of Stat2 in HeLa-V and HeLa-SV41V cells. (A) HeLa-V cells were incubated with 0 (0.3% dimethyl sulfoxide), 10, 30, or 90 μM MG132. After 3, 6, 9, or 18 h, the expression of Stat2 was analyzed by Western blotting (ECL). (B and C) HeLa-V (B) and HeLa-SV41V cells (C) were preincubated without or with the proteasome inhibitors MG132 (M; 10 μM) and lactacystin (L; 10 μM) for 3 h. Subsequently, hIFN-α, -β, or -γ (10 3 U) was added to the culture fluids of these cells. After 15 h, the expression of Stat1 and Stat2 was analyzed by Western blotting (ECL).
Figure Legend Snippet: Effects of proteasome inhibitors on the expression of Stat2 in HeLa-V and HeLa-SV41V cells. (A) HeLa-V cells were incubated with 0 (0.3% dimethyl sulfoxide), 10, 30, or 90 μM MG132. After 3, 6, 9, or 18 h, the expression of Stat2 was analyzed by Western blotting (ECL). (B and C) HeLa-V (B) and HeLa-SV41V cells (C) were preincubated without or with the proteasome inhibitors MG132 (M; 10 μM) and lactacystin (L; 10 μM) for 3 h. Subsequently, hIFN-α, -β, or -γ (10 3 U) was added to the culture fluids of these cells. After 15 h, the expression of Stat1 and Stat2 was analyzed by Western blotting (ECL).

Techniques Used: Expressing, Incubation, Western Blot

Expression of Stat1 and Stat2 in HeLa-CA, HeLa-P, HeLa-V, and HeLa-SV41V cells. HeLa (A), HeLa-CA (A), HeLa-P (B), HeLa-V (B) and HeLa-SV41V cells (C) were treated for 15 h without or with hIFN-α (10 3 and 10 4 U), hIFN-β (10 3 and 10 4 U) or hIFN-γ (10 3 U). Stat1 and Stat2 were detected by Western blotting. (D and E) Low labeling of Stat2 in HeLa-V cells with [ 35 S]methionine. HeLa, HeLa-P, and HeLa-V cells were cultured without or with IFN-β (10 3 U) for 15 h, and then the cells were labeled with [ 35 S]methionine (500 μCi/ml) for 30 min. The cell lysates were analyzed by immunoprecipitation using anti-Stat2 MAb (D), anti-Stat2 polyclonal antibody (E), or anti-Stat1 MAb (E) and SDS-PAGE.
Figure Legend Snippet: Expression of Stat1 and Stat2 in HeLa-CA, HeLa-P, HeLa-V, and HeLa-SV41V cells. HeLa (A), HeLa-CA (A), HeLa-P (B), HeLa-V (B) and HeLa-SV41V cells (C) were treated for 15 h without or with hIFN-α (10 3 and 10 4 U), hIFN-β (10 3 and 10 4 U) or hIFN-γ (10 3 U). Stat1 and Stat2 were detected by Western blotting. (D and E) Low labeling of Stat2 in HeLa-V cells with [ 35 S]methionine. HeLa, HeLa-P, and HeLa-V cells were cultured without or with IFN-β (10 3 U) for 15 h, and then the cells were labeled with [ 35 S]methionine (500 μCi/ml) for 30 min. The cell lysates were analyzed by immunoprecipitation using anti-Stat2 MAb (D), anti-Stat2 polyclonal antibody (E), or anti-Stat1 MAb (E) and SDS-PAGE.

Techniques Used: Expressing, Western Blot, Labeling, Cell Culture, Immunoprecipitation, SDS Page

(A) Presence of Stat2 mRNA in HeLa-V cells. HeLa, HeLa-P, and HeLa-V cells were cultured for 15 h, and then mRNAs were isolated. RT-PCR was carried out for the detection of Stat2 mRNA using total RNA (1 μg) isolated from HeLa, HeLa-P, and HeLa-V cells. (B) Presence of Stat2 mRNA in the cytoplasm of HeLa-V cells. HeLa-V, HeLa-P, and HeLa-SV41V cells were cultured for 15 h, and then mRNAs were isolated from cytoplasmic fractions of these cells. RT-PCR (30 cycles) was carried out for the detection of Stat2 mRNA using total RNA (1 μg). (C) Purification of hPIV-2 P and V proteins: Recombinantly expressed P and V proteins of hPIV-2 were analyzed by SDS–10% PAGE and then stained with Coomassie brilliant blue solution. M, marker proteins. (D) Suppression of in vitro translation of Stat2 mRNA by the V protein. Full-length luciferase, Stat1, or Stat2 mRNA was synthesized in vitro by using T7 polymerase. Luciferase cDNA and an in vitro translation kit were used. In vitro translation was carried out using a TNT Quick coupled transcription-translation system (Promega). The reaction (final volume, 50 μl) was performed in the absence (lane 2) or presence of purified P protein (lane 3, 2 μg/ml; lane 4, 0.6 μg/ml) or purified V protein (lane 5, 2 μg/ml; lane 6, 0.6 μg/ml) with [ 35 S]methionine (20 μCi). Lane 1, reaction without template. The products were analyzed by SDS–10% PAGE. (E) Suppression of in vitro translation of Stat2 mRNA by V protein. The specificity of the product was checked by Western blot using specific antibody. The reaction was performed in the absence (lane 2) or presence of purified P protein (lane 3, 20 μg/ml; lane 4, 2 μg/ml) or purified V protein of hPIV2 (lane 5, 20 μg/ml; lane 6, 2 μg/ml). Lane 1, reaction without template. The products were analyzed with Western blotting using anti-Stat2 MAb. (F) Absence of degradation of Stat2 incubated with the purified V protein: In vitro-translated and radioisotope-labeled Stat1 or Stat2 was incubated with either purified P or V protein (20 or 2 μg/ml) for 90 min at 30C. Subsequently, the samples were analyzed by SDS–10% PAGE.
Figure Legend Snippet: (A) Presence of Stat2 mRNA in HeLa-V cells. HeLa, HeLa-P, and HeLa-V cells were cultured for 15 h, and then mRNAs were isolated. RT-PCR was carried out for the detection of Stat2 mRNA using total RNA (1 μg) isolated from HeLa, HeLa-P, and HeLa-V cells. (B) Presence of Stat2 mRNA in the cytoplasm of HeLa-V cells. HeLa-V, HeLa-P, and HeLa-SV41V cells were cultured for 15 h, and then mRNAs were isolated from cytoplasmic fractions of these cells. RT-PCR (30 cycles) was carried out for the detection of Stat2 mRNA using total RNA (1 μg). (C) Purification of hPIV-2 P and V proteins: Recombinantly expressed P and V proteins of hPIV-2 were analyzed by SDS–10% PAGE and then stained with Coomassie brilliant blue solution. M, marker proteins. (D) Suppression of in vitro translation of Stat2 mRNA by the V protein. Full-length luciferase, Stat1, or Stat2 mRNA was synthesized in vitro by using T7 polymerase. Luciferase cDNA and an in vitro translation kit were used. In vitro translation was carried out using a TNT Quick coupled transcription-translation system (Promega). The reaction (final volume, 50 μl) was performed in the absence (lane 2) or presence of purified P protein (lane 3, 2 μg/ml; lane 4, 0.6 μg/ml) or purified V protein (lane 5, 2 μg/ml; lane 6, 0.6 μg/ml) with [ 35 S]methionine (20 μCi). Lane 1, reaction without template. The products were analyzed by SDS–10% PAGE. (E) Suppression of in vitro translation of Stat2 mRNA by V protein. The specificity of the product was checked by Western blot using specific antibody. The reaction was performed in the absence (lane 2) or presence of purified P protein (lane 3, 20 μg/ml; lane 4, 2 μg/ml) or purified V protein of hPIV2 (lane 5, 20 μg/ml; lane 6, 2 μg/ml). Lane 1, reaction without template. The products were analyzed with Western blotting using anti-Stat2 MAb. (F) Absence of degradation of Stat2 incubated with the purified V protein: In vitro-translated and radioisotope-labeled Stat1 or Stat2 was incubated with either purified P or V protein (20 or 2 μg/ml) for 90 min at 30C. Subsequently, the samples were analyzed by SDS–10% PAGE.

Techniques Used: Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Purification, Polyacrylamide Gel Electrophoresis, Staining, Marker, In Vitro, Luciferase, Synthesized, Western Blot, Incubation, Labeling

Effects of hPIV-2 infection of Stat2 degradation. (A) HeLa cells were preinfected or infected with hPIV-2 (CA strain) at an MOI of 5 for 30 min or 1, 2, 4, or 8 h, and then expression of Stat1 and Stat2 was analyzed by Western blotting. (B) Plot of the NIH Image analysis of the Stat2 bands in panel A. Data are expressed as percentages of the baseline (the value in the uninfected-cell lane). (C) Pulse-chase experiments with HeLa cells infected with hPIV-2 (CA strain). HeLa cells were labeled with [ 35 S]methionine (250 μCi/ml) for 2 h. Label was removed, and the cells were washed in normal medium and chased in the presence of MEM supplemented with 250 mM methionine for various times without or with hPIV-2 infection (MOI, 5). The cell lysates were analyzed by immunoprecipitation using anti-Stat2 MAb and SDS-PAGE. (D and E) Plot of the NIH Image analysis of the Stat2 bands in panel C. Data are expressed as percentages of the baseline (the value in the prechase lane [D] or the value in the 30-min chase lane [E]).
Figure Legend Snippet: Effects of hPIV-2 infection of Stat2 degradation. (A) HeLa cells were preinfected or infected with hPIV-2 (CA strain) at an MOI of 5 for 30 min or 1, 2, 4, or 8 h, and then expression of Stat1 and Stat2 was analyzed by Western blotting. (B) Plot of the NIH Image analysis of the Stat2 bands in panel A. Data are expressed as percentages of the baseline (the value in the uninfected-cell lane). (C) Pulse-chase experiments with HeLa cells infected with hPIV-2 (CA strain). HeLa cells were labeled with [ 35 S]methionine (250 μCi/ml) for 2 h. Label was removed, and the cells were washed in normal medium and chased in the presence of MEM supplemented with 250 mM methionine for various times without or with hPIV-2 infection (MOI, 5). The cell lysates were analyzed by immunoprecipitation using anti-Stat2 MAb and SDS-PAGE. (D and E) Plot of the NIH Image analysis of the Stat2 bands in panel C. Data are expressed as percentages of the baseline (the value in the prechase lane [D] or the value in the 30-min chase lane [E]).

Techniques Used: Infection, Expressing, Western Blot, Pulse Chase, Labeling, Immunoprecipitation, SDS Page

20) Product Images from "Oncostatin M Regulates Secretoglobin 3A1 and 3A2 Expression in a Bidirectional Manner"

Article Title: Oncostatin M Regulates Secretoglobin 3A1 and 3A2 Expression in a Bidirectional Manner

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2008-0062OC

Analysis of OSM signaling molecules. ( A ) RT-PCR analysis for the presence of OSM (OSMR) and gp130 receptors in mtCC cells. The size of PCR product is 511 bp for OSMR and 325 bp for gp130 with the primers used. RT(−) is a negative control. RNAs prepared from fetal lungs were used as a positive control. PCR product for OSMR using lung RNA without RT reaction (lung, RT−) was run on a separate gel, and the image was attached to those showing other PCR products. ( B ) mtCC cells were subjected to flow cytometry analysis for the presence of OSM receptor on the cell surface. The solid area was obtained with mtCC cells alone. The gray line represents mtCC cells incubated with anti-OSMR and anti-goat IgG-PE. Note that all cells are positive for OSM receptor. ( C ) Activation of STATs in mtCC cells by OSM. mtCC cells were treated with 100 ng/ml OSM for 5 minutes, and whole cell lysates were subjected to Western blot analysis using antibody against STAT1, phospho (p)STAT1, STAT3, pSTAT3, STAT5, and pSTAT5 with (+) and without (−) OSM.
Figure Legend Snippet: Analysis of OSM signaling molecules. ( A ) RT-PCR analysis for the presence of OSM (OSMR) and gp130 receptors in mtCC cells. The size of PCR product is 511 bp for OSMR and 325 bp for gp130 with the primers used. RT(−) is a negative control. RNAs prepared from fetal lungs were used as a positive control. PCR product for OSMR using lung RNA without RT reaction (lung, RT−) was run on a separate gel, and the image was attached to those showing other PCR products. ( B ) mtCC cells were subjected to flow cytometry analysis for the presence of OSM receptor on the cell surface. The solid area was obtained with mtCC cells alone. The gray line represents mtCC cells incubated with anti-OSMR and anti-goat IgG-PE. Note that all cells are positive for OSM receptor. ( C ) Activation of STATs in mtCC cells by OSM. mtCC cells were treated with 100 ng/ml OSM for 5 minutes, and whole cell lysates were subjected to Western blot analysis using antibody against STAT1, phospho (p)STAT1, STAT3, pSTAT3, STAT5, and pSTAT5 with (+) and without (−) OSM.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Positive Control, Flow Cytometry, Cytometry, Incubation, Activation Assay, Western Blot

Binding of STATs to the proximal SBE of Scgb3a1 promoter. Electrophoretic mobility shift analysis was performed using a probe containing the proximal SBE and nuclear extracts prepared from mtCC ( A ) and NIH3T3 cells ( B ) that had been treated with 100 ng/ml of OSM for 30 minutes with and without addition of various anti-STAT antibodies as indicated. IFN-γ–treated nuclear extract was used to demonstrate the position of STAT1-specific band ( A , left panel , lane 2 , indicated by an arrow ). The band(s) produced by OSM treatment are indicated by an asterisk in the left panel and an arrow in the right panel in A . In B , the bands corresponding to STAT1, STAT3, and STAT5 are indicated on the right .
Figure Legend Snippet: Binding of STATs to the proximal SBE of Scgb3a1 promoter. Electrophoretic mobility shift analysis was performed using a probe containing the proximal SBE and nuclear extracts prepared from mtCC ( A ) and NIH3T3 cells ( B ) that had been treated with 100 ng/ml of OSM for 30 minutes with and without addition of various anti-STAT antibodies as indicated. IFN-γ–treated nuclear extract was used to demonstrate the position of STAT1-specific band ( A , left panel , lane 2 , indicated by an arrow ). The band(s) produced by OSM treatment are indicated by an asterisk in the left panel and an arrow in the right panel in A . In B , the bands corresponding to STAT1, STAT3, and STAT5 are indicated on the right .

Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Produced

21) Product Images from "The C Proteins of Human Parainfluenza Virus Type 1 Block IFN Signaling by Binding and Retaining Stat1 in Perinuclear Aggregates at the Late Endosome"

Article Title: The C Proteins of Human Parainfluenza Virus Type 1 Block IFN Signaling by Binding and Retaining Stat1 in Perinuclear Aggregates at the Late Endosome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028382

Co-localization of Stat1 and mannose6-phosphate receptor in Vero cells. Vero cells were treated as described for Figure 6 . Cells were stained for Stat1 (red) and M6PR (green). Z-stacks of Figure 8 are shown in the Videos S9 , S10 , S11 , S12 .
Figure Legend Snippet: Co-localization of Stat1 and mannose6-phosphate receptor in Vero cells. Vero cells were treated as described for Figure 6 . Cells were stained for Stat1 (red) and M6PR (green). Z-stacks of Figure 8 are shown in the Videos S9 , S10 , S11 , S12 .

Techniques Used: Staining

Co-localization of Stat1 and HPIV1 C proteins in Vero cells. Vero cells were mock-infected or infected with WT or F170S HPIV1 at an MOI of 1 TCID 50 /cell. After 48 h, cells were mock-treated (−IFN) or treated with 1000 IU/ml of IFN-β (+IFN) for 30 min. Cells were subsequently fixed, permeabilized, and stained for HPIV1 C proteins (red) and endogenous Stat1 protein (green). Z-stacks of Figure 6 are shown in the Videos S1 , S2 , S3 , S4 .
Figure Legend Snippet: Co-localization of Stat1 and HPIV1 C proteins in Vero cells. Vero cells were mock-infected or infected with WT or F170S HPIV1 at an MOI of 1 TCID 50 /cell. After 48 h, cells were mock-treated (−IFN) or treated with 1000 IU/ml of IFN-β (+IFN) for 30 min. Cells were subsequently fixed, permeabilized, and stained for HPIV1 C proteins (red) and endogenous Stat1 protein (green). Z-stacks of Figure 6 are shown in the Videos S1 , S2 , S3 , S4 .

Techniques Used: Infection, Staining

Co-immunoprecipitation of WT HPIV1 C protein and Stat1. 293 T cells were transfected with pcDNA3.1(+) plasmids expressing myc-tagged C′ WT or C′ F170S protein, or untagged CAT as a negative control. After 48 h, cells were mock-treated (IFN−) or treated (IFN+) with 1000 IU/ml of IFN-β for 30 min. Cell lysates were subjected to immunoprecipitation with anti-myc antibodies. Whole cell lysates and precipitates were separated on SDS-PAGE gels and analyzed by Western blot with antibodies against pStat1, Stat1, or the C protein, as indicated at the left. The experiment was carried out three times with comparable outcomes.
Figure Legend Snippet: Co-immunoprecipitation of WT HPIV1 C protein and Stat1. 293 T cells were transfected with pcDNA3.1(+) plasmids expressing myc-tagged C′ WT or C′ F170S protein, or untagged CAT as a negative control. After 48 h, cells were mock-treated (IFN−) or treated (IFN+) with 1000 IU/ml of IFN-β for 30 min. Cell lysates were subjected to immunoprecipitation with anti-myc antibodies. Whole cell lysates and precipitates were separated on SDS-PAGE gels and analyzed by Western blot with antibodies against pStat1, Stat1, or the C protein, as indicated at the left. The experiment was carried out three times with comparable outcomes.

Techniques Used: Immunoprecipitation, Transfection, Expressing, Negative Control, SDS Page, Western Blot

Intracellular localization of Stat2 in WT or F170S HPIV1-infected Vero cells following IFN treatment. Cells were infected and analyzed as described in the legend to Figure 3 except that the antibodies against Stat1 were replaced with antibodies against Stat2 (red). Representative fields are shown. Overall, 2% of the WT HPIV1-infected cells and 100% of the F170S HPIV1-infected cells showed nuclear Stat1 following IFN-β treatment.
Figure Legend Snippet: Intracellular localization of Stat2 in WT or F170S HPIV1-infected Vero cells following IFN treatment. Cells were infected and analyzed as described in the legend to Figure 3 except that the antibodies against Stat1 were replaced with antibodies against Stat2 (red). Representative fields are shown. Overall, 2% of the WT HPIV1-infected cells and 100% of the F170S HPIV1-infected cells showed nuclear Stat1 following IFN-β treatment.

Techniques Used: Infection

Intracellular localization of Stat1 in WT or F170S HPIV1-infected Vero cells following IFN treatment. Vero cells were mock-infected or infected with WT or F170S HPIV1 at an MOI of 1 TCID 50 /cell, and 48 h later were mock-treated (-IFN) or treated (+IFN) with 1000 IU/ml of IFN-β for 1 h. Cells were fixed, permeabilized, immunostained with antibodies for HPIV1 surface proteins (green) and Stat1 (red), stained with DAPI to visualize nuclei (blue), and analyzed by confocal microscopy. Representative fields are shown. Overall, 2% of the WT HPIV1-infected cells and 82% of the F170S HPIV1-infected cells showed nuclear Stat1 following IFN-β treatment.
Figure Legend Snippet: Intracellular localization of Stat1 in WT or F170S HPIV1-infected Vero cells following IFN treatment. Vero cells were mock-infected or infected with WT or F170S HPIV1 at an MOI of 1 TCID 50 /cell, and 48 h later were mock-treated (-IFN) or treated (+IFN) with 1000 IU/ml of IFN-β for 1 h. Cells were fixed, permeabilized, immunostained with antibodies for HPIV1 surface proteins (green) and Stat1 (red), stained with DAPI to visualize nuclei (blue), and analyzed by confocal microscopy. Representative fields are shown. Overall, 2% of the WT HPIV1-infected cells and 82% of the F170S HPIV1-infected cells showed nuclear Stat1 following IFN-β treatment.

Techniques Used: Infection, Staining, Confocal Microscopy

Western blot of total and phosphorylated Stat1 and Stat2 in WT or F170S HPIV1-infected Vero cells following treatment with IFN-α, -β, or -γ. Vero cells were mock-infected or infected with WT HPIV1, F170S HPIV1, or HPIV2 at an MOI of 5 TCID 50 /cell. After 48 h, cells were mock-treated or treated for 30 min with 1000 IU/ml of the indicated IFN. A) Western blots were probed for total or phosphorylated (p)Stat1 and Stat2, as well as for the HPIV1 C protein and HPIV2 P protein. Alpha-tubulin was used as loading control. B) Extended exposure (over night) of the top panel in Figure 2A [“pStat1 (Tyr701)”], showing that a low level of pStat1 is detected in cells infected with WT HPIV1 in the absence of IFN treatment.
Figure Legend Snippet: Western blot of total and phosphorylated Stat1 and Stat2 in WT or F170S HPIV1-infected Vero cells following treatment with IFN-α, -β, or -γ. Vero cells were mock-infected or infected with WT HPIV1, F170S HPIV1, or HPIV2 at an MOI of 5 TCID 50 /cell. After 48 h, cells were mock-treated or treated for 30 min with 1000 IU/ml of the indicated IFN. A) Western blots were probed for total or phosphorylated (p)Stat1 and Stat2, as well as for the HPIV1 C protein and HPIV2 P protein. Alpha-tubulin was used as loading control. B) Extended exposure (over night) of the top panel in Figure 2A [“pStat1 (Tyr701)”], showing that a low level of pStat1 is detected in cells infected with WT HPIV1 in the absence of IFN treatment.

Techniques Used: Western Blot, Infection

22) Product Images from "The C Proteins of Human Parainfluenza Virus Type 1 Block IFN Signaling by Binding and Retaining Stat1 in Perinuclear Aggregates at the Late Endosome"

Article Title: The C Proteins of Human Parainfluenza Virus Type 1 Block IFN Signaling by Binding and Retaining Stat1 in Perinuclear Aggregates at the Late Endosome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028382

Co-localization of Stat1 and mannose6-phosphate receptor in Vero cells. Vero cells were treated as described for Figure 6 . Cells were stained for Stat1 (red) and M6PR (green). Z-stacks of Figure 8 are shown in the Videos S9 , S10 , S11 , S12 .
Figure Legend Snippet: Co-localization of Stat1 and mannose6-phosphate receptor in Vero cells. Vero cells were treated as described for Figure 6 . Cells were stained for Stat1 (red) and M6PR (green). Z-stacks of Figure 8 are shown in the Videos S9 , S10 , S11 , S12 .

Techniques Used: Staining

Co-localization of Stat1 and HPIV1 C proteins in Vero cells. Vero cells were mock-infected or infected with WT or F170S HPIV1 at an MOI of 1 TCID 50 /cell. After 48 h, cells were mock-treated (−IFN) or treated with 1000 IU/ml of IFN-β (+IFN) for 30 min. Cells were subsequently fixed, permeabilized, and stained for HPIV1 C proteins (red) and endogenous Stat1 protein (green). Z-stacks of Figure 6 are shown in the Videos S1 , S2 , S3 , S4 .
Figure Legend Snippet: Co-localization of Stat1 and HPIV1 C proteins in Vero cells. Vero cells were mock-infected or infected with WT or F170S HPIV1 at an MOI of 1 TCID 50 /cell. After 48 h, cells were mock-treated (−IFN) or treated with 1000 IU/ml of IFN-β (+IFN) for 30 min. Cells were subsequently fixed, permeabilized, and stained for HPIV1 C proteins (red) and endogenous Stat1 protein (green). Z-stacks of Figure 6 are shown in the Videos S1 , S2 , S3 , S4 .

Techniques Used: Infection, Staining

Co-immunoprecipitation of WT HPIV1 C protein and Stat1. 293 T cells were transfected with pcDNA3.1(+) plasmids expressing myc-tagged C′ WT or C′ F170S protein, or untagged CAT as a negative control. After 48 h, cells were mock-treated (IFN−) or treated (IFN+) with 1000 IU/ml of IFN-β for 30 min. Cell lysates were subjected to immunoprecipitation with anti-myc antibodies. Whole cell lysates and precipitates were separated on SDS-PAGE gels and analyzed by Western blot with antibodies against pStat1, Stat1, or the C protein, as indicated at the left. The experiment was carried out three times with comparable outcomes.
Figure Legend Snippet: Co-immunoprecipitation of WT HPIV1 C protein and Stat1. 293 T cells were transfected with pcDNA3.1(+) plasmids expressing myc-tagged C′ WT or C′ F170S protein, or untagged CAT as a negative control. After 48 h, cells were mock-treated (IFN−) or treated (IFN+) with 1000 IU/ml of IFN-β for 30 min. Cell lysates were subjected to immunoprecipitation with anti-myc antibodies. Whole cell lysates and precipitates were separated on SDS-PAGE gels and analyzed by Western blot with antibodies against pStat1, Stat1, or the C protein, as indicated at the left. The experiment was carried out three times with comparable outcomes.

Techniques Used: Immunoprecipitation, Transfection, Expressing, Negative Control, SDS Page, Western Blot

Intracellular localization of Stat2 in WT or F170S HPIV1-infected Vero cells following IFN treatment. Cells were infected and analyzed as described in the legend to Figure 3 except that the antibodies against Stat1 were replaced with antibodies against Stat2 (red). Representative fields are shown. Overall, 2% of the WT HPIV1-infected cells and 100% of the F170S HPIV1-infected cells showed nuclear Stat1 following IFN-β treatment.
Figure Legend Snippet: Intracellular localization of Stat2 in WT or F170S HPIV1-infected Vero cells following IFN treatment. Cells were infected and analyzed as described in the legend to Figure 3 except that the antibodies against Stat1 were replaced with antibodies against Stat2 (red). Representative fields are shown. Overall, 2% of the WT HPIV1-infected cells and 100% of the F170S HPIV1-infected cells showed nuclear Stat1 following IFN-β treatment.

Techniques Used: Infection

Intracellular localization of Stat1 in WT or F170S HPIV1-infected Vero cells following IFN treatment. Vero cells were mock-infected or infected with WT or F170S HPIV1 at an MOI of 1 TCID 50 /cell, and 48 h later were mock-treated (-IFN) or treated (+IFN) with 1000 IU/ml of IFN-β for 1 h. Cells were fixed, permeabilized, immunostained with antibodies for HPIV1 surface proteins (green) and Stat1 (red), stained with DAPI to visualize nuclei (blue), and analyzed by confocal microscopy. Representative fields are shown. Overall, 2% of the WT HPIV1-infected cells and 82% of the F170S HPIV1-infected cells showed nuclear Stat1 following IFN-β treatment.
Figure Legend Snippet: Intracellular localization of Stat1 in WT or F170S HPIV1-infected Vero cells following IFN treatment. Vero cells were mock-infected or infected with WT or F170S HPIV1 at an MOI of 1 TCID 50 /cell, and 48 h later were mock-treated (-IFN) or treated (+IFN) with 1000 IU/ml of IFN-β for 1 h. Cells were fixed, permeabilized, immunostained with antibodies for HPIV1 surface proteins (green) and Stat1 (red), stained with DAPI to visualize nuclei (blue), and analyzed by confocal microscopy. Representative fields are shown. Overall, 2% of the WT HPIV1-infected cells and 82% of the F170S HPIV1-infected cells showed nuclear Stat1 following IFN-β treatment.

Techniques Used: Infection, Staining, Confocal Microscopy

Western blot of total and phosphorylated Stat1 and Stat2 in WT or F170S HPIV1-infected Vero cells following treatment with IFN-α, -β, or -γ. Vero cells were mock-infected or infected with WT HPIV1, F170S HPIV1, or HPIV2 at an MOI of 5 TCID 50 /cell. After 48 h, cells were mock-treated or treated for 30 min with 1000 IU/ml of the indicated IFN. A) Western blots were probed for total or phosphorylated (p)Stat1 and Stat2, as well as for the HPIV1 C protein and HPIV2 P protein. Alpha-tubulin was used as loading control. B) Extended exposure (over night) of the top panel in Figure 2A [“pStat1 (Tyr701)”], showing that a low level of pStat1 is detected in cells infected with WT HPIV1 in the absence of IFN treatment.
Figure Legend Snippet: Western blot of total and phosphorylated Stat1 and Stat2 in WT or F170S HPIV1-infected Vero cells following treatment with IFN-α, -β, or -γ. Vero cells were mock-infected or infected with WT HPIV1, F170S HPIV1, or HPIV2 at an MOI of 5 TCID 50 /cell. After 48 h, cells were mock-treated or treated for 30 min with 1000 IU/ml of the indicated IFN. A) Western blots were probed for total or phosphorylated (p)Stat1 and Stat2, as well as for the HPIV1 C protein and HPIV2 P protein. Alpha-tubulin was used as loading control. B) Extended exposure (over night) of the top panel in Figure 2A [“pStat1 (Tyr701)”], showing that a low level of pStat1 is detected in cells infected with WT HPIV1 in the absence of IFN treatment.

Techniques Used: Western Blot, Infection

23) Product Images from "Suppression of Interferon Lambda Signaling by SOCS-1 Results in Their Excessive Production during Influenza Virus Infection"

Article Title: Suppression of Interferon Lambda Signaling by SOCS-1 Results in Their Excessive Production during Influenza Virus Infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003845

Forced activation of STAT1 causes a significant decrease in IFN-λ expression during IAV infection. ( A ) A549 cell lines stably expressing STAT1-WT, STAT1-2C or empty vector (EV) were treated with or without IL-29 (50 ng/ml) for 45 min. Cell lysates were analyzed by Western blot using indicated antibodies. ( B–D ) A549 cell lines described in (A) were infected with or without WSN virus for 15 h. Subsequently, the cell lysates were analyzed by Western blot probed with indicated antibodies (B), and the protein levels of IL-29 in the cell culture supernatants were examined by ELISA (C). IL-29 levels produced by infected cells expressing EV were set to 100%. Plotted are the average results from three independent experiments. The error bars represent the S.E. mRNA levels of OAS-2, Mx1, IL-28A/B and IL-29 were measured by RT-PCR (D). ( E ) IFN-λ levels and OAS-2 and Mx1 levels in (D) were quantitated by densitometry, and normalized to GAPDH levels as described in Figure 2D . Plotted are the average levels from three independent experiments. The error bars represent the S.E. Statistical significance of change was determined by Student's t-test (*P
Figure Legend Snippet: Forced activation of STAT1 causes a significant decrease in IFN-λ expression during IAV infection. ( A ) A549 cell lines stably expressing STAT1-WT, STAT1-2C or empty vector (EV) were treated with or without IL-29 (50 ng/ml) for 45 min. Cell lysates were analyzed by Western blot using indicated antibodies. ( B–D ) A549 cell lines described in (A) were infected with or without WSN virus for 15 h. Subsequently, the cell lysates were analyzed by Western blot probed with indicated antibodies (B), and the protein levels of IL-29 in the cell culture supernatants were examined by ELISA (C). IL-29 levels produced by infected cells expressing EV were set to 100%. Plotted are the average results from three independent experiments. The error bars represent the S.E. mRNA levels of OAS-2, Mx1, IL-28A/B and IL-29 were measured by RT-PCR (D). ( E ) IFN-λ levels and OAS-2 and Mx1 levels in (D) were quantitated by densitometry, and normalized to GAPDH levels as described in Figure 2D . Plotted are the average levels from three independent experiments. The error bars represent the S.E. Statistical significance of change was determined by Student's t-test (*P

Techniques Used: Activation Assay, Expressing, Infection, Stable Transfection, Plasmid Preparation, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Produced, Reverse Transcription Polymerase Chain Reaction

IAV inhibits IL-29-induced STAT1 phosphorylation in A549 cells. ( A ) A549 cells were treated with IL-29 at final concentration of 3, 6, 12, 25, and 50 ng/ml for 45 min, followed by immunoblotting with indicated antibodies. ( B, C ) A549 cells infected with WSN (MOI = 1) for 15 h (WSN+) or non-infected (WSN−) were stimulated with human IL-28A (B) or IL-29 (50 ng/ml) (C) for indicated time. Cell lysates were analyzed by Western blotting using indicated antibodies. ( D ) Levels of phosphorylated STAT1 in (C) were quantitated by densitometry, and normalized to STAT1 expression and control β-actin levels. In each experiment, the highest level of STAT1 phosphorylation is 100. Plotted are the average levels from three independent experiments. The error bars represent the S.E. ( E ) A549 cells were infected with WSN (MOI = 1), lysed at the 0, 5, 10, 15 and 20 h p.i., and analyzed by Western blotting using indicated antibodies. ( F ) A549 cells were either stimulated by supernatant (SN) culture medium from IAV-infected cells in (E) or infected with WSN for 1 h, followed by Western blotting with indicated antibodies.
Figure Legend Snippet: IAV inhibits IL-29-induced STAT1 phosphorylation in A549 cells. ( A ) A549 cells were treated with IL-29 at final concentration of 3, 6, 12, 25, and 50 ng/ml for 45 min, followed by immunoblotting with indicated antibodies. ( B, C ) A549 cells infected with WSN (MOI = 1) for 15 h (WSN+) or non-infected (WSN−) were stimulated with human IL-28A (B) or IL-29 (50 ng/ml) (C) for indicated time. Cell lysates were analyzed by Western blotting using indicated antibodies. ( D ) Levels of phosphorylated STAT1 in (C) were quantitated by densitometry, and normalized to STAT1 expression and control β-actin levels. In each experiment, the highest level of STAT1 phosphorylation is 100. Plotted are the average levels from three independent experiments. The error bars represent the S.E. ( E ) A549 cells were infected with WSN (MOI = 1), lysed at the 0, 5, 10, 15 and 20 h p.i., and analyzed by Western blotting using indicated antibodies. ( F ) A549 cells were either stimulated by supernatant (SN) culture medium from IAV-infected cells in (E) or infected with WSN for 1 h, followed by Western blotting with indicated antibodies.

Techniques Used: Concentration Assay, Infection, Western Blot, Expressing

IAV infection induces robust expression of SOCS-1, resulting in decreased phosphorylation of STAT1. ( A ) Quantitative real-time RT-PCR was performed to examine the expression of SOCS-1 in A549 infected with WSN for indicated time. ( B ) Lysates from cells in (A) were analyzed for the protein levels of SOCS-1, as detected by Western blotting with indicated antibodies. ( C ) A549 cells were infected by WSN for indicated time. Supernatants (SN) derived from these cells were used to stimulate the native A549 for 2 h. Both infected cells and supernatants-stimulated cells were lysed and analyzed for SOCS-1 expression by RT-PCR. ( D ) SOCS-1 levels in (C) were quantitated by densitometry, and normalized to GAPDH levels as described in Figure 2D . Plotted are the average levels from three independent experiments. The error bars represent the S.E. ( E ) A549 cells expressing shRNAs targeting either SOCS-1 or control luciferase (Luc) were infected with WSN for 15 h. Western blotting was performed to determine the interference efficiency. Treatment with SOCS-1-shRNA#2 caused approximately 75% reduction in SOCS-1 expression quantitated by densitometry. Thus, SOCS-1-shRNA#2 was used in this study. ( F ) SOCS-1-ablated or control A549 cells were infected with WSN for the indicated time. Cell lysates were analyzed by Western blot probed with the antibodies as indicated. ( G ) Levels of phosphorylated STAT1 in (F) were quantitated by densitometry, and normalized to control β-actin levels as described in Figure 2D . Plotted are the average levels from three independent experiments. The error bars represent the S.E.
Figure Legend Snippet: IAV infection induces robust expression of SOCS-1, resulting in decreased phosphorylation of STAT1. ( A ) Quantitative real-time RT-PCR was performed to examine the expression of SOCS-1 in A549 infected with WSN for indicated time. ( B ) Lysates from cells in (A) were analyzed for the protein levels of SOCS-1, as detected by Western blotting with indicated antibodies. ( C ) A549 cells were infected by WSN for indicated time. Supernatants (SN) derived from these cells were used to stimulate the native A549 for 2 h. Both infected cells and supernatants-stimulated cells were lysed and analyzed for SOCS-1 expression by RT-PCR. ( D ) SOCS-1 levels in (C) were quantitated by densitometry, and normalized to GAPDH levels as described in Figure 2D . Plotted are the average levels from three independent experiments. The error bars represent the S.E. ( E ) A549 cells expressing shRNAs targeting either SOCS-1 or control luciferase (Luc) were infected with WSN for 15 h. Western blotting was performed to determine the interference efficiency. Treatment with SOCS-1-shRNA#2 caused approximately 75% reduction in SOCS-1 expression quantitated by densitometry. Thus, SOCS-1-shRNA#2 was used in this study. ( F ) SOCS-1-ablated or control A549 cells were infected with WSN for the indicated time. Cell lysates were analyzed by Western blot probed with the antibodies as indicated. ( G ) Levels of phosphorylated STAT1 in (F) were quantitated by densitometry, and normalized to control β-actin levels as described in Figure 2D . Plotted are the average levels from three independent experiments. The error bars represent the S.E.

Techniques Used: Infection, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Luciferase, shRNA

Disruption of cytokine signaling pathway results in robust activation of NF-κB during IAV infection. ( A ) 293T cells were co-transfected with pNF-κB-Luc and pRL-TK for 10 h and then infected with WSN virus at indicated MOI for 15 h. Luciferase activity in cell lysates was measured and displayed as the mean ± SD of relative luciferase units normalized to Renilla luciferase activity from three independent experiments. ( B, C ) A549 cells were infected with WSN virus as described in (A). RT-PCR was performed to examine the expression of indicated genes (B), and Western blotting was performed using indicated antibodies (C). ( D ) A549 cells expressing shRNAs targeting SOCS-1 or luciferase were infected with or without WSN virus for 15 h, followed by Western blotting with indicated antibodies. ( E ) 293T cells were co-transfected with pNF-κB-Luc, pRL-TK and either SOCS-1 shRNA expressing vector or control for 10 h and then infected with WSN virus for 15 h. Luciferase activity was analyzed as described in (A). ( F ) A549 cells expressing STAT1-WT, STAT1-2C or control were infected with or without WSN virus and analyzed by Western blotting with indicated antibodies. ( G ) Experiments were carried out as described in (E). Shown are results from experiments using cells expressing STAT1-WT, STAT1-2C or control. ( H ) A549 cells stably expressing SOCS-1 shRNA or control were infected with or without WSN virus for 15 h. Immunofluorescence staining was performed using an anti-p65 antibody to detect translocation of NF-κB. The nuclei were stained with DAPI. Bar, 10 µm. ( I ) Experiments were carried out as described in (H). Shown are results from experiments using cells expressing STAT1-WT, STAT1-2C or control. Bar, 10 µm.
Figure Legend Snippet: Disruption of cytokine signaling pathway results in robust activation of NF-κB during IAV infection. ( A ) 293T cells were co-transfected with pNF-κB-Luc and pRL-TK for 10 h and then infected with WSN virus at indicated MOI for 15 h. Luciferase activity in cell lysates was measured and displayed as the mean ± SD of relative luciferase units normalized to Renilla luciferase activity from three independent experiments. ( B, C ) A549 cells were infected with WSN virus as described in (A). RT-PCR was performed to examine the expression of indicated genes (B), and Western blotting was performed using indicated antibodies (C). ( D ) A549 cells expressing shRNAs targeting SOCS-1 or luciferase were infected with or without WSN virus for 15 h, followed by Western blotting with indicated antibodies. ( E ) 293T cells were co-transfected with pNF-κB-Luc, pRL-TK and either SOCS-1 shRNA expressing vector or control for 10 h and then infected with WSN virus for 15 h. Luciferase activity was analyzed as described in (A). ( F ) A549 cells expressing STAT1-WT, STAT1-2C or control were infected with or without WSN virus and analyzed by Western blotting with indicated antibodies. ( G ) Experiments were carried out as described in (E). Shown are results from experiments using cells expressing STAT1-WT, STAT1-2C or control. ( H ) A549 cells stably expressing SOCS-1 shRNA or control were infected with or without WSN virus for 15 h. Immunofluorescence staining was performed using an anti-p65 antibody to detect translocation of NF-κB. The nuclei were stained with DAPI. Bar, 10 µm. ( I ) Experiments were carried out as described in (H). Shown are results from experiments using cells expressing STAT1-WT, STAT1-2C or control. Bar, 10 µm.

Techniques Used: Activation Assay, Infection, Transfection, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, shRNA, Plasmid Preparation, Stable Transfection, Immunofluorescence, Staining, Translocation Assay

Inhibition of cytokine-mediated STAT1 activation by SOCS-1 contributes to overproduction of IFN-λ during IAV infection. ( A ) A549 cells expressing SOCS-1, empty vector (EV) or shRNAs targeting SOCS-1 or luciferase (Luc) were treated with or without IL-29 (50 ng/ml) for 45 min. Cell lysates were analyzed by Western blotting using indicated antibodies. ( B, C ) Luc or SOCS-1 knockdown A549 cells were infected without (B) or with (C) WSN virus for 15 h and then treated with IL-29 for indicated time. Shown are immunoblots of the cell lysates probed with indicated antibodies. ( D, E ) SOCS-1-ablated or control A549 cells were infected with or without WSN virus for 15 h. Subsequently, IL-29 levels in the supernatants from the cell culture were examined by ELISA. IL-29 levels produced by infected control cells were set to 100%. Plotted are the average results from three independent experiments and the error bars represent the S.E. (D). mRNA levels of OAS-2, Mx1, IL-28A/B, and IL-29 were examined by RT-PCR (E). ( F ) Levels of these mRNAs in (E) were quantitated by densitometry, and normalized to GAPDH levels as described in Figure 2D . mRNA level in infected control cells is 100. Plotted are the average results from three independent experiments. The error bars represent the S.E. Statistical significance of change was determined by Student's t-test (*P
Figure Legend Snippet: Inhibition of cytokine-mediated STAT1 activation by SOCS-1 contributes to overproduction of IFN-λ during IAV infection. ( A ) A549 cells expressing SOCS-1, empty vector (EV) or shRNAs targeting SOCS-1 or luciferase (Luc) were treated with or without IL-29 (50 ng/ml) for 45 min. Cell lysates were analyzed by Western blotting using indicated antibodies. ( B, C ) Luc or SOCS-1 knockdown A549 cells were infected without (B) or with (C) WSN virus for 15 h and then treated with IL-29 for indicated time. Shown are immunoblots of the cell lysates probed with indicated antibodies. ( D, E ) SOCS-1-ablated or control A549 cells were infected with or without WSN virus for 15 h. Subsequently, IL-29 levels in the supernatants from the cell culture were examined by ELISA. IL-29 levels produced by infected control cells were set to 100%. Plotted are the average results from three independent experiments and the error bars represent the S.E. (D). mRNA levels of OAS-2, Mx1, IL-28A/B, and IL-29 were examined by RT-PCR (E). ( F ) Levels of these mRNAs in (E) were quantitated by densitometry, and normalized to GAPDH levels as described in Figure 2D . mRNA level in infected control cells is 100. Plotted are the average results from three independent experiments. The error bars represent the S.E. Statistical significance of change was determined by Student's t-test (*P

Techniques Used: Inhibition, Activation Assay, Infection, Expressing, Plasmid Preparation, Luciferase, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Produced, Reverse Transcription Polymerase Chain Reaction

24) Product Images from "PRDM16 represses the type I interferon response in adipocytes to promote mitochondrial and thermogenic programing"

Article Title: PRDM16 represses the type I interferon response in adipocytes to promote mitochondrial and thermogenic programing

Journal: The EMBO Journal

doi: 10.15252/embj.201695588

PRDM16 represses ISGs through direct binding at gene promoters ChIP‐seq stack‐height profiles in reads per million (RPM) for PRDM16 and H3K27 acetylation (Ac) at Ifi44 and Oas3 in Prdm16 KO brown adipocyte precursors that express PRDM16 or a control (Ctl) retrovirus. ChIP‐qPCR analysis of PRDM16 binding at ISG promoters/enhancers in control (Ctl) or PRDM16‐expressing brown preadipose cells. Ins1 and 18S were used as non‐specific binding site controls. Data represent ( n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. ** P ≤ 0.01 (Student's t ‐test). Western blot analysis of STAT1 and PRDM16 protein levels in Prdm16 KO brown preadipose cells transduced with retroviral vectors that express different forms of PRDM16: wild‐type (WT), CtBP‐binding mutant (CtBP1/2), PR‐domain deletion mutant (∆PR), DNA‐binding mutant (R998Q), or empty vector (Ctl). Loading control, actin. Relative mRNA levels of ISGs in cells from (C). Data represent ( n = 3) mean ± standard deviation. Source data are available online for this figure.
Figure Legend Snippet: PRDM16 represses ISGs through direct binding at gene promoters ChIP‐seq stack‐height profiles in reads per million (RPM) for PRDM16 and H3K27 acetylation (Ac) at Ifi44 and Oas3 in Prdm16 KO brown adipocyte precursors that express PRDM16 or a control (Ctl) retrovirus. ChIP‐qPCR analysis of PRDM16 binding at ISG promoters/enhancers in control (Ctl) or PRDM16‐expressing brown preadipose cells. Ins1 and 18S were used as non‐specific binding site controls. Data represent ( n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. ** P ≤ 0.01 (Student's t ‐test). Western blot analysis of STAT1 and PRDM16 protein levels in Prdm16 KO brown preadipose cells transduced with retroviral vectors that express different forms of PRDM16: wild‐type (WT), CtBP‐binding mutant (CtBP1/2), PR‐domain deletion mutant (∆PR), DNA‐binding mutant (R998Q), or empty vector (Ctl). Loading control, actin. Relative mRNA levels of ISGs in cells from (C). Data represent ( n = 3) mean ± standard deviation. Source data are available online for this figure.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, CTL Assay, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation, Western Blot, Transduction, Mutagenesis, Plasmid Preparation

PRDM16 blocks type I IFN signaling downstream of IFNAR receptor Relative mRNA levels of Prdm16 , Irf7 , Ifi44 , and Stat1 in WT and R26 Cre+ inguinal precursors treated with increasing doses of recombinant mouse IFNα. Relative mRNA levels of ISGs in brown preadipocytes treated with vehicle, anti‐IFNAR (αIFNAR) neutralizing antibody, mouse IFNα, or a combination of αIFNAR and IFNα. Data information: Data represent ( n = 3) mean ± standard deviation. * P ≤ 0.05, ** P ≤ 0.01 (Student's t ‐test).
Figure Legend Snippet: PRDM16 blocks type I IFN signaling downstream of IFNAR receptor Relative mRNA levels of Prdm16 , Irf7 , Ifi44 , and Stat1 in WT and R26 Cre+ inguinal precursors treated with increasing doses of recombinant mouse IFNα. Relative mRNA levels of ISGs in brown preadipocytes treated with vehicle, anti‐IFNAR (αIFNAR) neutralizing antibody, mouse IFNα, or a combination of αIFNAR and IFNα. Data information: Data represent ( n = 3) mean ± standard deviation. * P ≤ 0.05, ** P ≤ 0.01 (Student's t ‐test).

Techniques Used: Recombinant, Standard Deviation

PRDM16 opposes type I IFN signaling in vivo A–D Prdm16 fl/fl (WT) and Myf5 Cre ; Prdm16 fl/fl (KO) mice treated with IFNα or phosphate‐buffered saline (PBS) for 2 weeks prior to analysis of brown adipose tissue (BAT). Experimental groups: WT+PBS ( n = 4), KO+PBS ( n = 3), WT+IFN ( n = 6), KO+IFN ( n = 4). (A) Western blot analysis of PRDM16, STAT1, and GAPDH (loading control) protein levels. (B) qPCR analysis of Ifi44 and Stat1 mRNA levels. Data are presented as mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01 (paired two‐way ANOVA). (C) Hematoxylin and eosin (H E) and anti‐UCP1 immunohistochemical staining. Scale bar = 50 μm. (D) Relative mRNA levels of brown fat‐specific genes ( Ucp1 , Cidea ) and mitochondrial genes ( mt‐Co1 , mt‐CytB ). Data are presented as mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01 (paired two‐way ANOVA). E Volume of O 2 (VO 2 ) consumed before and after norepinephrine injection. Experimental groups: WT+PBS ( n = 9), KO+PBS ( n = 6), WT+IFN ( n = 6), KO+IFN ( n = 7). Data are presented as mean ± SEM. ** P ≤ 0.01 (paired two‐way ANOVA). Source data are available online for this figure.
Figure Legend Snippet: PRDM16 opposes type I IFN signaling in vivo A–D Prdm16 fl/fl (WT) and Myf5 Cre ; Prdm16 fl/fl (KO) mice treated with IFNα or phosphate‐buffered saline (PBS) for 2 weeks prior to analysis of brown adipose tissue (BAT). Experimental groups: WT+PBS ( n = 4), KO+PBS ( n = 3), WT+IFN ( n = 6), KO+IFN ( n = 4). (A) Western blot analysis of PRDM16, STAT1, and GAPDH (loading control) protein levels. (B) qPCR analysis of Ifi44 and Stat1 mRNA levels. Data are presented as mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01 (paired two‐way ANOVA). (C) Hematoxylin and eosin (H E) and anti‐UCP1 immunohistochemical staining. Scale bar = 50 μm. (D) Relative mRNA levels of brown fat‐specific genes ( Ucp1 , Cidea ) and mitochondrial genes ( mt‐Co1 , mt‐CytB ). Data are presented as mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01 (paired two‐way ANOVA). E Volume of O 2 (VO 2 ) consumed before and after norepinephrine injection. Experimental groups: WT+PBS ( n = 9), KO+PBS ( n = 6), WT+IFN ( n = 6), KO+IFN ( n = 7). Data are presented as mean ± SEM. ** P ≤ 0.01 (paired two‐way ANOVA). Source data are available online for this figure.

Techniques Used: In Vivo, Mouse Assay, Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining, Injection

PRDM16 blocks type I IFN signaling downstream of IFNAR receptor Relative mRNA levels of IFN‐stimulated genes (ISGs) in Prdm16 KO brown adipocyte precursors infected with control (Ctl) or PRDM16 retrovirus. Data represent ( n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. * P ≤ 0.05, ** P ≤ 0.01 (Student's t ‐test). Western blot analysis of FLAG, phosphorylated STAT1 (pSTAT1), STAT1, phosphorylated STAT2 (pSTAT2), STAT3, and tubulin (loading control) protein in Prdm16 KO precursors infected with control (Ctl) or FLAG‐PRDM16 retrovirus. Relative mRNA levels of ISGs in control (Ctl) and PRDM16‐expressing preadipocytes +/− recombinant mouse IFNα. Data represent ( n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. * P ≤ 0.05, ** P ≤ 0.01 (Student's t ‐test). Relative mRNA levels of ISGs in WT and R26 Cre+ inguinal preadipocytes treated with 4OHT and vehicle or anti‐IFNAR1 neutralizing antibody (αIFNAR1) for 4 days. Data represent ( n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. * P ≤ 0.05, ** P ≤ 0.01 (paired two‐way ANOVA). Western blot analysis of PRDM16, STAT1, and actin (loading control) protein in brown adipocytes expressing gR26 (control) and gPrdm16 CRISPR/Cas9 constructs and treated +/− αIFNAR1 throughout differentiation. Relative mRNA levels of brown‐selective ( Ucp1 , Cidea , Pgc1a ) and mitochondrial ( mt‐Co1 , mt‐CytB , mt‐Nd1 ) genes in brown adipocytes expressing gR26 and gPrdm16 CRISPR/Cas9 constructs +/− αIFNAR1 throughout differentiation. Data represent ( n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. * P ≤ 0.05, ** P ≤ 0.01 (paired two‐way ANOVA). Source data are available online for this figure.
Figure Legend Snippet: PRDM16 blocks type I IFN signaling downstream of IFNAR receptor Relative mRNA levels of IFN‐stimulated genes (ISGs) in Prdm16 KO brown adipocyte precursors infected with control (Ctl) or PRDM16 retrovirus. Data represent ( n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. * P ≤ 0.05, ** P ≤ 0.01 (Student's t ‐test). Western blot analysis of FLAG, phosphorylated STAT1 (pSTAT1), STAT1, phosphorylated STAT2 (pSTAT2), STAT3, and tubulin (loading control) protein in Prdm16 KO precursors infected with control (Ctl) or FLAG‐PRDM16 retrovirus. Relative mRNA levels of ISGs in control (Ctl) and PRDM16‐expressing preadipocytes +/− recombinant mouse IFNα. Data represent ( n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. * P ≤ 0.05, ** P ≤ 0.01 (Student's t ‐test). Relative mRNA levels of ISGs in WT and R26 Cre+ inguinal preadipocytes treated with 4OHT and vehicle or anti‐IFNAR1 neutralizing antibody (αIFNAR1) for 4 days. Data represent ( n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. * P ≤ 0.05, ** P ≤ 0.01 (paired two‐way ANOVA). Western blot analysis of PRDM16, STAT1, and actin (loading control) protein in brown adipocytes expressing gR26 (control) and gPrdm16 CRISPR/Cas9 constructs and treated +/− αIFNAR1 throughout differentiation. Relative mRNA levels of brown‐selective ( Ucp1 , Cidea , Pgc1a ) and mitochondrial ( mt‐Co1 , mt‐CytB , mt‐Nd1 ) genes in brown adipocytes expressing gR26 and gPrdm16 CRISPR/Cas9 constructs +/− αIFNAR1 throughout differentiation. Data represent ( n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. * P ≤ 0.05, ** P ≤ 0.01 (paired two‐way ANOVA). Source data are available online for this figure.

Techniques Used: Infection, CTL Assay, Standard Deviation, Western Blot, Expressing, Recombinant, CRISPR, Construct

Type I IFN disrupts mitochondrial structure and function in adipocytes Brown adipocytes were treated with 1,000 U/ml mouse IFNα or vehicle (Ctl) throughout differentiation. Relative expression levels of ISGs. Data represent ( n = 3) mean ± standard deviation. ** P ≤ 0.01 (Student's t ‐test). Oil Red O staining of lipid droplets and relative mRNA levels of pan‐adipogenic genes ( Fabp4 , Pparg2 ). Data represent ( n = 3) mean ± standard deviation. Western blot analysis of STAT1, UCP1, and actin (loading control) protein levels. Relative mRNA levels of brown fat‐selective ( Ucp1 , Cidea , Pgc1a ) and mitochondrial ( Cox7a1 , mt‐Co1 , mt‐Cytb , mt‐Nd1 ) genes. Data represent ( n = 3) mean ± standard deviation and are consistent with duplicate independent experiments. * P ≤ 0.05, ** P ≤ 0.01 (Student's t ‐test). Western blot analysis of mitochondrial complex proteins and actin (loading control). Relative ratio of mitochondrial DNA (mt‐Co1) to nuclear DNA (Ndufv1) ( n = 6). Data are presented as mean ± standard deviation. Transmission electron micrograph of representative brown adipocytes showing mitochondria (M), lipid droplets (L), and nuclei (N). Scale bar = 500 nm. Relative oxygen consumption rates of adipocytes ( n = 6). Data are presented as mean ± standard deviation. * P ≤ 0.05 (Student's t ‐test). Relative mRNA levels of brown‐selective genes ( Ucp1 , Cidea , Pgc1a ) and mitochondrial genes ( mt‐Co1 , mt‐CytB , mt‐Nd1 ) in brown adipocytes infected with control (Ctl) or PRDM16 retrovirus +/− mouse IFNα. Data represent ( n = 3) mean ± standard deviation. * P ≤ 0.05, ** P ≤ 0.01 (paired two‐way ANOVA). Source data are available online for this figure.
Figure Legend Snippet: Type I IFN disrupts mitochondrial structure and function in adipocytes Brown adipocytes were treated with 1,000 U/ml mouse IFNα or vehicle (Ctl) throughout differentiation. Relative expression levels of ISGs. Data represent ( n = 3) mean ± standard deviation. ** P ≤ 0.01 (Student's t ‐test). Oil Red O staining of lipid droplets and relative mRNA levels of pan‐adipogenic genes ( Fabp4 , Pparg2 ). Data represent ( n = 3) mean ± standard deviation. Western blot analysis of STAT1, UCP1, and actin (loading control) protein levels. Relative mRNA levels of brown fat‐selective ( Ucp1 , Cidea , Pgc1a ) and mitochondrial ( Cox7a1 , mt‐Co1 , mt‐Cytb , mt‐Nd1 ) genes. Data represent ( n = 3) mean ± standard deviation and are consistent with duplicate independent experiments. * P ≤ 0.05, ** P ≤ 0.01 (Student's t ‐test). Western blot analysis of mitochondrial complex proteins and actin (loading control). Relative ratio of mitochondrial DNA (mt‐Co1) to nuclear DNA (Ndufv1) ( n = 6). Data are presented as mean ± standard deviation. Transmission electron micrograph of representative brown adipocytes showing mitochondria (M), lipid droplets (L), and nuclei (N). Scale bar = 500 nm. Relative oxygen consumption rates of adipocytes ( n = 6). Data are presented as mean ± standard deviation. * P ≤ 0.05 (Student's t ‐test). Relative mRNA levels of brown‐selective genes ( Ucp1 , Cidea , Pgc1a ) and mitochondrial genes ( mt‐Co1 , mt‐CytB , mt‐Nd1 ) in brown adipocytes infected with control (Ctl) or PRDM16 retrovirus +/− mouse IFNα. Data represent ( n = 3) mean ± standard deviation. * P ≤ 0.05, ** P ≤ 0.01 (paired two‐way ANOVA). Source data are available online for this figure.

Techniques Used: CTL Assay, Expressing, Standard Deviation, Staining, Western Blot, Transmission Assay, Infection

25) Product Images from "COOPERATIVITY OF THE MUC1 ONCOPROTEIN AND STAT1 PATHWAY IN POOR PROGNOSIS HUMAN BREAST CANCER"

Article Title: COOPERATIVITY OF THE MUC1 ONCOPROTEIN AND STAT1 PATHWAY IN POOR PROGNOSIS HUMAN BREAST CANCER

Journal: Oncogene

doi: 10.1038/onc.2009.391

MUC1-C associates with the STAT1 transcription complex A. Soluble chromatin from MCF-10A cells left untreated or treated with IFNγ for 24 h was immunoprecipitated with anti-STAT1. The final DNA extractions were amplified by PCR with pairs of primers that cover the STAT binding site (SBS; −689 to −414) and the control region (CR; +4524 to +4745) in the MUC1 promoter. B. Soluble chromatin from MCF-10A cells left untreated or treated with IFNγ for 24 h was immunoprecipitated with anti-MUC1-C and analyzed for SBS and CR sequences. C. Lysates from ZR-75-1 cells were immunoprecipitated with anti-MUC1-C or a control IgG. The precipitates were immunoblotted with the indicated antibodies. D. Soluble chromatin from ZR-75-1/vector and ZR-75-1/MUC1siRNA cells was precipitated anti-STAT1 and analyzed for MUC1 promoter SBS and CR sequences. In re-ChIP analysis, the anti-STAT1 precipitates were released, reimmunoprecipitated with anti-MUC1-C and then analyzed for MUC1 promoter sequences.
Figure Legend Snippet: MUC1-C associates with the STAT1 transcription complex A. Soluble chromatin from MCF-10A cells left untreated or treated with IFNγ for 24 h was immunoprecipitated with anti-STAT1. The final DNA extractions were amplified by PCR with pairs of primers that cover the STAT binding site (SBS; −689 to −414) and the control region (CR; +4524 to +4745) in the MUC1 promoter. B. Soluble chromatin from MCF-10A cells left untreated or treated with IFNγ for 24 h was immunoprecipitated with anti-MUC1-C and analyzed for SBS and CR sequences. C. Lysates from ZR-75-1 cells were immunoprecipitated with anti-MUC1-C or a control IgG. The precipitates were immunoblotted with the indicated antibodies. D. Soluble chromatin from ZR-75-1/vector and ZR-75-1/MUC1siRNA cells was precipitated anti-STAT1 and analyzed for MUC1 promoter SBS and CR sequences. In re-ChIP analysis, the anti-STAT1 precipitates were released, reimmunoprecipitated with anti-MUC1-C and then analyzed for MUC1 promoter sequences.

Techniques Used: Immunoprecipitation, Amplification, Polymerase Chain Reaction, Binding Assay, Plasmid Preparation, Chromatin Immunoprecipitation

STAT1 pathway activation linked to MUC1 expression is associated with increased risk of death A. An additional expressional database derived from 155 breast tumors was analyzed for expression of MUC1 (MUC1+) and STAT1 (STAT1+). Coexpression was detectable in 25 tumors (16.1%). Hierarchical clustering for expression of the STAT1 pathway (24 genes) was performed for the MUC1+ tumors. Relative expression values are in log2 scale. Activation of the STAT1 pathway (STAT1P+) was significantly associated with MUC1 expression (P
Figure Legend Snippet: STAT1 pathway activation linked to MUC1 expression is associated with increased risk of death A. An additional expressional database derived from 155 breast tumors was analyzed for expression of MUC1 (MUC1+) and STAT1 (STAT1+). Coexpression was detectable in 25 tumors (16.1%). Hierarchical clustering for expression of the STAT1 pathway (24 genes) was performed for the MUC1+ tumors. Relative expression values are in log2 scale. Activation of the STAT1 pathway (STAT1P+) was significantly associated with MUC1 expression (P

Techniques Used: Activation Assay, Expressing, Derivative Assay

Functional gene network analysis links MUC1 and STAT1 A. Genes differentially expressed in 3Y1/MUC1-CD cells growing in vivo as compared to in vitro were analyzed using Ingenuity Pathway Analysis to identify functional networks. The gene network containing MUC1 and STAT1 as shown is associated with genes involved in cellular growth and inflammation. Red signifies up-regulation. Green signifies down-regulation. B. 3Y1/vector (open bars) and 3Y1/MUC1-CD (solid bars) were left untreated and treated with IFNγ for 24 h. Total RNA was analyzed by quantitative RT-PCR for expression of the indicated genes. The results are expressed as the fold-induction (mean±SD) for IFNγ-treated as compared to untreated cells. The differences between values obtained for the 3Y1/vector and 3Y1/MUC1-CD cells were significant at p values ranging from 0.035 to 0.00078.
Figure Legend Snippet: Functional gene network analysis links MUC1 and STAT1 A. Genes differentially expressed in 3Y1/MUC1-CD cells growing in vivo as compared to in vitro were analyzed using Ingenuity Pathway Analysis to identify functional networks. The gene network containing MUC1 and STAT1 as shown is associated with genes involved in cellular growth and inflammation. Red signifies up-regulation. Green signifies down-regulation. B. 3Y1/vector (open bars) and 3Y1/MUC1-CD (solid bars) were left untreated and treated with IFNγ for 24 h. Total RNA was analyzed by quantitative RT-PCR for expression of the indicated genes. The results are expressed as the fold-induction (mean±SD) for IFNγ-treated as compared to untreated cells. The differences between values obtained for the 3Y1/vector and 3Y1/MUC1-CD cells were significant at p values ranging from 0.035 to 0.00078.

Techniques Used: Functional Assay, In Vivo, In Vitro, Plasmid Preparation, Quantitative RT-PCR, Expressing

MUC1-C interacts directly with STAT1 A. Lysates from 3Y1/MUC1-CD cells were immunoprecipitated with anti-MUC1-C or a control IgG. The precipitates were immunoblotted with the indicated antibodies. B. Amino acid sequence of MUC1-CD and MUC1-CD (mSRM) (upper panel). GST, GST-MUC1-CD, GST-MUC1-CD (1–45) and GST-MUC1-CD (46–72) bound to glutathione beads were incubated with purified recombinant STAT1 (lower left). GST, GST-MUC1-CD and GST-MUC1-CD (mSRM) bound to gluthatione beads were incubated with recombinant STAT1 (lower right). The adsorbates were immunoblotted with anti-STAT1. Input of the GST and GST-MUC1-CD proteins was assessed by Coomassie blue staining. C. Structure of STAT1 (upper panel). GST, GST-STAT1 (full length; amino acids 1–750), GST-STAT1 (N-terminal; amino acids 1–324), GST-STAT1 (DBD; amino acids 295–491) and GST-STAT1 (C-terminal; amino acids 464–750) bound to glutathione beads were incubated with purified MUC1-CD. Adsorbates were immunoblotted with anti-MUC1-C. Input of GST and GST-STAT1 fusion proteins was assessed by Coomassie blue staining.
Figure Legend Snippet: MUC1-C interacts directly with STAT1 A. Lysates from 3Y1/MUC1-CD cells were immunoprecipitated with anti-MUC1-C or a control IgG. The precipitates were immunoblotted with the indicated antibodies. B. Amino acid sequence of MUC1-CD and MUC1-CD (mSRM) (upper panel). GST, GST-MUC1-CD, GST-MUC1-CD (1–45) and GST-MUC1-CD (46–72) bound to glutathione beads were incubated with purified recombinant STAT1 (lower left). GST, GST-MUC1-CD and GST-MUC1-CD (mSRM) bound to gluthatione beads were incubated with recombinant STAT1 (lower right). The adsorbates were immunoblotted with anti-STAT1. Input of the GST and GST-MUC1-CD proteins was assessed by Coomassie blue staining. C. Structure of STAT1 (upper panel). GST, GST-STAT1 (full length; amino acids 1–750), GST-STAT1 (N-terminal; amino acids 1–324), GST-STAT1 (DBD; amino acids 295–491) and GST-STAT1 (C-terminal; amino acids 464–750) bound to glutathione beads were incubated with purified MUC1-CD. Adsorbates were immunoblotted with anti-MUC1-C. Input of GST and GST-STAT1 fusion proteins was assessed by Coomassie blue staining.

Techniques Used: Immunoprecipitation, Sequencing, Incubation, Purification, Recombinant, Staining

Induction of MUC1 by IFNγ in MCF-10A cells is conferred by both STAT1 and MUC1 A. MCF-10A cells were treated with IFNγ for the indicated times. Lysates were immunoblotted with the indicated antibodies (left). MUC1 and, as a control, β-actin mRNA levels were determined by RT-PCR (right). B. MCF-10A cells left untreated or treated with IFNγ for 24 h were immunoprecitated with anti-STAT1. The precipitates were immunoblotted with the indicated antibodies. C. MCF-10A cells were transfected with control or STAT1 siRNA pools for 24 h and then left untreated or stimulated with IFNγ for 24. Lysates were immunoblotted with the indicated antibodies. D. MCF-10A cells were left untreated and treated with 5 μM GO-201 or CP-1 for 3 days and then stimulated with IFNγ for 24 h. Whole cell lysates were immunoblotted with the indicated antibodies. NS: non-specific band. E. MCF-10A cells were transfected with control or MUC1siRNA pools for 72 h. The transfected cells were left untreated or stimulated with IFNγ for 24 h. Total cellular RNA was analyzed for IFITM1 expression by real-time RT-PCR. The results are expressed as the fold activation (mean±SD from three experiments) compared with that obtained with cells transfected with the control siRNA and left untreated (assigned a value of 1).
Figure Legend Snippet: Induction of MUC1 by IFNγ in MCF-10A cells is conferred by both STAT1 and MUC1 A. MCF-10A cells were treated with IFNγ for the indicated times. Lysates were immunoblotted with the indicated antibodies (left). MUC1 and, as a control, β-actin mRNA levels were determined by RT-PCR (right). B. MCF-10A cells left untreated or treated with IFNγ for 24 h were immunoprecitated with anti-STAT1. The precipitates were immunoblotted with the indicated antibodies. C. MCF-10A cells were transfected with control or STAT1 siRNA pools for 24 h and then left untreated or stimulated with IFNγ for 24. Lysates were immunoblotted with the indicated antibodies. D. MCF-10A cells were left untreated and treated with 5 μM GO-201 or CP-1 for 3 days and then stimulated with IFNγ for 24 h. Whole cell lysates were immunoblotted with the indicated antibodies. NS: non-specific band. E. MCF-10A cells were transfected with control or MUC1siRNA pools for 72 h. The transfected cells were left untreated or stimulated with IFNγ for 24 h. Total cellular RNA was analyzed for IFITM1 expression by real-time RT-PCR. The results are expressed as the fold activation (mean±SD from three experiments) compared with that obtained with cells transfected with the control siRNA and left untreated (assigned a value of 1).

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

Coexpression of MUC1 and the STAT1 pathway is associated with reduced recurrence-free survival A. An expressional database derived from 327 breast tumors was analyzed for expression of MUC1 (MUC1+) and STAT1 (STAT1+). Coexpression was detectable in 45 tumors (13.8%). Hierarchical clustering for expression of the STAT1 pathway (24 genes) was performed for the entire database. Relative expression values are in log2 scale. Activation of the STAT1 pathway (STAT1P+) was significantly associated with MUC1 expression (49 tumors; Fisher’s exact test, P=0.0003). B. Kaplan-Meier curves for recurrence-free survival were determined for patients with MUC1+/STAT1P+ tumors (n=49; red curve) compared to those patients without coexpression (n=257; blue curve). C. Kaplan-Meier curves were determined for recurrence-free survival of patients with grade 2/3 tumors that are MUC1+/STAT1P+ (n=32; red curve) compared to those patients with grade 2/3 tumors without coexpression (n=169; blue curve).
Figure Legend Snippet: Coexpression of MUC1 and the STAT1 pathway is associated with reduced recurrence-free survival A. An expressional database derived from 327 breast tumors was analyzed for expression of MUC1 (MUC1+) and STAT1 (STAT1+). Coexpression was detectable in 45 tumors (13.8%). Hierarchical clustering for expression of the STAT1 pathway (24 genes) was performed for the entire database. Relative expression values are in log2 scale. Activation of the STAT1 pathway (STAT1P+) was significantly associated with MUC1 expression (49 tumors; Fisher’s exact test, P=0.0003). B. Kaplan-Meier curves for recurrence-free survival were determined for patients with MUC1+/STAT1P+ tumors (n=49; red curve) compared to those patients without coexpression (n=257; blue curve). C. Kaplan-Meier curves were determined for recurrence-free survival of patients with grade 2/3 tumors that are MUC1+/STAT1P+ (n=32; red curve) compared to those patients with grade 2/3 tumors without coexpression (n=169; blue curve).

Techniques Used: Derivative Assay, Expressing, Activation Assay

MUC1-CD activates transcription of STAT1 and STAT1-dependent genes A. 3Y1/vector (clones A and B) and 3Y1/MUC1-CD (clones A and B) cells growing in vitro were analyzed for expression of the indicated genes in the IFNγ/STAT1 pathway. B. 3Y1/MUC1-CD cells growing in vitro and as tumors in nude mice were analyzed for expression of the indicated IFNγ/STAT1 target genes. Values are in log2 scale and represent expression relative to the average value of the gene across samples.
Figure Legend Snippet: MUC1-CD activates transcription of STAT1 and STAT1-dependent genes A. 3Y1/vector (clones A and B) and 3Y1/MUC1-CD (clones A and B) cells growing in vitro were analyzed for expression of the indicated genes in the IFNγ/STAT1 pathway. B. 3Y1/MUC1-CD cells growing in vitro and as tumors in nude mice were analyzed for expression of the indicated IFNγ/STAT1 target genes. Values are in log2 scale and represent expression relative to the average value of the gene across samples.

Techniques Used: Plasmid Preparation, In Vitro, Expressing, Mouse Assay

26) Product Images from "Vitamin D Counteracts Mycobacterium tuberculosis-Induced Cathelicidin Downregulation in Dendritic Cells and Allows Th1 Differentiation and IFNγ Secretion"

Article Title: Vitamin D Counteracts Mycobacterium tuberculosis-Induced Cathelicidin Downregulation in Dendritic Cells and Allows Th1 Differentiation and IFNγ Secretion

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00656

Vitamin D does not prevent Th1 differentiation. Relative TBX21 (A) , IL-12Rβ2 (B) , and IFNγ (C) expression in T cells activated for 24, 48, or 72 h in Th1-polarizing medium in the presence or absence of 100 nM 25(OH)D 3 . Data are normalized to unstimulated T cells (mean + SEM, n = 6). Representative Western blots (lower panel) and quantification (upper panel) of phosphorylated STAT1 and total STAT1 (D) and phosphorylated STAT4 and total STAT4 (E) with GAPDH as loading control from T cells activated for 0, 24, 48, and 72 h in Th1-polarizing medium in the presence or absence of 100 nM 25(OH)D 3 . Western blots including protein ladder are shown in the Figure S2 in Supplementary Material.
Figure Legend Snippet: Vitamin D does not prevent Th1 differentiation. Relative TBX21 (A) , IL-12Rβ2 (B) , and IFNγ (C) expression in T cells activated for 24, 48, or 72 h in Th1-polarizing medium in the presence or absence of 100 nM 25(OH)D 3 . Data are normalized to unstimulated T cells (mean + SEM, n = 6). Representative Western blots (lower panel) and quantification (upper panel) of phosphorylated STAT1 and total STAT1 (D) and phosphorylated STAT4 and total STAT4 (E) with GAPDH as loading control from T cells activated for 0, 24, 48, and 72 h in Th1-polarizing medium in the presence or absence of 100 nM 25(OH)D 3 . Western blots including protein ladder are shown in the Figure S2 in Supplementary Material.

Techniques Used: Expressing, Western Blot

27) Product Images from "WSX1 Expression in Tumors Induces Immune Tolerance via Suppression of Effector Immune Cells"

Article Title: WSX1 Expression in Tumors Induces Immune Tolerance via Suppression of Effector Immune Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0019072

IL27-independent and WSX1-dependent tumor growth promotion. ( a ) Comparison of IL27 signaling function between WSX1- and DN-WSX1 cells. Equal amounts of cell extracts of LLC cells expressing GFP, WSX1, or DN-WSX1 were treated with IL27 for 10 minutes (10′), overnight (24 hr), or left untreated (-), and then analyzed using WB techniques and probed with pSTAT1 and STAT1 antibodies. ( b ) Comparison of tumor growth between LLC-GFP, LLC-WSX1, and LLC-MUT in wildtype C57Bl/6 mice (N = 5). Representative of two independent experiments. ( c ) Comparison of tumor growth between LLC-GFP and LLC-WSX1 in TCCR −/− mice (N = 4). ( d ) Comparison of tumor growth between LLC-WSX1 and LLC-MUT in TCCR −/− mice (N = 4). Points, mean; bars, SE. *, P
Figure Legend Snippet: IL27-independent and WSX1-dependent tumor growth promotion. ( a ) Comparison of IL27 signaling function between WSX1- and DN-WSX1 cells. Equal amounts of cell extracts of LLC cells expressing GFP, WSX1, or DN-WSX1 were treated with IL27 for 10 minutes (10′), overnight (24 hr), or left untreated (-), and then analyzed using WB techniques and probed with pSTAT1 and STAT1 antibodies. ( b ) Comparison of tumor growth between LLC-GFP, LLC-WSX1, and LLC-MUT in wildtype C57Bl/6 mice (N = 5). Representative of two independent experiments. ( c ) Comparison of tumor growth between LLC-GFP and LLC-WSX1 in TCCR −/− mice (N = 4). ( d ) Comparison of tumor growth between LLC-WSX1 and LLC-MUT in TCCR −/− mice (N = 4). Points, mean; bars, SE. *, P

Techniques Used: Expressing, Western Blot, Mouse Assay

28) Product Images from "Ginkgo biloba extract reduces high-glucose-induced endothelial adhesion by inhibiting the redox-dependent interleukin-6 pathways"

Article Title: Ginkgo biloba extract reduces high-glucose-induced endothelial adhesion by inhibiting the redox-dependent interleukin-6 pathways

Journal: Cardiovascular Diabetology

doi: 10.1186/1475-2840-11-49

GBE inhibits high-glucose-induced activation of STAT1 and STAT3 as well as ICAM-1 accumulation in HAECs. (A) HAECs were incubated in high glucose for 3 days, followed by incubation with GBE, fludarabine (STAT1 inhibitor;20 μmol/l), or piceatannol (10 μmol/l) for 1 day. (B) HAECs were incubated in high glucose for 3 days, followed by incubation with GBE, fludarabine, piceatannol, fludarabine plus GBE, or piceatannol plus GBE for 1 day. (C and D) EMSAs for STAT1 and STAT3 were performed using nuclear extracts from human aortic endothelial cells that were cultured in high glucose or mannitol for 3 days, followed by treatment with GBE. Quantification of STAT1 and STAT3 activation in HAECs treated with high glucose combined with GBE is also shown. N = 6 in each set of experiment. * p
Figure Legend Snippet: GBE inhibits high-glucose-induced activation of STAT1 and STAT3 as well as ICAM-1 accumulation in HAECs. (A) HAECs were incubated in high glucose for 3 days, followed by incubation with GBE, fludarabine (STAT1 inhibitor;20 μmol/l), or piceatannol (10 μmol/l) for 1 day. (B) HAECs were incubated in high glucose for 3 days, followed by incubation with GBE, fludarabine, piceatannol, fludarabine plus GBE, or piceatannol plus GBE for 1 day. (C and D) EMSAs for STAT1 and STAT3 were performed using nuclear extracts from human aortic endothelial cells that were cultured in high glucose or mannitol for 3 days, followed by treatment with GBE. Quantification of STAT1 and STAT3 activation in HAECs treated with high glucose combined with GBE is also shown. N = 6 in each set of experiment. * p

Techniques Used: Activation Assay, Incubation, Cell Culture

29) Product Images from "STAT1 deficiency in the heart protects against myocardial infarction by enhancing autophagy"

Article Title: STAT1 deficiency in the heart protects against myocardial infarction by enhancing autophagy

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2011.01323.x

(A) Electron scanning microscopy of wt (STAT +/+ ) and STAT1 −/− hearts subjected to ex vivo I/R. As indicated by the arrow heads STAT1 −/− hearts subjected to I/R contained more number of double-membrane autophagosomes than the wt STAT1 +/+ hearts. (B) wt (STAT1 +/+ ) and STAT1 −/− hearts were subjected to ex vivo I/R. LC3-I and LC3-II Western blotting of LC3-II. Levels appeared raised during ischaemia in the STAT1 −/− animals. (C) Western blot analysis of Beclin 1 levels is enhanced during ischaemia in STAT1 −/− compared to wts. Densitometry demonstrated significant increase of Beclin 1 protein levels in STAT1 −/− hearts (lower panel). (D) Western blots analysis of Atg12 levels in wt (STAT +/+ ) and STAT1 −/− hearts subjected to ex vivo I/R. Densitometry demonstrated significant increase of Atg12 protein levels in STAT1 −/− hearts (lower panel).
Figure Legend Snippet: (A) Electron scanning microscopy of wt (STAT +/+ ) and STAT1 −/− hearts subjected to ex vivo I/R. As indicated by the arrow heads STAT1 −/− hearts subjected to I/R contained more number of double-membrane autophagosomes than the wt STAT1 +/+ hearts. (B) wt (STAT1 +/+ ) and STAT1 −/− hearts were subjected to ex vivo I/R. LC3-I and LC3-II Western blotting of LC3-II. Levels appeared raised during ischaemia in the STAT1 −/− animals. (C) Western blot analysis of Beclin 1 levels is enhanced during ischaemia in STAT1 −/− compared to wts. Densitometry demonstrated significant increase of Beclin 1 protein levels in STAT1 −/− hearts (lower panel). (D) Western blots analysis of Atg12 levels in wt (STAT +/+ ) and STAT1 −/− hearts subjected to ex vivo I/R. Densitometry demonstrated significant increase of Atg12 protein levels in STAT1 −/− hearts (lower panel).

Techniques Used: Microscopy, Ex Vivo, Western Blot

(A) Simulated I/R of isolated primary cardiac myocytes showed LC3-II protein levels being induced from 90 min. reperfusion. Western blot analysis at various time-points and immunoblotting with the indicated antibodies (B) IFN-γ-STAT1 activation reduced and epigallocatechin-3-gallate-reduce STAT1 activation enhanced conversion of LC3-I to II following simulated I/R of isolated primary cardiac myocytes. Primary cardiac myocytes were treated with either IFN-γ (50 ng/ml) or epigallocatechin-3-gallate (EGCG 50 ng/ml) and exposed to simulated I/R and cells harvested after 180 min. and processed for Western blot with the indicated antibodies (lower panel). Upper panel shows densitometric analysis of LC3-I over LC3-II ratio. (C) Enhanced autophagic flux in STAT1-deficient (ST1 −/− ) cells following serum starvation (SS) using the ployQ80 luciferase reporter assay. Wt (ST1 +/+ or STAT1-deficient (ST1 −/− ) MEF cells were transfected with polyQ80luc and subjected to serum starvation (SSQ80) or control conditions (NDQ80). At 6 hrs of nutrient deprivation, no significant difference in autophagic turnover of the polyQ80 aggregates was seen. (D) After 18 hrs of nutrient deprivation the ST1 −/− MEFs showed nearly 59% decrease in polyQ80 luciferase activity, whereas the ST1 +/+ MEFs showed no statistically significant decrease in polyQ80 luciferase activity The data above show pooled data from three experiments with the NDQ80 set at 100% and the data analysed using a paired Student’s t-test to compare NDQ80 versus SSQ80.
Figure Legend Snippet: (A) Simulated I/R of isolated primary cardiac myocytes showed LC3-II protein levels being induced from 90 min. reperfusion. Western blot analysis at various time-points and immunoblotting with the indicated antibodies (B) IFN-γ-STAT1 activation reduced and epigallocatechin-3-gallate-reduce STAT1 activation enhanced conversion of LC3-I to II following simulated I/R of isolated primary cardiac myocytes. Primary cardiac myocytes were treated with either IFN-γ (50 ng/ml) or epigallocatechin-3-gallate (EGCG 50 ng/ml) and exposed to simulated I/R and cells harvested after 180 min. and processed for Western blot with the indicated antibodies (lower panel). Upper panel shows densitometric analysis of LC3-I over LC3-II ratio. (C) Enhanced autophagic flux in STAT1-deficient (ST1 −/− ) cells following serum starvation (SS) using the ployQ80 luciferase reporter assay. Wt (ST1 +/+ or STAT1-deficient (ST1 −/− ) MEF cells were transfected with polyQ80luc and subjected to serum starvation (SSQ80) or control conditions (NDQ80). At 6 hrs of nutrient deprivation, no significant difference in autophagic turnover of the polyQ80 aggregates was seen. (D) After 18 hrs of nutrient deprivation the ST1 −/− MEFs showed nearly 59% decrease in polyQ80 luciferase activity, whereas the ST1 +/+ MEFs showed no statistically significant decrease in polyQ80 luciferase activity The data above show pooled data from three experiments with the NDQ80 set at 100% and the data analysed using a paired Student’s t-test to compare NDQ80 versus SSQ80.

Techniques Used: Isolation, Western Blot, Activation Assay, Luciferase, Reporter Assay, Transfection, Activity Assay

(A) Mice lacking STAT1 (STAT1 −/− ) showed significantly smaller infarct areas than wt littermates when subjected to ex vivo I/R. (B) Inhibiting autophagy using 3-methyladenine (3-MA, 5 nM) in STAT1 −/− hearts abrogated the protective effects of STAT1. 3-MA treatment also enhanced the infarct size in wt animals. (C) Pre-treatment with the mTOR inhibitor, rapamycin (Rapa, 5 nM), followed by ex vivo I/R reduced infarct size in wt animals suggesting activation of autophagy elicits cardioprotective effects.
Figure Legend Snippet: (A) Mice lacking STAT1 (STAT1 −/− ) showed significantly smaller infarct areas than wt littermates when subjected to ex vivo I/R. (B) Inhibiting autophagy using 3-methyladenine (3-MA, 5 nM) in STAT1 −/− hearts abrogated the protective effects of STAT1. 3-MA treatment also enhanced the infarct size in wt animals. (C) Pre-treatment with the mTOR inhibitor, rapamycin (Rapa, 5 nM), followed by ex vivo I/R reduced infarct size in wt animals suggesting activation of autophagy elicits cardioprotective effects.

Techniques Used: Mouse Assay, Ex Vivo, Activation Assay

30) Product Images from "Enterovirus 71 Disrupts Interferon Signaling by Reducing the Level of Interferon Receptor 1"

Article Title: Enterovirus 71 Disrupts Interferon Signaling by Reducing the Level of Interferon Receptor 1

Journal: Journal of Virology

doi: 10.1128/JVI.06687-11

EV71 inhibited phosphorylation of STAT1/STAT2. RD cells (A and C) or HeLa cells (B and D) were infected with EV71 at an MOI of 1 or 10 for 9 h, and cells were left untreated (−) or treated (+) with IFN-α2b (A and B) or IFN-β (C
Figure Legend Snippet: EV71 inhibited phosphorylation of STAT1/STAT2. RD cells (A and C) or HeLa cells (B and D) were infected with EV71 at an MOI of 1 or 10 for 9 h, and cells were left untreated (−) or treated (+) with IFN-α2b (A and B) or IFN-β (C

Techniques Used: Infection

31) Product Images from "Two Modes of the Axonal Interferon Response Limit Alphaherpesvirus Neuroinvasion"

Article Title: Two Modes of the Axonal Interferon Response Limit Alphaherpesvirus Neuroinvasion

Journal: mBio

doi: 10.1128/mBio.02145-15

Biphasic immune activation against alphaherpesvirus spread from epithelial cells to the PNS neurons. Alphaherpesvirus infection of epithelial cells induces production and secretion of various cytokines, including IFN-β. Exposure of innervating nerve termini to IFN-β limits the transport of alphaherpesviruses. In the first phase (1), STAT1 is phosphorylated and retained in axons, which may cause the reduction of Clip2 proteins that limit the transport of virus capsids into the connected cell body. If the virus infection is not contained in the first phase and the infection progresses without effective control, innate and adaptive immune cells are activated (e.g., natural killer and T cells). These cells produce copious amounts of cytokines, including IFN-γ. In the second phase (2), exposure of axons to IFN-γ results in the nuclear translocation of p-STAT1, which may activate expression of numerous ISGs. In this phase, the transport of virus capsids is limited through reduction of Clip2, while the replication of virus in the cell bodies is also restricted.
Figure Legend Snippet: Biphasic immune activation against alphaherpesvirus spread from epithelial cells to the PNS neurons. Alphaherpesvirus infection of epithelial cells induces production and secretion of various cytokines, including IFN-β. Exposure of innervating nerve termini to IFN-β limits the transport of alphaherpesviruses. In the first phase (1), STAT1 is phosphorylated and retained in axons, which may cause the reduction of Clip2 proteins that limit the transport of virus capsids into the connected cell body. If the virus infection is not contained in the first phase and the infection progresses without effective control, innate and adaptive immune cells are activated (e.g., natural killer and T cells). These cells produce copious amounts of cytokines, including IFN-γ. In the second phase (2), exposure of axons to IFN-γ results in the nuclear translocation of p-STAT1, which may activate expression of numerous ISGs. In this phase, the transport of virus capsids is limited through reduction of Clip2, while the replication of virus in the cell bodies is also restricted.

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

The observed antiviral effect of IFN requires the IFN receptors. (A to D) Mouse SCG neurons were treated with a nonspecific control antibody (Ab) or IFNAR antibody or not treated with antibody prior to the addition of IFN-β for 24 h. (E to H) SCG neurons from WT or IFNAR and IFNGR double knockout mice were treated with IFN-β or IFN-γ for 24 h (or the indicated time) or not treated with IFN (No Treatment). (A and E) Immunoblotting of p-STAT1 in noncompartmentalized mouse SCG neurons. (B, C, F, and G) After axonal IFN treatment, PRV-180 was added to the N compartment. Movement of fluorescent particles in the N compartment was recorded at ~1.5 fps. (B and F) Stack images of representative movies. Each dot represents a stalled particle, and each line represents a moving particle (bars, 10 µm). (C and G) Quantification of the percentage of moving particles. The results are normalized to the values for no-treatment samples. Data are shown as means plus SEMs. Statistical significance by Student’s t test ( n ≥ 3) is indicated as follows: ns, not significant; *, P
Figure Legend Snippet: The observed antiviral effect of IFN requires the IFN receptors. (A to D) Mouse SCG neurons were treated with a nonspecific control antibody (Ab) or IFNAR antibody or not treated with antibody prior to the addition of IFN-β for 24 h. (E to H) SCG neurons from WT or IFNAR and IFNGR double knockout mice were treated with IFN-β or IFN-γ for 24 h (or the indicated time) or not treated with IFN (No Treatment). (A and E) Immunoblotting of p-STAT1 in noncompartmentalized mouse SCG neurons. (B, C, F, and G) After axonal IFN treatment, PRV-180 was added to the N compartment. Movement of fluorescent particles in the N compartment was recorded at ~1.5 fps. (B and F) Stack images of representative movies. Each dot represents a stalled particle, and each line represents a moving particle (bars, 10 µm). (C and G) Quantification of the percentage of moving particles. The results are normalized to the values for no-treatment samples. Data are shown as means plus SEMs. Statistical significance by Student’s t test ( n ≥ 3) is indicated as follows: ns, not significant; *, P

Techniques Used: Double Knockout, Mouse Assay

Axonal IFN-β treatment induces local antiviral responses in axons. (A) Immunoblotting of p-STAT1 in cell bodies in the S compartment and axons in the N compartment after axonal IFN-β or IFN-γ treatment for 24 h. (B) Immunoblotting of p-STAT1 in axons in the absence (−) or presence of IFN-β treatment in the N compartment for 1, 6, or 24 h. (C) Immunofluorescence staining of p-STAT1 in neuron cell bodies after IFN-β or IFN-γ treatment in the N compartment for 24 h. CellTracker Orange (CMRA) labeling in cell bodies indicates that they have axonal connection to the N compartment. The numbers of cells visualized were 78, 118, and 117 for no-treatment, IFN-β-treated, and IFN-γ-treated samples, respectively. Representative images are shown (bars, 20 µm). (D and E) q-PCR quantification of Mx1, IFIT1, or GBP2 expression level in rat SCG with S- or N-compartment treatment of IFN-β (D) or IFN-γ (E). Raw threshold cycle ( C T ) values were normalized to the values for internal control β-actin, and the Δ C T value was calculated by subtracting the normalized C T value of the experimental condition from that of no-treatment sample. Data are shown as means plus SEMs. Statistical significance by Student’s t test ( n = 3) is indicated as follows: ns, not significant; *, P
Figure Legend Snippet: Axonal IFN-β treatment induces local antiviral responses in axons. (A) Immunoblotting of p-STAT1 in cell bodies in the S compartment and axons in the N compartment after axonal IFN-β or IFN-γ treatment for 24 h. (B) Immunoblotting of p-STAT1 in axons in the absence (−) or presence of IFN-β treatment in the N compartment for 1, 6, or 24 h. (C) Immunofluorescence staining of p-STAT1 in neuron cell bodies after IFN-β or IFN-γ treatment in the N compartment for 24 h. CellTracker Orange (CMRA) labeling in cell bodies indicates that they have axonal connection to the N compartment. The numbers of cells visualized were 78, 118, and 117 for no-treatment, IFN-β-treated, and IFN-γ-treated samples, respectively. Representative images are shown (bars, 20 µm). (D and E) q-PCR quantification of Mx1, IFIT1, or GBP2 expression level in rat SCG with S- or N-compartment treatment of IFN-β (D) or IFN-γ (E). Raw threshold cycle ( C T ) values were normalized to the values for internal control β-actin, and the Δ C T value was calculated by subtracting the normalized C T value of the experimental condition from that of no-treatment sample. Data are shown as means plus SEMs. Statistical significance by Student’s t test ( n = 3) is indicated as follows: ns, not significant; *, P

Techniques Used: Immunofluorescence, Staining, Labeling, Polymerase Chain Reaction, Expressing

32) Product Images from "Mapuera virus, a rubulavirus that inhibits interferon signalling in a wide variety of mammalian cells without degrading STATs"

Article Title: Mapuera virus, a rubulavirus that inhibits interferon signalling in a wide variety of mammalian cells without degrading STATs

Journal: The Journal of General Virology

doi: 10.1099/vir.0.82579-0

IFN signalling in bat cells. (a) Luciferase reporter assay. Tb1-Lu cells were transfected as described in the legend to Fig. 1(c) . NiV/V was used as a positive control. Forty-eight hours p.i., the cells were induced or not for 6 h with bat IFN supernatant, and the lysates analysed for luciferase activity. Data are plotted as described in the legend to Fig. 1(c) . (b) STAT degradation. Tb1-Lu cells were infected with MPRV, PIV5 or hPIV2 at 10 m.o.i. or mock-infected. The lysates were analysed by Western blot with antibodies against STAT1, STAT2 and against the P proteins of the different viruses. More than 95 % of the cells were infected at the time of harvest.
Figure Legend Snippet: IFN signalling in bat cells. (a) Luciferase reporter assay. Tb1-Lu cells were transfected as described in the legend to Fig. 1(c) . NiV/V was used as a positive control. Forty-eight hours p.i., the cells were induced or not for 6 h with bat IFN supernatant, and the lysates analysed for luciferase activity. Data are plotted as described in the legend to Fig. 1(c) . (b) STAT degradation. Tb1-Lu cells were infected with MPRV, PIV5 or hPIV2 at 10 m.o.i. or mock-infected. The lysates were analysed by Western blot with antibodies against STAT1, STAT2 and against the P proteins of the different viruses. More than 95 % of the cells were infected at the time of harvest.

Techniques Used: Luciferase, Reporter Assay, Transfection, Positive Control, Activity Assay, Infection, Western Blot

STAT nuclear translocation. Hep2 cells were transfected with a construct encoding myc-tagged MPRV/V. At 48 h p.i., the cells were stimulated with 10 5 IU human IFN- α ml −1 for 70 min or left untreated. The cells were analysed by immunofluorescence with antibodies against (a) the V protein of MPRV (red) and STAT1 (green), or against (b) the V protein of MPRV (red) and STAT2 (green).
Figure Legend Snippet: STAT nuclear translocation. Hep2 cells were transfected with a construct encoding myc-tagged MPRV/V. At 48 h p.i., the cells were stimulated with 10 5 IU human IFN- α ml −1 for 70 min or left untreated. The cells were analysed by immunofluorescence with antibodies against (a) the V protein of MPRV (red) and STAT1 (green), or against (b) the V protein of MPRV (red) and STAT2 (green).

Techniques Used: Translocation Assay, Transfection, Construct, Immunofluorescence

STATs in the presence of MPRV/V. (a) STAT degradation. 2fTGH cells were infected with MPRV strain BeAnn 370284 at 10 m.o.i. or mock-infected. They were stimulated with 3.2×10 4 U human IFN α ml −1 at 16 h p.i. or left untreated. At 25 h p.i., the cells were harvested and the lysates were analysed by Western blot probed with monoclonal antibodies against human STAT1, STAT2, STAT3 and cellular actin, as well as with an antiserum raised against the P and V proteins of MPRV. More than 95 % of the cells were infected at the time of harvest, as determined by immunofluorescence. (b) STAT phosphorylation. 2fTGH and Vero cells were infected as above and stimulated for 20 min with 2.5×10 3 U human IFN- α ml −1 at 22 h p.i. or left untreated. Cells lysates were analysed by Western blot probed with antibodies against human Tyr701-phosphorylated STAT1 or Tyr689-phosphorylated STAT2.
Figure Legend Snippet: STATs in the presence of MPRV/V. (a) STAT degradation. 2fTGH cells were infected with MPRV strain BeAnn 370284 at 10 m.o.i. or mock-infected. They were stimulated with 3.2×10 4 U human IFN α ml −1 at 16 h p.i. or left untreated. At 25 h p.i., the cells were harvested and the lysates were analysed by Western blot probed with monoclonal antibodies against human STAT1, STAT2, STAT3 and cellular actin, as well as with an antiserum raised against the P and V proteins of MPRV. More than 95 % of the cells were infected at the time of harvest, as determined by immunofluorescence. (b) STAT phosphorylation. 2fTGH and Vero cells were infected as above and stimulated for 20 min with 2.5×10 3 U human IFN- α ml −1 at 22 h p.i. or left untreated. Cells lysates were analysed by Western blot probed with antibodies against human Tyr701-phosphorylated STAT1 or Tyr689-phosphorylated STAT2.

Techniques Used: Infection, Western Blot, Immunofluorescence

Interaction of MPRV/V with cellular factors. Purified GST-tagged V proteins or GST alone were coupled to glutathione beads and incubated with human DDB1, STAT1 or STAT2 expressed in the baculovirus system. Proteins bound to beads were separated on a 4–12 % gradient SDS-PAGE gel and visualized by Coomassie staining. Bands corresponding to DDB1, STAT1 and STAT2 are marked by asterisks.
Figure Legend Snippet: Interaction of MPRV/V with cellular factors. Purified GST-tagged V proteins or GST alone were coupled to glutathione beads and incubated with human DDB1, STAT1 or STAT2 expressed in the baculovirus system. Proteins bound to beads were separated on a 4–12 % gradient SDS-PAGE gel and visualized by Coomassie staining. Bands corresponding to DDB1, STAT1 and STAT2 are marked by asterisks.

Techniques Used: Purification, Incubation, SDS Page, Staining

33) Product Images from "Janus Kinase 1 Is Essential for Inflammatory Cytokine Signaling and Mammary Gland Remodeling"

Article Title: Janus Kinase 1 Is Essential for Inflammatory Cytokine Signaling and Mammary Gland Remodeling

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00999-15

JAK1 is essential for the activation of STAT1, STAT3, and STAT6 in embryonic fibroblasts and in mammary epithelial cells. (A) Immunoblot analysis to assess the expression of JAK1 and tyrosine phosphorylation of STAT1, STAT3, STAT5, and STAT6 during mammary
Figure Legend Snippet: JAK1 is essential for the activation of STAT1, STAT3, and STAT6 in embryonic fibroblasts and in mammary epithelial cells. (A) Immunoblot analysis to assess the expression of JAK1 and tyrosine phosphorylation of STAT1, STAT3, STAT5, and STAT6 during mammary

Techniques Used: Activation Assay, Expressing

34) Product Images from "IL-21 Contributes to JAK3/STAT3 Activation and Promotes Cell Growth in ALK-Positive Anaplastic Large Cell Lymphoma"

Article Title: IL-21 Contributes to JAK3/STAT3 Activation and Promotes Cell Growth in ALK-Positive Anaplastic Large Cell Lymphoma

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.080982

Effects of rIL-21 on JAK3, STAT3, and STAT1. A: Western blot studies revealed that treatment of Karpas 299 recombinant IL-21 protein (10 ng/ml) for 30 minutes increased the levels of pJAK3 and pSTAT3. No detectable change in pSTAT1 was noted. B: rIL-21-induced
Figure Legend Snippet: Effects of rIL-21 on JAK3, STAT3, and STAT1. A: Western blot studies revealed that treatment of Karpas 299 recombinant IL-21 protein (10 ng/ml) for 30 minutes increased the levels of pJAK3 and pSTAT3. No detectable change in pSTAT1 was noted. B: rIL-21-induced

Techniques Used: Western Blot, Recombinant

35) Product Images from "Interferon Regulatory Factors IRF5 and IRF7 Inhibit Growth and Induce Senescence in Immortal Li-Fraumeni Fibroblasts"

Article Title: Interferon Regulatory Factors IRF5 and IRF7 Inhibit Growth and Induce Senescence in Immortal Li-Fraumeni Fibroblasts

Journal:

doi: 10.1158/1541-7786.MCR-07-0114

Western blot of 041 PC senescent cells. Western blots for phospho-STAT1 (Tyr 701 ; p-STAT1) , STAT1, IRF7, and IRF5 were done on senescing MDAH041 cells compared with early nonsenescing cells (p13). Tubulin was used as a loading control. 041 PC, MDAH041
Figure Legend Snippet: Western blot of 041 PC senescent cells. Western blots for phospho-STAT1 (Tyr 701 ; p-STAT1) , STAT1, IRF7, and IRF5 were done on senescing MDAH041 cells compared with early nonsenescing cells (p13). Tubulin was used as a loading control. 041 PC, MDAH041

Techniques Used: Western Blot

Western blot analysis of IRF5 and/or IRF7 overexpression in immortal MDAH041 and MDAH087-1 cells. The immortal MDAH041 and MDAH087-1 cells were stably transfected with pCMV-IRF5 and/or pCMV-IRF7. Protein expression levels of IRF5, IRF7, and STAT1 were
Figure Legend Snippet: Western blot analysis of IRF5 and/or IRF7 overexpression in immortal MDAH041 and MDAH087-1 cells. The immortal MDAH041 and MDAH087-1 cells were stably transfected with pCMV-IRF5 and/or pCMV-IRF7. Protein expression levels of IRF5, IRF7, and STAT1 were

Techniques Used: Western Blot, Over Expression, Stable Transfection, Transfection, Expressing

36) Product Images from "Subtype and Regional-Specific Neuroinflammation in Sporadic Creutzfeldt–Jakob Disease"

Article Title: Subtype and Regional-Specific Neuroinflammation in Sporadic Creutzfeldt–Jakob Disease

Journal: Frontiers in Aging Neuroscience

doi: 10.3389/fnagi.2014.00198

Activation of the inflammatory-related signaling pathways NFκB/IKK and JAK/STAT in sCJD is shown . (A) Western blot analysis of NFκB-p65, p-NFκB-p65, and IκBα in the frontal cortex and cerebellum of control, sCJD MM1, and sCJD VV2 cases. Four representative cases are shown. GAPDH immunostaining was used to normalize total protein loading. Densitometry values of western blots result from the analysis of 15 control (Con), 15 MM1, and 15 VV2 cases. Region- and subtype-dependent significant increased expression is found in sCJD when compared to controls. * p > 0.05, ** p > 0.005 control versus sCJD; # p > 0.05 sCJD MM1 versus VV2. AU: arbitrary units. (B) ELISA of p-STAT1 and p-STAT3 in the frontal cortex of control (Con), sCJD MM1 (MM1), and sCJD VV2 (VV2) cases. Total values result from the analysis of eight controls, eight MM1, and eight VV2 cases. (C) Western blotting of STAT1 and STAT3 in the frontal cortex of control (Con), sCJD MM1, and sCJD VV2 cases. Three representative cases of a total of 15 cases analyzed are shown. GAPDH immunostaining was used to normalize total protein loading. * p > 0.05, ** p > 0.0 05 control versus sCJD; # p > 0.05 sCJD MM1 versus sCJD VV2. AU: arbitrary units.
Figure Legend Snippet: Activation of the inflammatory-related signaling pathways NFκB/IKK and JAK/STAT in sCJD is shown . (A) Western blot analysis of NFκB-p65, p-NFκB-p65, and IκBα in the frontal cortex and cerebellum of control, sCJD MM1, and sCJD VV2 cases. Four representative cases are shown. GAPDH immunostaining was used to normalize total protein loading. Densitometry values of western blots result from the analysis of 15 control (Con), 15 MM1, and 15 VV2 cases. Region- and subtype-dependent significant increased expression is found in sCJD when compared to controls. * p > 0.05, ** p > 0.005 control versus sCJD; # p > 0.05 sCJD MM1 versus VV2. AU: arbitrary units. (B) ELISA of p-STAT1 and p-STAT3 in the frontal cortex of control (Con), sCJD MM1 (MM1), and sCJD VV2 (VV2) cases. Total values result from the analysis of eight controls, eight MM1, and eight VV2 cases. (C) Western blotting of STAT1 and STAT3 in the frontal cortex of control (Con), sCJD MM1, and sCJD VV2 cases. Three representative cases of a total of 15 cases analyzed are shown. GAPDH immunostaining was used to normalize total protein loading. * p > 0.05, ** p > 0.0 05 control versus sCJD; # p > 0.05 sCJD MM1 versus sCJD VV2. AU: arbitrary units.

Techniques Used: Activation Assay, Western Blot, Immunostaining, Expressing, Enzyme-linked Immunosorbent Assay

37) Product Images from "Identification of Host-Chromosome Binding Sites and Candidate Gene Targets for Kaposi's Sarcoma-Associated Herpesvirus LANA"

Article Title: Identification of Host-Chromosome Binding Sites and Candidate Gene Targets for Kaposi's Sarcoma-Associated Herpesvirus LANA

Journal: Journal of Virology

doi: 10.1128/JVI.07216-11

Example of LANA and STAT1 binding peaks near transcriptional start sites of cellular genes. The UCSC genome browser was used to map LANA peaks (black) and STAT1 IFN-γ-stimulated (blue, top) and unstimulated (blue, bottom) peaks to cellular genes for TAP1 (A), IQGAP3 (B), FBXO4 (C), and PARL (D). RefSeq annotated transcripts are indicated below each ChIP-Seq peak.
Figure Legend Snippet: Example of LANA and STAT1 binding peaks near transcriptional start sites of cellular genes. The UCSC genome browser was used to map LANA peaks (black) and STAT1 IFN-γ-stimulated (blue, top) and unstimulated (blue, bottom) peaks to cellular genes for TAP1 (A), IQGAP3 (B), FBXO4 (C), and PARL (D). RefSeq annotated transcripts are indicated below each ChIP-Seq peak.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation

Ectopic expression of LANA inhibits IFN-γ-induced TAP1 gene expression. (A) KSHV-negative DG75 was transfected with control vector or with FLAG-LANA expression vector and pmaxGFP. At 24 h after transfection, the cells were induced with 5 ng of human recombinant IFN-γ/ml for another 24 h. The GFP-positive cells were then sorted out and assayed by RT-PCR for IFN-γ-induced transcription of TAP1, PSMB9, PARL, FBXO4, IQGAP3, and NIPBL, as indicated. (B) BCBL1 cells were stimulated or unstimulated with IFN-γ for 30 min and then subjected to immunoprecipitation with antibodies to IgG, LANA, or STAT1. Immunoprecipitates were analyzed by Western blotting for LANA and then reprobed for STAT1 or STAT3 as indicated. (C) DG75 cells were transfected with control vector or with FLAG-LANA expression vector and pmaxGFP. At 24 h after transfection, GFP-positive cells were sorted, then treated or not treated with 5 ng of IFN-γ for 30 min/ml, and assayed by ChIP with antibodies to IgG and STAT1 at the TAP1/PSMB9 site.
Figure Legend Snippet: Ectopic expression of LANA inhibits IFN-γ-induced TAP1 gene expression. (A) KSHV-negative DG75 was transfected with control vector or with FLAG-LANA expression vector and pmaxGFP. At 24 h after transfection, the cells were induced with 5 ng of human recombinant IFN-γ/ml for another 24 h. The GFP-positive cells were then sorted out and assayed by RT-PCR for IFN-γ-induced transcription of TAP1, PSMB9, PARL, FBXO4, IQGAP3, and NIPBL, as indicated. (B) BCBL1 cells were stimulated or unstimulated with IFN-γ for 30 min and then subjected to immunoprecipitation with antibodies to IgG, LANA, or STAT1. Immunoprecipitates were analyzed by Western blotting for LANA and then reprobed for STAT1 or STAT3 as indicated. (C) DG75 cells were transfected with control vector or with FLAG-LANA expression vector and pmaxGFP. At 24 h after transfection, GFP-positive cells were sorted, then treated or not treated with 5 ng of IFN-γ for 30 min/ml, and assayed by ChIP with antibodies to IgG and STAT1 at the TAP1/PSMB9 site.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Recombinant, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation

38) Product Images from "Two different STAT1 gain-of-function mutations lead to diverse IFN-γ-mediated gene expression"

Article Title: Two different STAT1 gain-of-function mutations lead to diverse IFN-γ-mediated gene expression

Journal: NPJ Genomic Medicine

doi: 10.1038/s41525-018-0063-6

Abnormal STAT1 function and gene expression. a IL-17 (left panel), IFN-γ (middle panel) and IL-2 (right panel) secretion by PBMCs following phytohemagglutinin (PHA) stimulation for 48 h. Levels of indicated cytokines were determined by ELISA in triplicate samples. NS not stimulated. Western blot of activating STAT1 phosphorylation (anti-pTyr701) following stimulation with b IL-27 (10 µg/µL) and c IFN-α (8.5 ng/μL) in patient and control T-cell lysates. Anti-actin was used as a loading control. d STAT1 phosphorylation in STAT1 wild-type (WT), H629Y- and R274G-transfected U3A cells, following stimulation with IFN-γ (100 ng/mL). e Increase in phosphorylated STAT1 in the nuclear fraction of transfected U3A cells following IFN-γ stimulation. Anti-tubulin was used as the cytoplasmic marker and anti-RCC1 as the nuclear marker. Cyt cytoplasmic, Nuc nuclear. f Venn diagram of 1.5-fold change in IFN-γ-inducible genes. Diagram shows microarray analysis of upregulated genes following IFN-γ stimulation (100 ng/mL for 8 h) in U3A cells transfected with WT, H629Y or R274G STAT1. g U3A cells transfected as indicated with WT, H629Y or R274G STAT1 were stimulated for 8 h with IFN-γ (100 ng/mL) and mRNA levels of CXCL10, CXCL9, GBP1 and IRF1 were determined by q-PCR. Expression data, normalised to the levels of the house-keeping gene GAPDH , are presented as means and standard deviations from a total of 3–6 experiments. * P
Figure Legend Snippet: Abnormal STAT1 function and gene expression. a IL-17 (left panel), IFN-γ (middle panel) and IL-2 (right panel) secretion by PBMCs following phytohemagglutinin (PHA) stimulation for 48 h. Levels of indicated cytokines were determined by ELISA in triplicate samples. NS not stimulated. Western blot of activating STAT1 phosphorylation (anti-pTyr701) following stimulation with b IL-27 (10 µg/µL) and c IFN-α (8.5 ng/μL) in patient and control T-cell lysates. Anti-actin was used as a loading control. d STAT1 phosphorylation in STAT1 wild-type (WT), H629Y- and R274G-transfected U3A cells, following stimulation with IFN-γ (100 ng/mL). e Increase in phosphorylated STAT1 in the nuclear fraction of transfected U3A cells following IFN-γ stimulation. Anti-tubulin was used as the cytoplasmic marker and anti-RCC1 as the nuclear marker. Cyt cytoplasmic, Nuc nuclear. f Venn diagram of 1.5-fold change in IFN-γ-inducible genes. Diagram shows microarray analysis of upregulated genes following IFN-γ stimulation (100 ng/mL for 8 h) in U3A cells transfected with WT, H629Y or R274G STAT1. g U3A cells transfected as indicated with WT, H629Y or R274G STAT1 were stimulated for 8 h with IFN-γ (100 ng/mL) and mRNA levels of CXCL10, CXCL9, GBP1 and IRF1 were determined by q-PCR. Expression data, normalised to the levels of the house-keeping gene GAPDH , are presented as means and standard deviations from a total of 3–6 experiments. * P

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Marker, Microarray, Polymerase Chain Reaction

39) Product Images from "Noncanonical Effects of IRF9 in Intestinal Inflammation: More than Type I and Type III Interferons"

Article Title: Noncanonical Effects of IRF9 in Intestinal Inflammation: More than Type I and Type III Interferons

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01498-14

CXCL10 expression in the colon is not dependent on IFN-I and -III signaling but requires IRF9 and Stat1. (A) qPCR analysis of IRF7, IFIT2, ISG15, CXCL9, and CXCL10 expression in colon tissue from 5-day-DSS-treated mice, normalized to the OAZ1 housekeeping gene ( n = 6 to 14/genotype). (B) CXCL10 protein levels in healthy and inflamed colon tissue were determined by bead assay ( n = 5 to 13/genotype). Data are presented as means ± SEM. *, P
Figure Legend Snippet: CXCL10 expression in the colon is not dependent on IFN-I and -III signaling but requires IRF9 and Stat1. (A) qPCR analysis of IRF7, IFIT2, ISG15, CXCL9, and CXCL10 expression in colon tissue from 5-day-DSS-treated mice, normalized to the OAZ1 housekeeping gene ( n = 6 to 14/genotype). (B) CXCL10 protein levels in healthy and inflamed colon tissue were determined by bead assay ( n = 5 to 13/genotype). Data are presented as means ± SEM. *, P

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

), a complex designated ISGF3 II , containing phosphorylated Stat1, unphosphorylated Stat2, and IRF9, may explain the Stat2 dependence. A contribution by Stat1-IRF9 complexes or GAF at this stage is likely but speculative.
Figure Legend Snippet: ), a complex designated ISGF3 II , containing phosphorylated Stat1, unphosphorylated Stat2, and IRF9, may explain the Stat2 dependence. A contribution by Stat1-IRF9 complexes or GAF at this stage is likely but speculative.

Techniques Used:

Stat-IRF9 association with IFN-responsive elements of the CXCL10 gene. (A) Representation of the CXCL10 gene binding sites for Stat1 and Stat2 as determined by ChIP-Seq. ChIP with antibodies to Stat1 and Stat2 was performed using BMM after stimulation with IFN-β for 2 h, followed by next-generation sequencing. (B) BMM with indicated genotypes, treated with IFN-γ for the indicated times, were subjected to ChIP with antibodies to Stat1 and Stat2. E1 and E2 IFN response regions were amplified by qPCR. (C) Left and middle panels, amplification of the Mx promoter containing the ISRE region after ChIP with antibodies against IRF9 and re-ChIP with Stat1 and Stat2 antibodies. Macrophages were treated with IFN-β for the indicated time. Right panel, amplification of the CXCL10 promoter region after ChIP with an antibody against Stat1 and reprecipitation with an antibody against IRF9 from chromatin of macrophages with indicated genotypes, treated for 4 h with IFN-γ. Three technical replicates from one of three independent experiments are shown. n.d., not detectable.
Figure Legend Snippet: Stat-IRF9 association with IFN-responsive elements of the CXCL10 gene. (A) Representation of the CXCL10 gene binding sites for Stat1 and Stat2 as determined by ChIP-Seq. ChIP with antibodies to Stat1 and Stat2 was performed using BMM after stimulation with IFN-β for 2 h, followed by next-generation sequencing. (B) BMM with indicated genotypes, treated with IFN-γ for the indicated times, were subjected to ChIP with antibodies to Stat1 and Stat2. E1 and E2 IFN response regions were amplified by qPCR. (C) Left and middle panels, amplification of the Mx promoter containing the ISRE region after ChIP with antibodies against IRF9 and re-ChIP with Stat1 and Stat2 antibodies. Macrophages were treated with IFN-β for the indicated time. Right panel, amplification of the CXCL10 promoter region after ChIP with an antibody against Stat1 and reprecipitation with an antibody against IRF9 from chromatin of macrophages with indicated genotypes, treated for 4 h with IFN-γ. Three technical replicates from one of three independent experiments are shown. n.d., not detectable.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Next-Generation Sequencing, Amplification, Real-time Polymerase Chain Reaction

40) Product Images from "Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN-? target genes"

Article Title: Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN-? target genes

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

doi:

Identification of the STAT1-binding segment of BRCA1. Six GST-BRCA1 fusion proteins (aa 1–324; 260–553; 502–802; 758–1,064; 1,005–1,313; and 1,314–1,863) were generated in E. coli , and used for an in vitro binding assay (from lanes 4 to 9, respectively). GST protein alone was used for lane 3. Approximately equal amounts of each GST-fusion protein, bound to glutathione-Sepharose beads, were incubated with an extract of IFN-γ-treated (10 ng/ml, 15 min) 293T cells. Bound proteins were recovered and separated electrophoretically. Total lysate (20 μg) was loaded as a control (lane 1). Lane 2 contains no protein. The separated proteins were immunoblotted for STAT1. This STAT1 antiserum recognizes a 91-kDa (STAT1α), not 84-kDa (STAT1β), protein bound to GST-BRCA1 (aa 502–802) in lane 6, showing that STAT1α specifically binds to BRCA1.
Figure Legend Snippet: Identification of the STAT1-binding segment of BRCA1. Six GST-BRCA1 fusion proteins (aa 1–324; 260–553; 502–802; 758–1,064; 1,005–1,313; and 1,314–1,863) were generated in E. coli , and used for an in vitro binding assay (from lanes 4 to 9, respectively). GST protein alone was used for lane 3. Approximately equal amounts of each GST-fusion protein, bound to glutathione-Sepharose beads, were incubated with an extract of IFN-γ-treated (10 ng/ml, 15 min) 293T cells. Bound proteins were recovered and separated electrophoretically. Total lysate (20 μg) was loaded as a control (lane 1). Lane 2 contains no protein. The separated proteins were immunoblotted for STAT1. This STAT1 antiserum recognizes a 91-kDa (STAT1α), not 84-kDa (STAT1β), protein bound to GST-BRCA1 (aa 502–802) in lane 6, showing that STAT1α specifically binds to BRCA1.

Techniques Used: Binding Assay, Generated, In Vitro, Incubation

STAT1α and BRCA1 form a complex both in vitro and in vivo . ( A and B ) Coimmunoprecipitation of endogenous BRCA1 and STAT1α from IFN-γ-treated 293T cells. BRCA1 was immunoprecipitated from 1.2 mg of IFN-γ-treated (10 ng/ml, 12 h) (lane 3) or untreated (lane 4) cells. Total cell lysates (20 μg) were also analyzed for the positive control of protein expression (lanes 1 and 2). Samples were separated by 6% SDS/PAGE, and immunoblotted with ( A ) anti-STAT1 antibody or ( B ) anti-BRCA1 antibody . ( C ) Copurification of STAT1 with BRCA1. GST-BRCA1 and FLAG-tagged STAT1α or STAT1αSer-727A were cotransfected into 293T cells. For controls, separate plates were transfected with GST vector alone or GST-BRCA1 expression vector with or without FLAG-tagged STAT1α expression vector in the combinations shown at the bottom of the panel. Cells were treated with IFN-γ as indicated (10 ng/ml, 12 h) (lanes 3–6), and GST or GST-BRCA1 was purified from extracts with glutathione beads. Samples were separated by 6% SDS/PAGE, and immunoblotted with anti-FLAG antibody. ( D , E , and F ) Detection of exogenously expressed proteins. Total cell lysates (20 μg) were separated by 6% ( D and E ) or 10% ( F ) SDS/PAGE, and the expression level of FLAG-tagged STAT1α ( D ), GST-BRCA1 ( E ), and GST ( F ) were confirmed by immunoblot analysis by using anti-FLAG or anti-GST antibody.
Figure Legend Snippet: STAT1α and BRCA1 form a complex both in vitro and in vivo . ( A and B ) Coimmunoprecipitation of endogenous BRCA1 and STAT1α from IFN-γ-treated 293T cells. BRCA1 was immunoprecipitated from 1.2 mg of IFN-γ-treated (10 ng/ml, 12 h) (lane 3) or untreated (lane 4) cells. Total cell lysates (20 μg) were also analyzed for the positive control of protein expression (lanes 1 and 2). Samples were separated by 6% SDS/PAGE, and immunoblotted with ( A ) anti-STAT1 antibody or ( B ) anti-BRCA1 antibody . ( C ) Copurification of STAT1 with BRCA1. GST-BRCA1 and FLAG-tagged STAT1α or STAT1αSer-727A were cotransfected into 293T cells. For controls, separate plates were transfected with GST vector alone or GST-BRCA1 expression vector with or without FLAG-tagged STAT1α expression vector in the combinations shown at the bottom of the panel. Cells were treated with IFN-γ as indicated (10 ng/ml, 12 h) (lanes 3–6), and GST or GST-BRCA1 was purified from extracts with glutathione beads. Samples were separated by 6% SDS/PAGE, and immunoblotted with anti-FLAG antibody. ( D , E , and F ) Detection of exogenously expressed proteins. Total cell lysates (20 μg) were separated by 6% ( D and E ) or 10% ( F ) SDS/PAGE, and the expression level of FLAG-tagged STAT1α ( D ), GST-BRCA1 ( E ), and GST ( F ) were confirmed by immunoblot analysis by using anti-FLAG or anti-GST antibody.

Techniques Used: In Vitro, In Vivo, Immunoprecipitation, Positive Control, Expressing, SDS Page, Copurification, Transfection, Plasmid Preparation, Purification

Mammalian two-hybrid analysis demonstrates functional interaction between BRCA1 and STAT1. Both BRCA1 (502–802) segment fused with GAL4AD (AD-BRCA1) and full-length BRCA1 (BRCA1) can hyperactivate a STAT1α C-terminal transcriptional activation domain fused with GAL4 DBD (DBD-STAT). The reporter plasmids contain four copies of GAL4-binding sites upstream of the luciferase gene. Each construct shown at the bottom of the panel was expressed in 293T cells transiently with (+) or without (−) IFN-γ treatment (10 ng/ml, 6 h). Relative activity was normalized with a β-galactosidase control. In all cases, luciferase activity was determined at 48 h posttransfection.
Figure Legend Snippet: Mammalian two-hybrid analysis demonstrates functional interaction between BRCA1 and STAT1. Both BRCA1 (502–802) segment fused with GAL4AD (AD-BRCA1) and full-length BRCA1 (BRCA1) can hyperactivate a STAT1α C-terminal transcriptional activation domain fused with GAL4 DBD (DBD-STAT). The reporter plasmids contain four copies of GAL4-binding sites upstream of the luciferase gene. Each construct shown at the bottom of the panel was expressed in 293T cells transiently with (+) or without (−) IFN-γ treatment (10 ng/ml, 6 h). Relative activity was normalized with a β-galactosidase control. In all cases, luciferase activity was determined at 48 h posttransfection.

Techniques Used: Functional Assay, Activation Assay, Binding Assay, Luciferase, Construct, Activity Assay

Related Articles

Immunoprecipitation:

Article Title: Stat1 Acetylation Inhibits Inducible Nitric Oxide Synthase Expression in Interferon-γ Treated RAW264.7 Murine Macrophages
Article Snippet: .. Purified chromatin was immunoprecipitated using 10 µg of anti-NF-κB p65 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Stat1 (Santa Cruz Biotechnology, Santa Cruz, CA) or 5 µl of rabbit nonimmune serum; eluted DNA fragments were purified to serve as templates. .. The input fraction corresponded to 0.1 and 0.05% of the chromatin solution before immunoprecipitation.

Incubation:

Article Title: Altered Levels of STAT1 and STAT3 Influence the Neuronal Response to Interferon Gamma
Article Snippet: .. After three washes in PBS-T (5 min each), the blots were incubated in secondary antibody solution (goat anti-rabbit horseradish peroxidase (HRP; 1:1000; Vector Laboratories Inc.) for anti-STAT1 and anti-STAT3, goat anti-mouse HRP (1:2000; Santa Cruz Biotechnology Inc.) for anti-STAT1pY701 and anti-STAT3pY705; all diluted in PBS-T) for 1 h at room temperature. .. The blots were washed as described above, incubated in ECL detection solution (Amersham Biosciences, Little Chalfont, Buckinghamshire UK), and exposed to autoradiography film until a suitable image was obtained.

Article Title: Dysregulation of TLR3 Impairs the Innate Immune Response to West Nile Virus in the Elderly ▿
Article Snippet: .. Equal protein concentrations of the supernatants were incubated overnight with 5 μg of anti-STAT1 antibody. .. A fraction of the supernatant was immunoblotted to ensure that equal amounts of STAT1 protein were used in the immunoprecipitation.

Purification:

Article Title: Stat1 Acetylation Inhibits Inducible Nitric Oxide Synthase Expression in Interferon-γ Treated RAW264.7 Murine Macrophages
Article Snippet: .. Purified chromatin was immunoprecipitated using 10 µg of anti-NF-κB p65 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Stat1 (Santa Cruz Biotechnology, Santa Cruz, CA) or 5 µl of rabbit nonimmune serum; eluted DNA fragments were purified to serve as templates. .. The input fraction corresponded to 0.1 and 0.05% of the chromatin solution before immunoprecipitation.

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    Santa Cruz Biotechnology anti stat1
    Other TFs or signaling proteins are present in MAM but not in mitochondria. Western blot analysis of the Percoll density centrifugation result showed that TFs or signaling proteins including <t>STAT1,</t> MAPKs, AMPK, AKT, mTOR, and RELA could only be found in the MAM fractions but not in pure mitochondria. Mito.C., crude mitochondrial fraction; Mito.P., pure mitochondrial fraction; MAM.H, heavy MAM; MAM.L, light MAM.
    Anti Stat1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cell signaling technology inc anti stat1
    Localized <t>STAT1</t> activation in Ccr2 −/− tumors a. Comparison of STAT1 activation in tumors from Ccr2 +/+ or Ccr2 −/− cancer cells during the growth-restricted phase, by Western blot analysis (left). Each lane contains a protein sample from a tumor from a different mouse. Quantifications of protein expression normalized to β-actin levels (right) (mean +/− SEM, n=4 for Ccr2 +/+ , n=5 for Ccr2 −/− ). b. Representative photomicrographs of p-STAT1 (top) and p-STAT3 (bottom) staining in sections of tumors from Ccr2 +/+ or Ccr2 −/− cancer cells. Scale bar=100 μm. c. Quantification of the immunohistochemical staining in (b), using H-score, shows higher STAT1 phosphorylation in tumors from Ccr2 −/− than from Ccr2 +/+ cancer cells, but similar levels of STAT3 phosphorylation (mean +/− SEM, Student’s t-test; n= 5).
    Anti Stat1, supplied by cell signaling technology inc, used in various techniques. Bioz Stars score: 94/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Other TFs or signaling proteins are present in MAM but not in mitochondria. Western blot analysis of the Percoll density centrifugation result showed that TFs or signaling proteins including STAT1, MAPKs, AMPK, AKT, mTOR, and RELA could only be found in the MAM fractions but not in pure mitochondria. Mito.C., crude mitochondrial fraction; Mito.P., pure mitochondrial fraction; MAM.H, heavy MAM; MAM.L, light MAM.

    Journal: bioRxiv

    Article Title: STAT3 localizes in mitochondria-associated ER membranes instead of in mitochondria

    doi: 10.1101/2019.12.18.880567

    Figure Lengend Snippet: Other TFs or signaling proteins are present in MAM but not in mitochondria. Western blot analysis of the Percoll density centrifugation result showed that TFs or signaling proteins including STAT1, MAPKs, AMPK, AKT, mTOR, and RELA could only be found in the MAM fractions but not in pure mitochondria. Mito.C., crude mitochondrial fraction; Mito.P., pure mitochondrial fraction; MAM.H, heavy MAM; MAM.L, light MAM.

    Article Snippet: Samples were then transferred to PVDF membrane (Thermo) and immunoblotted using anti-NDUFA9 (Invitrogen), anti-NDUFS3 (Invitrogen), anti-NDUFA13 (Invitrogen), anti-ATP5A (Invitrogen), anti-SDHA (CST), anti-VDAC (CST), anti-HSP60 (CST), anti-PHB1 (CST), anti-PDH (CST), anti-GAPDH (Sigma), anti-GRP78 (SCBT), anti-ABCA1 (SCBT), anti-FLOT1 (CST), anti-STAT3 (CST), anti-STAT1 (SCBT), anti-AMPK (CST), anti-ERK1/2 (CST), anti-p38 (CST), anti-LC3 (CST), all diluted in 5% BSA:TBST at 1:1000, followed by appropriate HRP-conjugated secondary antibodies (Thermo) incubation (diluted in 5% non-fat milk:TBST at 1:10,000), and developed using the SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo).

    Techniques: Western Blot, Centrifugation

    ActD and flavopiridol inhibit STAT1 dephosphorylation independently of p53- and miRNA-mediated mechanisms. (A) Transcription inhibition by flavopiridol (FP) impairs STAT1 and STAT2 tyrosine dephosphorylation. BMDMs were either left untreated or pretreated

    Journal: Molecular and Cellular Biology

    Article Title: Promoter Occupancy of STAT1 in Interferon Responses Is Regulated by Processive Transcription

    doi: 10.1128/MCB.01097-14

    Figure Lengend Snippet: ActD and flavopiridol inhibit STAT1 dephosphorylation independently of p53- and miRNA-mediated mechanisms. (A) Transcription inhibition by flavopiridol (FP) impairs STAT1 and STAT2 tyrosine dephosphorylation. BMDMs were either left untreated or pretreated

    Article Snippet: For chromatin immunoprecipitation (ChIP), antibodies against STAT1 (2 μg/sample; catalog no. sc-346X; Santa Cruz), RNA polymerase II (RNAPII) (5 μg/sample; catalog no. sc-899; Santa Cruz), NF-κB (4 μg/sample; catalog no. sc-372; Santa Cruz), and an IgG control (preimmune serum [ ]; 2 μl/sample) were used.

    Techniques: De-Phosphorylation Assay, Inhibition

    Inhibition of transcription is permissive for Y701 dephosphorylation of nucleoplasmic STAT1. (A) STAT1 and STAT2 tyrosine dephosphorylation in the absence of Irf9. WT and Irf9 −/− BMDMs were stimulated with IFN-β for the indicated

    Journal: Molecular and Cellular Biology

    Article Title: Promoter Occupancy of STAT1 in Interferon Responses Is Regulated by Processive Transcription

    doi: 10.1128/MCB.01097-14

    Figure Lengend Snippet: Inhibition of transcription is permissive for Y701 dephosphorylation of nucleoplasmic STAT1. (A) STAT1 and STAT2 tyrosine dephosphorylation in the absence of Irf9. WT and Irf9 −/− BMDMs were stimulated with IFN-β for the indicated

    Article Snippet: For chromatin immunoprecipitation (ChIP), antibodies against STAT1 (2 μg/sample; catalog no. sc-346X; Santa Cruz), RNA polymerase II (RNAPII) (5 μg/sample; catalog no. sc-899; Santa Cruz), NF-κB (4 μg/sample; catalog no. sc-372; Santa Cruz), and an IgG control (preimmune serum [ ]; 2 μl/sample) were used.

    Techniques: Inhibition, De-Phosphorylation Assay

    Analysis of IFN-β-induced gene expression and STAT1 promoter occupancy in BMDMs expressing solely the STAT1β isoform. (A and B) IFN-β-induced transcription in WT and STAT1β BMDMs. Cells were stimulated with IFN-β;

    Journal: Molecular and Cellular Biology

    Article Title: Promoter Occupancy of STAT1 in Interferon Responses Is Regulated by Processive Transcription

    doi: 10.1128/MCB.01097-14

    Figure Lengend Snippet: Analysis of IFN-β-induced gene expression and STAT1 promoter occupancy in BMDMs expressing solely the STAT1β isoform. (A and B) IFN-β-induced transcription in WT and STAT1β BMDMs. Cells were stimulated with IFN-β;

    Article Snippet: For chromatin immunoprecipitation (ChIP), antibodies against STAT1 (2 μg/sample; catalog no. sc-346X; Santa Cruz), RNA polymerase II (RNAPII) (5 μg/sample; catalog no. sc-899; Santa Cruz), NF-κB (4 μg/sample; catalog no. sc-372; Santa Cruz), and an IgG control (preimmune serum [ ]; 2 μl/sample) were used.

    Techniques: Expressing

    Transcription inhibition by ActD prolongs STAT1 but not NF-κB occupancy at target promoters. (A and B) Transcription inhibition prolongs STAT1 occupancy at the Irf1 and Mx2 promoters. BMDMs were stimulated with IFN-β in the presence or

    Journal: Molecular and Cellular Biology

    Article Title: Promoter Occupancy of STAT1 in Interferon Responses Is Regulated by Processive Transcription

    doi: 10.1128/MCB.01097-14

    Figure Lengend Snippet: Transcription inhibition by ActD prolongs STAT1 but not NF-κB occupancy at target promoters. (A and B) Transcription inhibition prolongs STAT1 occupancy at the Irf1 and Mx2 promoters. BMDMs were stimulated with IFN-β in the presence or

    Article Snippet: For chromatin immunoprecipitation (ChIP), antibodies against STAT1 (2 μg/sample; catalog no. sc-346X; Santa Cruz), RNA polymerase II (RNAPII) (5 μg/sample; catalog no. sc-899; Santa Cruz), NF-κB (4 μg/sample; catalog no. sc-372; Santa Cruz), and an IgG control (preimmune serum [ ]; 2 μl/sample) were used.

    Techniques: Inhibition

    Tyrosine dephosphorylation of IFN-activated STAT1 is dependent on ongoing transcription. (A) Kinetics of STAT1 Y701 dephosphorylation after type I IFN stimulation. BMDMs were stimulated with IFN-β for the indicated times, and cell extracts were

    Journal: Molecular and Cellular Biology

    Article Title: Promoter Occupancy of STAT1 in Interferon Responses Is Regulated by Processive Transcription

    doi: 10.1128/MCB.01097-14

    Figure Lengend Snippet: Tyrosine dephosphorylation of IFN-activated STAT1 is dependent on ongoing transcription. (A) Kinetics of STAT1 Y701 dephosphorylation after type I IFN stimulation. BMDMs were stimulated with IFN-β for the indicated times, and cell extracts were

    Article Snippet: For chromatin immunoprecipitation (ChIP), antibodies against STAT1 (2 μg/sample; catalog no. sc-346X; Santa Cruz), RNA polymerase II (RNAPII) (5 μg/sample; catalog no. sc-899; Santa Cruz), NF-κB (4 μg/sample; catalog no. sc-372; Santa Cruz), and an IgG control (preimmune serum [ ]; 2 μl/sample) were used.

    Techniques: De-Phosphorylation Assay

    Processive transcription is required for downregulation of STAT1 promoter occupancy but not for Y701 dephosphorylation. (A) Pervanadate (Na 3 VO 4 ) inhibits the tyrosine dephosphorylation of STAT1 and STAT2. BMDMs were stimulated with IFN-β in the

    Journal: Molecular and Cellular Biology

    Article Title: Promoter Occupancy of STAT1 in Interferon Responses Is Regulated by Processive Transcription

    doi: 10.1128/MCB.01097-14

    Figure Lengend Snippet: Processive transcription is required for downregulation of STAT1 promoter occupancy but not for Y701 dephosphorylation. (A) Pervanadate (Na 3 VO 4 ) inhibits the tyrosine dephosphorylation of STAT1 and STAT2. BMDMs were stimulated with IFN-β in the

    Article Snippet: For chromatin immunoprecipitation (ChIP), antibodies against STAT1 (2 μg/sample; catalog no. sc-346X; Santa Cruz), RNA polymerase II (RNAPII) (5 μg/sample; catalog no. sc-899; Santa Cruz), NF-κB (4 μg/sample; catalog no. sc-372; Santa Cruz), and an IgG control (preimmune serum [ ]; 2 μl/sample) were used.

    Techniques: De-Phosphorylation Assay

    Transcription inhibition by flavopiridol and DRB prolongs STAT1 promoter occupancy. (A and B) Flavopiridol (FP) inhibits IFN-β-induced transcription. BMDMs that had been left untreated (w/o) or pretreated with FP for 15 min were stimulated with

    Journal: Molecular and Cellular Biology

    Article Title: Promoter Occupancy of STAT1 in Interferon Responses Is Regulated by Processive Transcription

    doi: 10.1128/MCB.01097-14

    Figure Lengend Snippet: Transcription inhibition by flavopiridol and DRB prolongs STAT1 promoter occupancy. (A and B) Flavopiridol (FP) inhibits IFN-β-induced transcription. BMDMs that had been left untreated (w/o) or pretreated with FP for 15 min were stimulated with

    Article Snippet: For chromatin immunoprecipitation (ChIP), antibodies against STAT1 (2 μg/sample; catalog no. sc-346X; Santa Cruz), RNA polymerase II (RNAPII) (5 μg/sample; catalog no. sc-899; Santa Cruz), NF-κB (4 μg/sample; catalog no. sc-372; Santa Cruz), and an IgG control (preimmune serum [ ]; 2 μl/sample) were used.

    Techniques: Inhibition

    Mnk1 and Mnk2 are not required for IFNγ-mediated engagement of STAT1 or activation of transcription via GAS elements. A , Mnk1/2 +/+ , Mnk1 −/− , Mnk2 −/− , and Mnk1/2 −/− MEFs were treated with IFNγ

    Journal: The Journal of Biological Chemistry

    Article Title: Essential Role for Mnk Kinases in Type II Interferon (IFN?) Signaling and Its Suppressive Effects on Normal Hematopoiesis *

    doi: 10.1074/jbc.M110.197921

    Figure Lengend Snippet: Mnk1 and Mnk2 are not required for IFNγ-mediated engagement of STAT1 or activation of transcription via GAS elements. A , Mnk1/2 +/+ , Mnk1 −/− , Mnk2 −/− , and Mnk1/2 −/− MEFs were treated with IFNγ

    Article Snippet: The antibodies against STAT1 and IRF-1 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Activation Assay

    Localized STAT1 activation in Ccr2 −/− tumors a. Comparison of STAT1 activation in tumors from Ccr2 +/+ or Ccr2 −/− cancer cells during the growth-restricted phase, by Western blot analysis (left). Each lane contains a protein sample from a tumor from a different mouse. Quantifications of protein expression normalized to β-actin levels (right) (mean +/− SEM, n=4 for Ccr2 +/+ , n=5 for Ccr2 −/− ). b. Representative photomicrographs of p-STAT1 (top) and p-STAT3 (bottom) staining in sections of tumors from Ccr2 +/+ or Ccr2 −/− cancer cells. Scale bar=100 μm. c. Quantification of the immunohistochemical staining in (b), using H-score, shows higher STAT1 phosphorylation in tumors from Ccr2 −/− than from Ccr2 +/+ cancer cells, but similar levels of STAT3 phosphorylation (mean +/− SEM, Student’s t-test; n= 5).

    Journal: bioRxiv

    Article Title: Cancer cell CCR2 orchestrates suppression of the adaptive immune response

    doi: 10.1101/390187

    Figure Lengend Snippet: Localized STAT1 activation in Ccr2 −/− tumors a. Comparison of STAT1 activation in tumors from Ccr2 +/+ or Ccr2 −/− cancer cells during the growth-restricted phase, by Western blot analysis (left). Each lane contains a protein sample from a tumor from a different mouse. Quantifications of protein expression normalized to β-actin levels (right) (mean +/− SEM, n=4 for Ccr2 +/+ , n=5 for Ccr2 −/− ). b. Representative photomicrographs of p-STAT1 (top) and p-STAT3 (bottom) staining in sections of tumors from Ccr2 +/+ or Ccr2 −/− cancer cells. Scale bar=100 μm. c. Quantification of the immunohistochemical staining in (b), using H-score, shows higher STAT1 phosphorylation in tumors from Ccr2 −/− than from Ccr2 +/+ cancer cells, but similar levels of STAT3 phosphorylation (mean +/− SEM, Student’s t-test; n= 5).

    Article Snippet: The membrane was incubated sequentially with different primary antibodies: anti-p65, anti-p-p65, anti-p-STAT1, anti-p-STAT3, and anti-STAT3 (all from Cell Signaling Technology), and anti-STAT1 and anti-beta-Actin (from Santa Cruz Biotechnology, Dallas, Tx).

    Techniques: Activation Assay, Western Blot, Expressing, Staining, Immunohistochemistry