Structured Review

Cell Signaling Technology Inc rabbit anti stat1
SFTSV inhibits exogenous Type I IFN signaling pathway. (A) SFTSV inhibits the phosphorylation of <t>STAT1</t> . Hela cells were infected with SFTSV(MOI = 1.0) for2 hours and cultured for 36 hours before 100IU/ml IFNα was added for 15 minutes,and total protein were collected for WB and densitometry analysis for p-STAT1. (B) SFTSV suppresses ISRE activity. Hela cells were co-transfected with ISRE-luc plasmid 2μg per well and pRL-TK plasmid 2ng per well, then Hela cells were infected with SFTSV (MOI = 1.0) for 2 hours and cultured for 24hours, then we used 100IU/ml IFNα to stimulated the Hela cells for 24 hours, and samples were collected for dual-luciferase reporter assay. (C) SFTSV down –regulates the expression of ISGs. Hela cells were infected with 1.0MOI SFTSV for2 hours and culturedfor24hours, then treated with 100IU/ml IFNα for 12 hours, and total RNAs were isolated with Trizolreagent, The levels of ISG15, MxA, OAS3, GAPDH mRNA were measured by quantitative RT-PCR. Data were presented as mean± SD, n = 3. **p
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1) Product Images from "Severe fever with thrombocytopenia syndrome virus inhibits exogenous Type I IFN signaling pathway through its NSs invitro"

Article Title: Severe fever with thrombocytopenia syndrome virus inhibits exogenous Type I IFN signaling pathway through its NSs invitro

Journal: PLoS ONE

doi: 10.1371/journal.pone.0172744

SFTSV inhibits exogenous Type I IFN signaling pathway. (A) SFTSV inhibits the phosphorylation of STAT1 . Hela cells were infected with SFTSV(MOI = 1.0) for2 hours and cultured for 36 hours before 100IU/ml IFNα was added for 15 minutes,and total protein were collected for WB and densitometry analysis for p-STAT1. (B) SFTSV suppresses ISRE activity. Hela cells were co-transfected with ISRE-luc plasmid 2μg per well and pRL-TK plasmid 2ng per well, then Hela cells were infected with SFTSV (MOI = 1.0) for 2 hours and cultured for 24hours, then we used 100IU/ml IFNα to stimulated the Hela cells for 24 hours, and samples were collected for dual-luciferase reporter assay. (C) SFTSV down –regulates the expression of ISGs. Hela cells were infected with 1.0MOI SFTSV for2 hours and culturedfor24hours, then treated with 100IU/ml IFNα for 12 hours, and total RNAs were isolated with Trizolreagent, The levels of ISG15, MxA, OAS3, GAPDH mRNA were measured by quantitative RT-PCR. Data were presented as mean± SD, n = 3. **p
Figure Legend Snippet: SFTSV inhibits exogenous Type I IFN signaling pathway. (A) SFTSV inhibits the phosphorylation of STAT1 . Hela cells were infected with SFTSV(MOI = 1.0) for2 hours and cultured for 36 hours before 100IU/ml IFNα was added for 15 minutes,and total protein were collected for WB and densitometry analysis for p-STAT1. (B) SFTSV suppresses ISRE activity. Hela cells were co-transfected with ISRE-luc plasmid 2μg per well and pRL-TK plasmid 2ng per well, then Hela cells were infected with SFTSV (MOI = 1.0) for 2 hours and cultured for 24hours, then we used 100IU/ml IFNα to stimulated the Hela cells for 24 hours, and samples were collected for dual-luciferase reporter assay. (C) SFTSV down –regulates the expression of ISGs. Hela cells were infected with 1.0MOI SFTSV for2 hours and culturedfor24hours, then treated with 100IU/ml IFNα for 12 hours, and total RNAs were isolated with Trizolreagent, The levels of ISG15, MxA, OAS3, GAPDH mRNA were measured by quantitative RT-PCR. Data were presented as mean± SD, n = 3. **p

Techniques Used: Infection, Cell Culture, Western Blot, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Expressing, Isolation, Quantitative RT-PCR

SFTSV inhibits exogenous IFNα-induced Jak/STAT signaling pathway through NSs. (A) NSs of SFTSV expresses in Hela cells successfully. Hela cells were transfected with empty vector pcDNA3.1 plasmid or NSs plasmid for 48hours, and total RNA were prepared with Trizol reagent, cDNA were harvested with reverse transcriptase for real time quantitative PCR. Protein samples were harvested for WB analyzed. (B) Expression of GFP protein in Hela cells. GFP-expressing plasmid DNA was transfected into Hela cells for 48 hours and GFP expression was observed under immunofluorescence microscope (200×). (C) NSs inhibits the phosphorylation of STAT1. Hela cells were transfected with NSs plasmid or empty vector pcDNA3.1 for 36 hours before IFNα was added to stimulate the cells for 15minutes. Total proteins were collected with protein lysis buffer for WB and densitometry analysis of western blot datafor p-STAT1. (D) NSs suppresses the ISRE activity . Hela cells were co-transfected with pISRE-luc plasmid and pRL-TK plasmid together with NSs plasmid, GFP-expressing plasmid or empty vector pcDNA3.1 plasmid for 24hours, and IFNα (100IU/mL)were added to the cells for 24hours, then samples were collected for dual-luciferase reporter assay. (E) NSs down-regulates the expression of several ISGs. Hela cells were transfectd with NSs plasmid or empty vectorpcDNA3.1 plasmid for 36hours, then we added IFNα (100IU/ml) to the cells for 12hours,RNA were collected with Trizol reagent, cDNA were used for real time quantitative PCR. Data were presented as mean± SD, n = 3.*p
Figure Legend Snippet: SFTSV inhibits exogenous IFNα-induced Jak/STAT signaling pathway through NSs. (A) NSs of SFTSV expresses in Hela cells successfully. Hela cells were transfected with empty vector pcDNA3.1 plasmid or NSs plasmid for 48hours, and total RNA were prepared with Trizol reagent, cDNA were harvested with reverse transcriptase for real time quantitative PCR. Protein samples were harvested for WB analyzed. (B) Expression of GFP protein in Hela cells. GFP-expressing plasmid DNA was transfected into Hela cells for 48 hours and GFP expression was observed under immunofluorescence microscope (200×). (C) NSs inhibits the phosphorylation of STAT1. Hela cells were transfected with NSs plasmid or empty vector pcDNA3.1 for 36 hours before IFNα was added to stimulate the cells for 15minutes. Total proteins were collected with protein lysis buffer for WB and densitometry analysis of western blot datafor p-STAT1. (D) NSs suppresses the ISRE activity . Hela cells were co-transfected with pISRE-luc plasmid and pRL-TK plasmid together with NSs plasmid, GFP-expressing plasmid or empty vector pcDNA3.1 plasmid for 24hours, and IFNα (100IU/mL)were added to the cells for 24hours, then samples were collected for dual-luciferase reporter assay. (E) NSs down-regulates the expression of several ISGs. Hela cells were transfectd with NSs plasmid or empty vectorpcDNA3.1 plasmid for 36hours, then we added IFNα (100IU/ml) to the cells for 12hours,RNA were collected with Trizol reagent, cDNA were used for real time quantitative PCR. Data were presented as mean± SD, n = 3.*p

Techniques Used: Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Microscopy, Lysis, Activity Assay, Luciferase, Reporter Assay

2) Product Images from "Combined genetic and epigenetic interferences with interferon signaling expose prostate cancer cells to viral infection"

Article Title: Combined genetic and epigenetic interferences with interferon signaling expose prostate cancer cells to viral infection

Journal: Oncotarget

doi: 10.18632/oncotarget.10313

Serially passaged EHDV (EHDV-TAU) differentially infects LNCaP and DU145 cells A. Schematic depiction of selection procedure. The single virion represents a selected clonal, plaque-purified virus; whereas the multiple virions represent diverse virus populations (quasispecies). B. Plaque assay analysis of the fold increase in titer for EHDV-TAU, compared to EHDV2-IBA, in LNCaP cells. Panel depicts typical images of the plaque assays; the dilution employed appears above the respective wells. Left inoculum (10 −3 dilution); right, dilutions (10 −3 and 10 −10 ) of the virions (of the indicated viruses) produced in LNCaP cells (60 h infection) C. Immunoblot analysis of NS3 production and STAT1 phosphorylation in EHDV-TAU-infected cells. Lysates (100 μg protein) of DU145 or LNCaP cells, infected or not with EHDV-TAU (0.05 pfu/cell, 45 h) were separated by SDS-PAGE, blotted and probed with antibodies against the indicated proteins. α-tubulin was used as a loading control. D-E Exogenous addition of IFNα blocks EHDV-TAU infection in DU145 cells, but not in LNCaP cells. D. Panels depict typical fields of DU145 and LNCaP cells, stained for DAPI (blue, left panels) and NS3 (red, right panels) under the indicated conditions: uninfected, EHDV-TAU-infected (45 hpi, 0.05 pfu/cell), or EHDV-TAU infected (45 hpi, 0.05 pfu/cell) treated with IFNα (200U/ml, 45 h). Bar, 100 μm. E. Graph depicts the mean ± SE percentage of NS3 positive cells. Quantification of percentage NS3 positive cells was from multiple (n=5) randomly selected fields, imaged under the same conditions as in (D). *, p
Figure Legend Snippet: Serially passaged EHDV (EHDV-TAU) differentially infects LNCaP and DU145 cells A. Schematic depiction of selection procedure. The single virion represents a selected clonal, plaque-purified virus; whereas the multiple virions represent diverse virus populations (quasispecies). B. Plaque assay analysis of the fold increase in titer for EHDV-TAU, compared to EHDV2-IBA, in LNCaP cells. Panel depicts typical images of the plaque assays; the dilution employed appears above the respective wells. Left inoculum (10 −3 dilution); right, dilutions (10 −3 and 10 −10 ) of the virions (of the indicated viruses) produced in LNCaP cells (60 h infection) C. Immunoblot analysis of NS3 production and STAT1 phosphorylation in EHDV-TAU-infected cells. Lysates (100 μg protein) of DU145 or LNCaP cells, infected or not with EHDV-TAU (0.05 pfu/cell, 45 h) were separated by SDS-PAGE, blotted and probed with antibodies against the indicated proteins. α-tubulin was used as a loading control. D-E Exogenous addition of IFNα blocks EHDV-TAU infection in DU145 cells, but not in LNCaP cells. D. Panels depict typical fields of DU145 and LNCaP cells, stained for DAPI (blue, left panels) and NS3 (red, right panels) under the indicated conditions: uninfected, EHDV-TAU-infected (45 hpi, 0.05 pfu/cell), or EHDV-TAU infected (45 hpi, 0.05 pfu/cell) treated with IFNα (200U/ml, 45 h). Bar, 100 μm. E. Graph depicts the mean ± SE percentage of NS3 positive cells. Quantification of percentage NS3 positive cells was from multiple (n=5) randomly selected fields, imaged under the same conditions as in (D). *, p

Techniques Used: Selection, Purification, Plaque Assay, Produced, Infection, SDS Page, Staining

Deficient JAK1 expression characterizes a subset of prostate cancer patient samples and LNCaP cells, and correlates with lack of IFN signaling A. Table depicts the prevalence of different JAK1 genetic contents (ranging from deep deletion to gain) in different subtypes of prostate cancer; in a cohort of 333 patients (TCGA, cBioPortal, [ 37 – 39 ]. Different shades of blue are indicative of number of patient samples in category. B. Chromatograms of portion of exon 5 sequence of JAK1 in DU145 cells and LNCaP cells. The sequence that appears above the chromatogram of the LNCaP cells represents the mutant sequence; arrow marks the site of frameshift mutations (insertion of A). The chromatogram of LNCaP cells shows the mixture of wild-type and mutated sequences. C. Chromatograms of portion of exon 9 sequence of JAK1 in DU145 cells and LNCaP cells. The sequence that appears above the chromatogram of the LNCaP cells represents the mutant sequence; arrow marks the site of frameshift mutations (insertion of C). The chromatogram of LNCaP cells shows the mixture of wild-type and mutated sequences. D. IFN-mediated STAT1 phosphorylation is defective in LNCaP cells. DU145 and LNCaP cells were incubated with IFNα (200 U/ml, 4 h). Cells were extracted, protein lysates were separated by SDS-PAGE and immunoblotted with antibodies against the indicated proteins. Actin served as a loading control. E. Nuclear localization of pSTAT1 is impaired in LNCaP cells. LNCaP and DU145 cells grown on glass coverslips were treated with IFN as in (D). Cells were fixed, permeabilized and stained with DAPI (blue) and anti-pSTAT1/Alexa555-Goat-anti-Rabbit antibodies (red). Cells were imaged by immunofluorescence microscopy. Micrographs depict typical fields of the different cell lines prior to, or following IFNα stimulation. Bar. 10 μm. F. IFN-mediated induction of ISGs is defective in LNCaP cells. Graph depicts the fold change in gene expression in DU145 (black) and LNCaP (grey) cells, following IFNα stimulation (200U/ml, 4h) as measured by qRT-PCR. Expression of ISGs in independent experiments (n=4) was normalized to measured expression levels of housekeeping gene (GAPDH). Expression levels in unstimulated cells were taken as 1. *, p
Figure Legend Snippet: Deficient JAK1 expression characterizes a subset of prostate cancer patient samples and LNCaP cells, and correlates with lack of IFN signaling A. Table depicts the prevalence of different JAK1 genetic contents (ranging from deep deletion to gain) in different subtypes of prostate cancer; in a cohort of 333 patients (TCGA, cBioPortal, [ 37 – 39 ]. Different shades of blue are indicative of number of patient samples in category. B. Chromatograms of portion of exon 5 sequence of JAK1 in DU145 cells and LNCaP cells. The sequence that appears above the chromatogram of the LNCaP cells represents the mutant sequence; arrow marks the site of frameshift mutations (insertion of A). The chromatogram of LNCaP cells shows the mixture of wild-type and mutated sequences. C. Chromatograms of portion of exon 9 sequence of JAK1 in DU145 cells and LNCaP cells. The sequence that appears above the chromatogram of the LNCaP cells represents the mutant sequence; arrow marks the site of frameshift mutations (insertion of C). The chromatogram of LNCaP cells shows the mixture of wild-type and mutated sequences. D. IFN-mediated STAT1 phosphorylation is defective in LNCaP cells. DU145 and LNCaP cells were incubated with IFNα (200 U/ml, 4 h). Cells were extracted, protein lysates were separated by SDS-PAGE and immunoblotted with antibodies against the indicated proteins. Actin served as a loading control. E. Nuclear localization of pSTAT1 is impaired in LNCaP cells. LNCaP and DU145 cells grown on glass coverslips were treated with IFN as in (D). Cells were fixed, permeabilized and stained with DAPI (blue) and anti-pSTAT1/Alexa555-Goat-anti-Rabbit antibodies (red). Cells were imaged by immunofluorescence microscopy. Micrographs depict typical fields of the different cell lines prior to, or following IFNα stimulation. Bar. 10 μm. F. IFN-mediated induction of ISGs is defective in LNCaP cells. Graph depicts the fold change in gene expression in DU145 (black) and LNCaP (grey) cells, following IFNα stimulation (200U/ml, 4h) as measured by qRT-PCR. Expression of ISGs in independent experiments (n=4) was normalized to measured expression levels of housekeeping gene (GAPDH). Expression levels in unstimulated cells were taken as 1. *, p

Techniques Used: Expressing, Sequencing, Mutagenesis, Incubation, SDS Page, Staining, Immunofluorescence, Microscopy, Quantitative RT-PCR

3) Product Images from "Regulation of STAT signaling in mouse bone marrow derived dendritic cells by respiratory syncytial virus"

Article Title: Regulation of STAT signaling in mouse bone marrow derived dendritic cells by respiratory syncytial virus

Journal: Virus research

doi: 10.1016/j.virusres.2011.01.007

Nuclear translocation of STAT1 in response to IFN-γ is not affected in RSV Infected BMDCs. Cellular localization of STAT1 in mock-, RSV-, or UV-killed RSV-infected BMDCs was analyzed by immunofluorescence microscopy in IFN-γ (100 U/ml
Figure Legend Snippet: Nuclear translocation of STAT1 in response to IFN-γ is not affected in RSV Infected BMDCs. Cellular localization of STAT1 in mock-, RSV-, or UV-killed RSV-infected BMDCs was analyzed by immunofluorescence microscopy in IFN-γ (100 U/ml

Techniques Used: Translocation Assay, Infection, Immunofluorescence, Microscopy

RSV infection impairs phosphorylation of STAT1 induced by IFN-β, but not IFN-γ. (A) BMDCs were infected with mock, live or UV-killed RSV for 24 h at an MOI of 1 and 5, followed by incubation with or without IFN-β (100 U/ml) for
Figure Legend Snippet: RSV infection impairs phosphorylation of STAT1 induced by IFN-β, but not IFN-γ. (A) BMDCs were infected with mock, live or UV-killed RSV for 24 h at an MOI of 1 and 5, followed by incubation with or without IFN-β (100 U/ml) for

Techniques Used: Infection, Incubation

Nuclear translocations of STAT1 and STAT2 in response to IFN-β are blocked in RSV-infected BMDCs. Cellular localization of STAT1 in mock-, RSV-, or UV-killed RSV-infected BMDCs was analyzed by immunofluorescence microscopy in IFN-β (100
Figure Legend Snippet: Nuclear translocations of STAT1 and STAT2 in response to IFN-β are blocked in RSV-infected BMDCs. Cellular localization of STAT1 in mock-, RSV-, or UV-killed RSV-infected BMDCs was analyzed by immunofluorescence microscopy in IFN-β (100

Techniques Used: Infection, Immunofluorescence, Microscopy

4) Product Images from "Interleukin-1β Signaling in Dendritic Cells Induces Antiviral Interferon Responses"

Article Title: Interleukin-1β Signaling in Dendritic Cells Induces Antiviral Interferon Responses

Journal: mBio

doi: 10.1128/mBio.00342-18

IL-1 signaling enhances antiviral responses. (A) WT or Il-1r −/− BMDCs were mock infected or infected with WNV at an MOI of 2.5. Expression of IFN-β and IFIT1 was measured by qRT-PCR at 24 and 48 h p.i. relative to that in matched, mock-treated controls. (B) Total cell WNV NS3, STAT1, and IFIT3 protein levels were measured by immunoblotting with GAPDH as a loading control (left). Densitometry analyses of STAT1 and IFIT3 protein abundance were compared against GAPDH abundance for each condition (right). The data are the averages of three independent experiments. Asterisks indicate values that are statistically significantly different between WT and Il - 1r −/− cells by unpaired t test (*, P
Figure Legend Snippet: IL-1 signaling enhances antiviral responses. (A) WT or Il-1r −/− BMDCs were mock infected or infected with WNV at an MOI of 2.5. Expression of IFN-β and IFIT1 was measured by qRT-PCR at 24 and 48 h p.i. relative to that in matched, mock-treated controls. (B) Total cell WNV NS3, STAT1, and IFIT3 protein levels were measured by immunoblotting with GAPDH as a loading control (left). Densitometry analyses of STAT1 and IFIT3 protein abundance were compared against GAPDH abundance for each condition (right). The data are the averages of three independent experiments. Asterisks indicate values that are statistically significantly different between WT and Il - 1r −/− cells by unpaired t test (*, P

Techniques Used: Infection, Expressing, Quantitative RT-PCR

5) Product Images from "Isocitrate dehydrogenase mutations suppress STAT1 and CD8+ T cell accumulation in gliomas"

Article Title: Isocitrate dehydrogenase mutations suppress STAT1 and CD8+ T cell accumulation in gliomas

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI90644

Decreased STAT1 protein levels in IDH-MUT and 2HG-treated tumor cell lines. ( A ) Western blotting was performed on GL261-WT and GL261-MUT cell lysates in the presence or absence of 1 μM IDH-C35 and 100 ng/ml recombinant murine IFN-γ. ( B ) Quantification of Western blot bands by ImageJ. Data represent the mean ± SD of band density/β-actin of 2 to 4 experiments. ( C ) Western blotting was performed on cell lines treated with 3 mM 2HG. Data shown represent GL261 cells treated with 2HG for 1, 3, or 5 days and NHA and SB28 cells treated with 2HG for 5 days. ( D ) ImageJ quantification of Western blot bands from C . Data represent the mean ± SD of band density/β-actin band density from 3 independent experiments. ( E ) ImageJ quantification of Western blot band densities of STAT1, p-STAT1, and IRF1, normalized to β-actin levels for NHA and SB28 cells treated with 2HG. Data are representative of 2 independent experiments with similar results.
Figure Legend Snippet: Decreased STAT1 protein levels in IDH-MUT and 2HG-treated tumor cell lines. ( A ) Western blotting was performed on GL261-WT and GL261-MUT cell lysates in the presence or absence of 1 μM IDH-C35 and 100 ng/ml recombinant murine IFN-γ. ( B ) Quantification of Western blot bands by ImageJ. Data represent the mean ± SD of band density/β-actin of 2 to 4 experiments. ( C ) Western blotting was performed on cell lines treated with 3 mM 2HG. Data shown represent GL261 cells treated with 2HG for 1, 3, or 5 days and NHA and SB28 cells treated with 2HG for 5 days. ( D ) ImageJ quantification of Western blot bands from C . Data represent the mean ± SD of band density/β-actin band density from 3 independent experiments. ( E ) ImageJ quantification of Western blot band densities of STAT1, p-STAT1, and IRF1, normalized to β-actin levels for NHA and SB28 cells treated with 2HG. Data are representative of 2 independent experiments with similar results.

Techniques Used: Western Blot, Recombinant

Decreased STAT1 positive cells in IDH-MUT compared with IDH-WT LGG tumor sections. ( A ) Representative images showing STAT1 staining on IDH-WT and IDH-MUT WHO grade III LGG samples. Scale bars: 80 μm and 8 μm. ( B ) Quantification of STAT1 + cells per area of tumor (mm 2 ) on IDH-WT (56 sections from 9 cases) and IDH-MUT (23 sections from 11 cases) tumors. Data represent the mean ± SD. Data obtained by 2-tailed unpaired t test. ( C ) RT-PCR analysis of Stat1 , Cxcl9 , and Cxcl10 expression in GL261 WT cells stimulated with 10 ng/ml recombinant murine IFN-γ for 12 hours, 36 hours after transfection with 1 μg plasmid encoding either scrambled shRNA (OriGene Technologies; TR30013) or 4 shRNAs targeting STAT1 (OriGene Technologies; TG502153). ( D ) CXCL10 ELISA was performed using supernatant from the cells described in C . Data shown in C and D represent the mean ± SD of 3 biologic replicate samples and are representative of 2 independent experiments; *** P
Figure Legend Snippet: Decreased STAT1 positive cells in IDH-MUT compared with IDH-WT LGG tumor sections. ( A ) Representative images showing STAT1 staining on IDH-WT and IDH-MUT WHO grade III LGG samples. Scale bars: 80 μm and 8 μm. ( B ) Quantification of STAT1 + cells per area of tumor (mm 2 ) on IDH-WT (56 sections from 9 cases) and IDH-MUT (23 sections from 11 cases) tumors. Data represent the mean ± SD. Data obtained by 2-tailed unpaired t test. ( C ) RT-PCR analysis of Stat1 , Cxcl9 , and Cxcl10 expression in GL261 WT cells stimulated with 10 ng/ml recombinant murine IFN-γ for 12 hours, 36 hours after transfection with 1 μg plasmid encoding either scrambled shRNA (OriGene Technologies; TR30013) or 4 shRNAs targeting STAT1 (OriGene Technologies; TG502153). ( D ) CXCL10 ELISA was performed using supernatant from the cells described in C . Data shown in C and D represent the mean ± SD of 3 biologic replicate samples and are representative of 2 independent experiments; *** P

Techniques Used: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Recombinant, Transfection, Plasmid Preparation, shRNA, Enzyme-linked Immunosorbent Assay

6) Product Images from "Monocarboxylate transporter 1 promotes classical microglial activation and pro-inflammatory effect via 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3"

Article Title: Monocarboxylate transporter 1 promotes classical microglial activation and pro-inflammatory effect via 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-019-1648-4

Knockdown of MCT1 inhibited LPS-stimulated classical microglial polarization and glycolysis rate in BV2 cells. a The interference efficiency of Lenti-siMCT1, Lenti-siMCT2, and Lenti-siMCT4, respectively, in BV2 cells (n = 6 per group and errors represent S.E.M; t test). b Quantification of the mRNA level of iNOS after treated with indicated lentivirus with or without LPS stimulation ( n = 6–7 per group). c Quantification of the mRNA levels of IL-1β , IL-6 , and STAT1 , respectively, in each group ( n = 5 per group). d Quantification of lactate levels in the cultured media in each group ( n = 4 per group). e Quantification of the mRNA level of PFKFB3 in each group ( n = 5–6 per group). f , g Representative immunoblots and relative quantification of iNOS, PFKFB3, and MCT1 in each group ( n = 6 per group). h , i The real-time extracellular acidification rate was measured by the sequential addition of glucose, oligomycin, and 2-DG in each group ( n = 5 per group). * p and # p
Figure Legend Snippet: Knockdown of MCT1 inhibited LPS-stimulated classical microglial polarization and glycolysis rate in BV2 cells. a The interference efficiency of Lenti-siMCT1, Lenti-siMCT2, and Lenti-siMCT4, respectively, in BV2 cells (n = 6 per group and errors represent S.E.M; t test). b Quantification of the mRNA level of iNOS after treated with indicated lentivirus with or without LPS stimulation ( n = 6–7 per group). c Quantification of the mRNA levels of IL-1β , IL-6 , and STAT1 , respectively, in each group ( n = 5 per group). d Quantification of lactate levels in the cultured media in each group ( n = 4 per group). e Quantification of the mRNA level of PFKFB3 in each group ( n = 5–6 per group). f , g Representative immunoblots and relative quantification of iNOS, PFKFB3, and MCT1 in each group ( n = 6 per group). h , i The real-time extracellular acidification rate was measured by the sequential addition of glucose, oligomycin, and 2-DG in each group ( n = 5 per group). * p and # p

Techniques Used: Cell Culture, Western Blot

Overexpression of PFKFB3 rescued MCT1 silence-induced suppression of classical microglial polarization in BV2 cells. a Representative immunoblots and relative quantification of Hif-1α in each group ( n = 4 per group and errors represent S.E.M). b The overexpression efficiency of Lenti-MCT1 and quantification of the mRNA level of PFKFB3 in each group ( n = 4 per group). KC7F2, an inhibitor of Hif-1α, 30 mM. c , Quantification of the mRNA level of iNOS , IL-1β , and IL-6 after treatment with Lenti-siMCT1 and Lenti-PFKFB3 with or without LPS ( n = 6–9 per group). d , e Representative immunoblots and relative quantification of iNOS and STAT1 protein, respectively, 24 h after LPS stimulation in each group ( n = 6 per group). f Representative immunoblots and relative quantification of STAT1 and p-STAT1 after stimulation with LPS for 3 h in each group ( n = 5–6 per group). * p , # p , and p
Figure Legend Snippet: Overexpression of PFKFB3 rescued MCT1 silence-induced suppression of classical microglial polarization in BV2 cells. a Representative immunoblots and relative quantification of Hif-1α in each group ( n = 4 per group and errors represent S.E.M). b The overexpression efficiency of Lenti-MCT1 and quantification of the mRNA level of PFKFB3 in each group ( n = 4 per group). KC7F2, an inhibitor of Hif-1α, 30 mM. c , Quantification of the mRNA level of iNOS , IL-1β , and IL-6 after treatment with Lenti-siMCT1 and Lenti-PFKFB3 with or without LPS ( n = 6–9 per group). d , e Representative immunoblots and relative quantification of iNOS and STAT1 protein, respectively, 24 h after LPS stimulation in each group ( n = 6 per group). f Representative immunoblots and relative quantification of STAT1 and p-STAT1 after stimulation with LPS for 3 h in each group ( n = 5–6 per group). * p , # p , and p

Techniques Used: Over Expression, Western Blot

7) Product Images from "E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts"

Article Title: E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-018-1713-x

STAT1/MyD88 complexes mediate E2F2 regulation of inflammatory cytokines. a – d STAT1/MyD88 complex is found in RASFs and MEFs. E2F2-silenced RASFs and E2f2 −/− MEFs were cultured with or without lipopolysaccharide (LPS; 10 μg/mL). Co-IP ( a , b ) was performed to test the binding. Confocal immunofluorescence ( c , d ) was performed to confirm the result (magnification 10 × 40, MyD88 (green) and STAT1 (red)). e – g Effect of STAT1/MyD88 complexes on the expression of cytokines. qRT-PCR was performed to detect expression of interleukin (IL)-1α ( e ), IL-1β ( f ), and tumor necrosis factor (TNF)-α ( g ) in STAT1/MyD88 knockdown RASFs with or without E2F2 overexpression in the presence of LPS (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. ** P
Figure Legend Snippet: STAT1/MyD88 complexes mediate E2F2 regulation of inflammatory cytokines. a – d STAT1/MyD88 complex is found in RASFs and MEFs. E2F2-silenced RASFs and E2f2 −/− MEFs were cultured with or without lipopolysaccharide (LPS; 10 μg/mL). Co-IP ( a , b ) was performed to test the binding. Confocal immunofluorescence ( c , d ) was performed to confirm the result (magnification 10 × 40, MyD88 (green) and STAT1 (red)). e – g Effect of STAT1/MyD88 complexes on the expression of cytokines. qRT-PCR was performed to detect expression of interleukin (IL)-1α ( e ), IL-1β ( f ), and tumor necrosis factor (TNF)-α ( g ) in STAT1/MyD88 knockdown RASFs with or without E2F2 overexpression in the presence of LPS (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. ** P

Techniques Used: Cell Culture, Co-Immunoprecipitation Assay, Binding Assay, Immunofluorescence, Expressing, Quantitative RT-PCR, Over Expression

STAT1 mediates E2F2 regulation of interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α expression. a – d Effect of E2F2 on STAT1. E2F2 was overexpressed by adenovirus infection or inhibited by small interfering RNA (siRNA) with or without lipopolysaccharide (LPS; 10 μg/mL). qRT-PCR ( a , b ) and Western blot ( c , d ) were performed to detect expression of STAT1. e Schematic representation of STAT1 promoters, primers for the ChIP assay, and the E2F2 binding motif in the STAT1 promoter. ChIP ( f ) and luciferase (Luc) reporter assays ( g ) were performed to show that E2F2 was recruited to the STAT1 gene promoter in RASFs in the presence of LPS. Nuclear and cytoplasmic proteins were fractionally extracted from E2F2 knocked-down RASFs ( h ) and E2f2 −/− MEFs ( i ). Effects of E2F2 on nuclear translocation of STAT1 were determined by Western blot. (Lamin A/C as a reference for nuclear extraction (N); Tubulin as a reference for cytoplasmic extraction (C).) Effect of E2F2 on nuclear translocation of STAT1 was observed using confocal fluorescence microscopy both in E2F2-silenced RASFs ( j ) and E2f2 −/− MEFs ( k ). STAT1 (green) was detected using anti-STAT1 antibody. Nuclei were stained with DAPI (blue). l In E2F2-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing STAT1 in the presence of LPS stimulation (10 μg/mL). m In STAT1-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing E2F2 in the presence of LPS stimulation (10 μg/mL). n In E2f2 −/− MEFs, expression of IL-1α, IL-1β, and TNF-α was detected using qRT-PCR after STAT1 overexpression in the presence of LPS stimulation (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. * P
Figure Legend Snippet: STAT1 mediates E2F2 regulation of interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α expression. a – d Effect of E2F2 on STAT1. E2F2 was overexpressed by adenovirus infection or inhibited by small interfering RNA (siRNA) with or without lipopolysaccharide (LPS; 10 μg/mL). qRT-PCR ( a , b ) and Western blot ( c , d ) were performed to detect expression of STAT1. e Schematic representation of STAT1 promoters, primers for the ChIP assay, and the E2F2 binding motif in the STAT1 promoter. ChIP ( f ) and luciferase (Luc) reporter assays ( g ) were performed to show that E2F2 was recruited to the STAT1 gene promoter in RASFs in the presence of LPS. Nuclear and cytoplasmic proteins were fractionally extracted from E2F2 knocked-down RASFs ( h ) and E2f2 −/− MEFs ( i ). Effects of E2F2 on nuclear translocation of STAT1 were determined by Western blot. (Lamin A/C as a reference for nuclear extraction (N); Tubulin as a reference for cytoplasmic extraction (C).) Effect of E2F2 on nuclear translocation of STAT1 was observed using confocal fluorescence microscopy both in E2F2-silenced RASFs ( j ) and E2f2 −/− MEFs ( k ). STAT1 (green) was detected using anti-STAT1 antibody. Nuclei were stained with DAPI (blue). l In E2F2-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing STAT1 in the presence of LPS stimulation (10 μg/mL). m In STAT1-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing E2F2 in the presence of LPS stimulation (10 μg/mL). n In E2f2 −/− MEFs, expression of IL-1α, IL-1β, and TNF-α was detected using qRT-PCR after STAT1 overexpression in the presence of LPS stimulation (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. * P

Techniques Used: Expressing, Infection, Small Interfering RNA, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Luciferase, Translocation Assay, Fluorescence, Microscopy, Staining, Over Expression

E2F2 participates in RA inflammation through STAT1 and PI3K/AKT/NF-κB pathways. RNA-seq was performed to screen target genes downstream of E2F2 in RASFs. a , b Heat maps indicate the most differentially expressed genes in RASFs with E2F2 knocked-down. Colored bands represent the change in gene expression: red, downregulation; blue, upregulation. c – e In-vitro verification of genes related to inflammation in RA was performed using qRT-PCR. mRNA levels of STAT1 ( c ), AKT1 ( d ), and AKT2 ( e ). f Western blot was performed to test inhibitory effects of siE2F2 on expression of E2F2, STAT1, AKT1, AKT2, p-AKT, and the p65 subunit of NF-κB. All results are presented as the mean ± SEM of three independent experiments performed in triplicate. NC knockdown scramble control, si small interfering
Figure Legend Snippet: E2F2 participates in RA inflammation through STAT1 and PI3K/AKT/NF-κB pathways. RNA-seq was performed to screen target genes downstream of E2F2 in RASFs. a , b Heat maps indicate the most differentially expressed genes in RASFs with E2F2 knocked-down. Colored bands represent the change in gene expression: red, downregulation; blue, upregulation. c – e In-vitro verification of genes related to inflammation in RA was performed using qRT-PCR. mRNA levels of STAT1 ( c ), AKT1 ( d ), and AKT2 ( e ). f Western blot was performed to test inhibitory effects of siE2F2 on expression of E2F2, STAT1, AKT1, AKT2, p-AKT, and the p65 subunit of NF-κB. All results are presented as the mean ± SEM of three independent experiments performed in triplicate. NC knockdown scramble control, si small interfering

Techniques Used: RNA Sequencing Assay, Expressing, In Vitro, Quantitative RT-PCR, Western Blot

8) Product Images from "Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein"

Article Title: Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein

Journal: Proteomics

doi: 10.1002/pmic.201300001

Inhibitory effects of JEV NS5 protein on IFNβ‐induced responses. (A) To analyze ISRE promoter activity, vector control and NS5‐expressing cells were transiently cotransfected with reporter plasmid containing firefly luciferase under control of ISRE and internal control reporter pRluc‐C1. After 4‐h IFNβ treatment, firefly luciferase and renilla luciferase were measured; firefly luciferase activity normalized to renilla luciferase activity is reported. (B) To analyze ISRE‐driven gene expression, vector control and NS5‐expressing cells were treated with or without 1000 U/mL IFNβ for 8 h; relative levels of PKR and OAS mRNAs were gauged by quantitative real‐time PCR, relative fold levels of PKR or OAS mRNA presented as ratio of PKR or OAS mRNA/GAPDH mRNA. (C) To analyze STAT1 phosphorylation, Western blot of lysates from cells treated with IFNβ for 0, 30, 60, or 120 min was performed by anti‐phospho‐STAT1 (Tyr701) and anti‐β actin antibody as internal control.
Figure Legend Snippet: Inhibitory effects of JEV NS5 protein on IFNβ‐induced responses. (A) To analyze ISRE promoter activity, vector control and NS5‐expressing cells were transiently cotransfected with reporter plasmid containing firefly luciferase under control of ISRE and internal control reporter pRluc‐C1. After 4‐h IFNβ treatment, firefly luciferase and renilla luciferase were measured; firefly luciferase activity normalized to renilla luciferase activity is reported. (B) To analyze ISRE‐driven gene expression, vector control and NS5‐expressing cells were treated with or without 1000 U/mL IFNβ for 8 h; relative levels of PKR and OAS mRNAs were gauged by quantitative real‐time PCR, relative fold levels of PKR or OAS mRNA presented as ratio of PKR or OAS mRNA/GAPDH mRNA. (C) To analyze STAT1 phosphorylation, Western blot of lysates from cells treated with IFNβ for 0, 30, 60, or 120 min was performed by anti‐phospho‐STAT1 (Tyr701) and anti‐β actin antibody as internal control.

Techniques Used: Activity Assay, Plasmid Preparation, Expressing, Luciferase, Real-time Polymerase Chain Reaction, Western Blot

Functional analysis of calcineurin with siRNA‐mediated gene silencing. To analyze subcellular localization of STAT1, vector control (A) and NS5‐expresing cells (B) transfected with control or calreticulin siRNA were tested using immunfluorescent staining with anti‐STAT1 antibodies. Relative mRNA levels of PKR (C) and IL‐4 (D) were measured by quantitative PCR and normalized by GAPDH mRNA, presented as relative ratio.
Figure Legend Snippet: Functional analysis of calcineurin with siRNA‐mediated gene silencing. To analyze subcellular localization of STAT1, vector control (A) and NS5‐expresing cells (B) transfected with control or calreticulin siRNA were tested using immunfluorescent staining with anti‐STAT1 antibodies. Relative mRNA levels of PKR (C) and IL‐4 (D) were measured by quantitative PCR and normalized by GAPDH mRNA, presented as relative ratio.

Techniques Used: Functional Assay, Plasmid Preparation, Transfection, Staining, Real-time Polymerase Chain Reaction

Effect of CsA on IFNβ‐induced phosphorylation and nuclear translocation of STAT1 in NS5‐expressing cells. (A) To analyze tyrosine phosphoryl‐ated STAT1, vector control and NS5‐expressing cells were treated singly or with both IFNβ and cyclosporine A for 0, 30, 60, or 120 min. Lysates were subjected to Western blot, probed with anti‐phospho‐STAT1 (Tyr701). For subcellular location of STAT1, vector control (B) and NS5‐expressing (C) cells were treated singly or with both CsA and IFNβ for 24 h, then washed, fixed, and reacted with anti‐STAT1 and FITC‐conjugated anti‐mouse IgG antibodies. Finally, cells were stained with DAPI for 10 min, imaging analyzed by immunofluorescent microscopy.
Figure Legend Snippet: Effect of CsA on IFNβ‐induced phosphorylation and nuclear translocation of STAT1 in NS5‐expressing cells. (A) To analyze tyrosine phosphoryl‐ated STAT1, vector control and NS5‐expressing cells were treated singly or with both IFNβ and cyclosporine A for 0, 30, 60, or 120 min. Lysates were subjected to Western blot, probed with anti‐phospho‐STAT1 (Tyr701). For subcellular location of STAT1, vector control (B) and NS5‐expressing (C) cells were treated singly or with both CsA and IFNβ for 24 h, then washed, fixed, and reacted with anti‐STAT1 and FITC‐conjugated anti‐mouse IgG antibodies. Finally, cells were stained with DAPI for 10 min, imaging analyzed by immunofluorescent microscopy.

Techniques Used: Translocation Assay, Expressing, Plasmid Preparation, Western Blot, Staining, Imaging, Microscopy

Functional characterization of calreticulin with siRNA‐mediated gene silencing. (A) To detect intracellular Ca 2+ , cells transfected with control or calreticulin siRNA were harvested 4 h posttreatment with or without IFNβ, stained with FLUO3/AM and analyzed by flow cytometry. To analyze subcellular localization of STAT1 (B, C) and NFAT‐1 (D, E), vector control (B, D) and NS5‐expressing cells (C, E) transfected with control or calreticulin siRNA were tested by immunfluorescent staining with anti‐STAT1 or anti‐NFAT‐1 antibodies.
Figure Legend Snippet: Functional characterization of calreticulin with siRNA‐mediated gene silencing. (A) To detect intracellular Ca 2+ , cells transfected with control or calreticulin siRNA were harvested 4 h posttreatment with or without IFNβ, stained with FLUO3/AM and analyzed by flow cytometry. To analyze subcellular localization of STAT1 (B, C) and NFAT‐1 (D, E), vector control (B, D) and NS5‐expressing cells (C, E) transfected with control or calreticulin siRNA were tested by immunfluorescent staining with anti‐STAT1 or anti‐NFAT‐1 antibodies.

Techniques Used: Functional Assay, Transfection, Staining, Flow Cytometry, Plasmid Preparation, Expressing

9) Product Images from "MicroRNA-146a Provides Feedback Regulation of Lyme Arthritis but Not Carditis during Infection with Borrelia burgdorferi"

Article Title: MicroRNA-146a Provides Feedback Regulation of Lyme Arthritis but Not Carditis during Infection with Borrelia burgdorferi

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004212

Bone marrow-derived macrophages from B6 miR-146a −/− mice are hyper-responsive to B. burgdorferi and have elevated levels of TRAF6. (A) Analysis of cytokine secretion of B. burgdorferi -treated BMDMs. Bone marrow-derived macrophages (BMDMs) from B6 (black) or B6 miR-146a −/− (white) mice were pre-treated for 4 hours in the presence or absence of recombinant mouse IL-10. Following pretreatment, cells were stimulated with B. burgdorferi for 24 hours. Cell supernatant was collected and secretion of IL-6, IL-1β, CXCL1, IL-12, TNF-α and IL-10 was measured by ELISA. (B) Immunoblot analysis of TRAF6, IRAK1 and STAT1 in B. burgdorferi -treated BMDMs from B6 (WT) or B6 miR-146a −/− (KO) mice. BMDMs were stimulated with media alone or B. burgdorferi for 24 hours and cells were lysed using NP-40. Quantification was determined based on band intensity, normalized to β-actin, with B6 (WT) value set to 1. (C) Traf6, Irak1 and Stat1 mRNA levels were quantified using qRT-PCR, normalized to β-actin . Significant differences between groups by ANOVA followed by Tukey's post-hoc test are indicated (*p
Figure Legend Snippet: Bone marrow-derived macrophages from B6 miR-146a −/− mice are hyper-responsive to B. burgdorferi and have elevated levels of TRAF6. (A) Analysis of cytokine secretion of B. burgdorferi -treated BMDMs. Bone marrow-derived macrophages (BMDMs) from B6 (black) or B6 miR-146a −/− (white) mice were pre-treated for 4 hours in the presence or absence of recombinant mouse IL-10. Following pretreatment, cells were stimulated with B. burgdorferi for 24 hours. Cell supernatant was collected and secretion of IL-6, IL-1β, CXCL1, IL-12, TNF-α and IL-10 was measured by ELISA. (B) Immunoblot analysis of TRAF6, IRAK1 and STAT1 in B. burgdorferi -treated BMDMs from B6 (WT) or B6 miR-146a −/− (KO) mice. BMDMs were stimulated with media alone or B. burgdorferi for 24 hours and cells were lysed using NP-40. Quantification was determined based on band intensity, normalized to β-actin, with B6 (WT) value set to 1. (C) Traf6, Irak1 and Stat1 mRNA levels were quantified using qRT-PCR, normalized to β-actin . Significant differences between groups by ANOVA followed by Tukey's post-hoc test are indicated (*p

Techniques Used: Derivative Assay, Mouse Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

10) Product Images from "Deciphering Molecular and Phenotypic Changes Associated with Early Autoimmune Disease in the Aire-Deficient Mouse Model of Sjögren’s Syndrome"

Article Title: Deciphering Molecular and Phenotypic Changes Associated with Early Autoimmune Disease in the Aire-Deficient Mouse Model of Sjögren’s Syndrome

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19113628

STAT signaling increased with disease progression in Aire-/- mice. ( A ) pSTAT3 is increased in CD68+ inflammatory macrophages and ( B ) NCKK1+ ductal cells of the inflamed LG by 5 wk and increases with disease progression. ( B ) IL6Ra expression is increased in infiltrating immune cells. ( C ) Increased levels of STAT1 protein in the Aire-/- LG were confirmed by Western blot. Data are expressed as mean ± SEM. n = 3–4 mice per group, and each sample is represented by a circle, square, or triangle within a group. * p
Figure Legend Snippet: STAT signaling increased with disease progression in Aire-/- mice. ( A ) pSTAT3 is increased in CD68+ inflammatory macrophages and ( B ) NCKK1+ ductal cells of the inflamed LG by 5 wk and increases with disease progression. ( B ) IL6Ra expression is increased in infiltrating immune cells. ( C ) Increased levels of STAT1 protein in the Aire-/- LG were confirmed by Western blot. Data are expressed as mean ± SEM. n = 3–4 mice per group, and each sample is represented by a circle, square, or triangle within a group. * p

Techniques Used: Mouse Assay, Expressing, Western Blot

11) Product Images from "Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication"

Article Title: Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication

Journal: Journal of Virology

doi: 10.1128/JVI.00217-17

Effect of blocking of type I interferon receptor signaling on CRT0066101. In order to determine if a blockade of the type I interferon receptor (IFNAR2) influenced the ability of CRT0066101 to inhibit viral replication, HeLa cells were left untreated (lane 1) or pretreated with DMSO (lanes 2 to 4) or 5 μM CRT0066101 (lane 5 to 7) for 1 h. Cells were subsequently infected with HRV16 for 1 h (lanes 2 to 7), and replication was allowed to proceed for a further 4 h. During the viral infection and replication periods, cells were treated with a blocking antibody to IFNAR2 alone (lane 3) or with an isotype-matched control antibody alone (lane 4) or cotreated with CRT0066101 and a blocking antibody (lane 6) or CRT0066101 with an isotype control (lane 7). As additional controls, cells were treated with 30 U/ml of IFN-β for 4 h (lanes 8 to 10), with no antibody (lane 8), with anti-IFNAR2 (lane 9), or with an isotype control antibody (lane 10). Cell extracts were prepared and analyzed by Western blotting with antibodies to pSTAT1 Y701, STAT1, HRV 2C, and LB1. The pSTAT1 Y701 blot is shown at high and low exposures, and data shown are from a representative experiment from three independent repeats.
Figure Legend Snippet: Effect of blocking of type I interferon receptor signaling on CRT0066101. In order to determine if a blockade of the type I interferon receptor (IFNAR2) influenced the ability of CRT0066101 to inhibit viral replication, HeLa cells were left untreated (lane 1) or pretreated with DMSO (lanes 2 to 4) or 5 μM CRT0066101 (lane 5 to 7) for 1 h. Cells were subsequently infected with HRV16 for 1 h (lanes 2 to 7), and replication was allowed to proceed for a further 4 h. During the viral infection and replication periods, cells were treated with a blocking antibody to IFNAR2 alone (lane 3) or with an isotype-matched control antibody alone (lane 4) or cotreated with CRT0066101 and a blocking antibody (lane 6) or CRT0066101 with an isotype control (lane 7). As additional controls, cells were treated with 30 U/ml of IFN-β for 4 h (lanes 8 to 10), with no antibody (lane 8), with anti-IFNAR2 (lane 9), or with an isotype control antibody (lane 10). Cell extracts were prepared and analyzed by Western blotting with antibodies to pSTAT1 Y701, STAT1, HRV 2C, and LB1. The pSTAT1 Y701 blot is shown at high and low exposures, and data shown are from a representative experiment from three independent repeats.

Techniques Used: Blocking Assay, Infection, Western Blot

Effect of CRT0066101 on interferon signaling. (A) Effect of CRT0066101 on STAT1 phosphorylation at residue Y701 in HeLa cells infected with HRV16. Cells were either untreated (lane 1) or treated with 30 U/ml IFN-β for 15 min (lane 2), the DMSO vehicle (lane 3), or increasing concentrations of CRT0066101 for 1 h, followed by a 6-h replication period. Cell extracts were prepared and analyzed by Western blotting with antibodies to pSTAT1 Y701, STAT1, HRV 2C, and LB1. Data shown are representative of results from three independent experiments. (B and C) To determine the effect of CRT0066101 on ISG expression, RNA was extracted from HRV16-infected HeLa cells after 20 h of culture in the presence of the DMSO vehicle or 1 μM, 2 μM, or 3.5 μM CRT0066101. UV-inactivated virus was included as a control. Viral replication was confirmed by measuring the levels of HRV16 RNA (HRV) (B) and OAS mRNA (C) as a representative ISG. The results are the means (±SEM) of data from four independent experiments, each performed in duplicate. Differences between infected DMSO-treated cells and infected CRT0066101-treated cells were determined by one-way ANOVA with Dunnett's post hoc analysis. *, P
Figure Legend Snippet: Effect of CRT0066101 on interferon signaling. (A) Effect of CRT0066101 on STAT1 phosphorylation at residue Y701 in HeLa cells infected with HRV16. Cells were either untreated (lane 1) or treated with 30 U/ml IFN-β for 15 min (lane 2), the DMSO vehicle (lane 3), or increasing concentrations of CRT0066101 for 1 h, followed by a 6-h replication period. Cell extracts were prepared and analyzed by Western blotting with antibodies to pSTAT1 Y701, STAT1, HRV 2C, and LB1. Data shown are representative of results from three independent experiments. (B and C) To determine the effect of CRT0066101 on ISG expression, RNA was extracted from HRV16-infected HeLa cells after 20 h of culture in the presence of the DMSO vehicle or 1 μM, 2 μM, or 3.5 μM CRT0066101. UV-inactivated virus was included as a control. Viral replication was confirmed by measuring the levels of HRV16 RNA (HRV) (B) and OAS mRNA (C) as a representative ISG. The results are the means (±SEM) of data from four independent experiments, each performed in duplicate. Differences between infected DMSO-treated cells and infected CRT0066101-treated cells were determined by one-way ANOVA with Dunnett's post hoc analysis. *, P

Techniques Used: Infection, Western Blot, Expressing

12) Product Images from "IFN/STAT signaling controls tumorigenesis and the drug response in colorectal cancer, et al. IFN/STAT signaling controls tumorigenesis and the drug response in colorectal cancer"

Article Title: IFN/STAT signaling controls tumorigenesis and the drug response in colorectal cancer, et al. IFN/STAT signaling controls tumorigenesis and the drug response in colorectal cancer

Journal: Cancer Science

doi: 10.1111/cas.13964

STAT1 disruption sensitized KRAS‐activated organoids to trametinib. (A) Disruption of STAT1. STAT1 knockout and control organoids were stained with anti STAT1 antibody. (B). GSEA of STAT1 KO organoids. NES (bar graph) and nominal P ‐value (line graph) are shown. (C) Response of STAT1‐deficient organoids. STAT1 KO (blue line) and control (black line) are shown. (D) Schematic representation of crosstalk between RAS signaling and IFN/STAT signaling. The phosphorylation status of STAT1 with (right side) or without (left side) trametinib treatment is illustrated. Activated RAS induces STAT1 phosphorylation and activates IFN/STAT signaling. Trametinib inhibits MEK activity, but does not abolish STAT1 phosphorylation, which confers resistance in CFAP organoids
Figure Legend Snippet: STAT1 disruption sensitized KRAS‐activated organoids to trametinib. (A) Disruption of STAT1. STAT1 knockout and control organoids were stained with anti STAT1 antibody. (B). GSEA of STAT1 KO organoids. NES (bar graph) and nominal P ‐value (line graph) are shown. (C) Response of STAT1‐deficient organoids. STAT1 KO (blue line) and control (black line) are shown. (D) Schematic representation of crosstalk between RAS signaling and IFN/STAT signaling. The phosphorylation status of STAT1 with (right side) or without (left side) trametinib treatment is illustrated. Activated RAS induces STAT1 phosphorylation and activates IFN/STAT signaling. Trametinib inhibits MEK activity, but does not abolish STAT1 phosphorylation, which confers resistance in CFAP organoids

Techniques Used: Knock-Out, Staining, Activity Assay

Disruption of Stat1 in a mouse model of FAP. (A) Stat1‐deficient mice were generated by introducing Stat1 sgRNA into zygotes carrying the Apc fl/fl , Lgr5‐CreERT2 +/− allele. (B) Immunohistochemical analysis of intestinal tumors. Apc fl/fl , Lgr5‐CreERT2, Stat1 wt/wt (Stat1 wt/wt ) or Apc fl/fl , Lgr5‐CreERT2, Stat1 null/null (Stat1 null/null ) mice were analyzed 26 days after administration of 4OHT. Immunohistochemical analysis is shown using an anti‐Stat1 antibody. Bar = 200 μm. (C) Disruption of Stat1 suppresses tumor formation. Tumors in the indicated mice (N = 6) were detected by immunohistochemistry using an anti‐β‐catenin antibody, and the tumor area was measured. ** P
Figure Legend Snippet: Disruption of Stat1 in a mouse model of FAP. (A) Stat1‐deficient mice were generated by introducing Stat1 sgRNA into zygotes carrying the Apc fl/fl , Lgr5‐CreERT2 +/− allele. (B) Immunohistochemical analysis of intestinal tumors. Apc fl/fl , Lgr5‐CreERT2, Stat1 wt/wt (Stat1 wt/wt ) or Apc fl/fl , Lgr5‐CreERT2, Stat1 null/null (Stat1 null/null ) mice were analyzed 26 days after administration of 4OHT. Immunohistochemical analysis is shown using an anti‐Stat1 antibody. Bar = 200 μm. (C) Disruption of Stat1 suppresses tumor formation. Tumors in the indicated mice (N = 6) were detected by immunohistochemistry using an anti‐β‐catenin antibody, and the tumor area was measured. ** P

Techniques Used: Mouse Assay, Generated, Immunohistochemistry

13) Product Images from "Adaptor Protein LNK Is a Negative Regulator of Brain Neural Stem Cell Proliferation after Stroke"

Article Title: Adaptor Protein LNK Is a Negative Regulator of Brain Neural Stem Cell Proliferation after Stroke

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0474-12.2012

STAT1 regulates expression of Lnk in NSPCs after stroke. A , Relative gene expression of Lnk in SVZ tissue from intact, stroke, and SE mice ipsilateral and contralateral to lesion and stimulating electrode, respectively. B , Occurrence of SP1, E2F1, STAT1, and STAT3 binding motifs (in bold) in mouse Lnk genomic region. C , Relative expression of transcription factor genes in SVZ tissue from intact mice or 7 d after stroke or SE. D , Lnk expression in neurospheres after transfection with CA- and DN-STAT1. E , Schematic representation of the murine Lnk locus with STAT1/3 binding motifs and primers used for ChIP. F , ChIP PCR analysis showing binding of STAT1 to genomic region of the Lnk gene and the positive control gene Irf1 . Genomic DNA immunoprecipitated with IgG, as a negative control, or STAT1 antibodies was used for PCR with primers spanning binding motifs of STAT1 in Lnk and Irf1 . G , Relative luciferase activity in 3T3 and U3A cell lines transfected with control (pGL4.10), Lnk (pLnk), mutated Lnk (pLnk-Mut), pLnk+pCA-STAT1, and pLnk+pCA-STAT3.
Figure Legend Snippet: STAT1 regulates expression of Lnk in NSPCs after stroke. A , Relative gene expression of Lnk in SVZ tissue from intact, stroke, and SE mice ipsilateral and contralateral to lesion and stimulating electrode, respectively. B , Occurrence of SP1, E2F1, STAT1, and STAT3 binding motifs (in bold) in mouse Lnk genomic region. C , Relative expression of transcription factor genes in SVZ tissue from intact mice or 7 d after stroke or SE. D , Lnk expression in neurospheres after transfection with CA- and DN-STAT1. E , Schematic representation of the murine Lnk locus with STAT1/3 binding motifs and primers used for ChIP. F , ChIP PCR analysis showing binding of STAT1 to genomic region of the Lnk gene and the positive control gene Irf1 . Genomic DNA immunoprecipitated with IgG, as a negative control, or STAT1 antibodies was used for PCR with primers spanning binding motifs of STAT1 in Lnk and Irf1 . G , Relative luciferase activity in 3T3 and U3A cell lines transfected with control (pGL4.10), Lnk (pLnk), mutated Lnk (pLnk-Mut), pLnk+pCA-STAT1, and pLnk+pCA-STAT3.

Techniques Used: Expressing, Mouse Assay, Binding Assay, Transfection, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Positive Control, Immunoprecipitation, Negative Control, Luciferase, Activity Assay

14) Product Images from "Cell Cycle Arrest in G2/M Phase Enhances Replication of Interferon-Sensitive Cytoplasmic RNA Viruses via Inhibition of Antiviral Gene Expression"

Article Title: Cell Cycle Arrest in G2/M Phase Enhances Replication of Interferon-Sensitive Cytoplasmic RNA Viruses via Inhibition of Antiviral Gene Expression

Journal: Journal of Virology

doi: 10.1128/JVI.01885-18

Induction of type I IFN signaling by viral and nonviral stimuli is inhibited in G 2 /M-arrested cells. Suit2 cells were treated for 25 h with the vehicle, paclitaxel, colchicine, or ruxolitinib at 500 nM. Cells were then treated with the vehicle (untreated), TransIT reagent (0.5%, vol/vol), poly(I:C) at 10 μg/ml plus TransIT reagent, IFN-α at 5,000 U/ml, or VSV at an MOI of 30 based on titration on BHK-21 cells. VSV was aspirated 1 h later, and medium was added to infected wells. Cells remained in treatment for a total of 4 h, after which total protein was isolated. Western blot results for STAT1 and -2 proteins and their phosphorylated forms are shown in addition to VSV proteins. GAPDH was used to confirm that protein loading was the same across the gel. Protein names and protein sizes in kilodaltons are indicated on the left and right, respectively.
Figure Legend Snippet: Induction of type I IFN signaling by viral and nonviral stimuli is inhibited in G 2 /M-arrested cells. Suit2 cells were treated for 25 h with the vehicle, paclitaxel, colchicine, or ruxolitinib at 500 nM. Cells were then treated with the vehicle (untreated), TransIT reagent (0.5%, vol/vol), poly(I:C) at 10 μg/ml plus TransIT reagent, IFN-α at 5,000 U/ml, or VSV at an MOI of 30 based on titration on BHK-21 cells. VSV was aspirated 1 h later, and medium was added to infected wells. Cells remained in treatment for a total of 4 h, after which total protein was isolated. Western blot results for STAT1 and -2 proteins and their phosphorylated forms are shown in addition to VSV proteins. GAPDH was used to confirm that protein loading was the same across the gel. Protein names and protein sizes in kilodaltons are indicated on the left and right, respectively.

Techniques Used: Titration, Infection, Isolation, Western Blot

Inhibition of the type I interferon response in Suit2 cells blocked in G 2 /M allows an increase in viral replication. Suit2 cells were treated with 500 nM paclitaxel (Pac) or 500 nM colchicine (Col) or remained untreated for 24 h and then infected with VSV-ΔM51 at different MOIs (0, 0.4, 2, 10, 50, and 250 PFU/cell based on the titer determined on BHK-21 cells) for 1, 8, or 24 h. Western blotting shows the expression of phospho-STAT1 (STAT1-P) at Y701, STAT1, VSV proteins (G, N/P, and M), and cyclin B1. Protein names and protein sizes in kilodaltons are indicated on the left and right, respectively. GAPDH was used to confirm that protein loading was the same across the gel.
Figure Legend Snippet: Inhibition of the type I interferon response in Suit2 cells blocked in G 2 /M allows an increase in viral replication. Suit2 cells were treated with 500 nM paclitaxel (Pac) or 500 nM colchicine (Col) or remained untreated for 24 h and then infected with VSV-ΔM51 at different MOIs (0, 0.4, 2, 10, 50, and 250 PFU/cell based on the titer determined on BHK-21 cells) for 1, 8, or 24 h. Western blotting shows the expression of phospho-STAT1 (STAT1-P) at Y701, STAT1, VSV proteins (G, N/P, and M), and cyclin B1. Protein names and protein sizes in kilodaltons are indicated on the left and right, respectively. GAPDH was used to confirm that protein loading was the same across the gel.

Techniques Used: Inhibition, Infection, Western Blot, Expressing

15) Product Images from "Lonicera japonica THUNB. Extract Inhibits Lipopolysaccharide-Stimulated Inflammatory Responses by Suppressing NF-κB Signaling in BV-2 Microglial Cells"

Article Title: Lonicera japonica THUNB. Extract Inhibits Lipopolysaccharide-Stimulated Inflammatory Responses by Suppressing NF-κB Signaling in BV-2 Microglial Cells

Journal: Journal of Medicinal Food

doi: 10.1089/jmf.2014.3341

LJ suppresses LPS-induced phosphorylation of JAK1 (A) and STAT1/3 (B, C) in BV-2 microglial cells. Cells were pretreated with the indicated concentrations of LJ for 30 min and then exposed to 100 ng/mL of LPS for 2 h. The expression
Figure Legend Snippet: LJ suppresses LPS-induced phosphorylation of JAK1 (A) and STAT1/3 (B, C) in BV-2 microglial cells. Cells were pretreated with the indicated concentrations of LJ for 30 min and then exposed to 100 ng/mL of LPS for 2 h. The expression

Techniques Used: Expressing

16) Product Images from "Obesity and Insulin Resistance Promote Atherosclerosis through an IFNγ-Regulated Macrophage Protein Network"

Article Title: Obesity and Insulin Resistance Promote Atherosclerosis through an IFNγ-Regulated Macrophage Protein Network

Journal: Cell reports

doi: 10.1016/j.celrep.2018.05.010

“Metabolic Disease Appropriate” Doses of IFN γ Specifically Target MSRN Proteins (A–D) Macrophages were treated with varying levels of IFNγ. (A) APOE levels. (B) p-STAT1/STAT1 levels. (C) Irf8 levels. (D) Number of P. aeruginosa remaining after incubation with macrophages. (E) Efficiency of STAT1 knockdown in macrophages treated with control or Stat1 siRNA. STAT1 levels were quantified 30 min after IFNγ exposure. (F and G) Effects of IFNγ on APOE levels and number of P. aeruginosa remaining after incubation with siRNA control or siRNA Stat1 macrophages (F) and WT or Irf1 −/− macrophages (G). Results are mean ± SEM. *p
Figure Legend Snippet: “Metabolic Disease Appropriate” Doses of IFN γ Specifically Target MSRN Proteins (A–D) Macrophages were treated with varying levels of IFNγ. (A) APOE levels. (B) p-STAT1/STAT1 levels. (C) Irf8 levels. (D) Number of P. aeruginosa remaining after incubation with macrophages. (E) Efficiency of STAT1 knockdown in macrophages treated with control or Stat1 siRNA. STAT1 levels were quantified 30 min after IFNγ exposure. (F and G) Effects of IFNγ on APOE levels and number of P. aeruginosa remaining after incubation with siRNA control or siRNA Stat1 macrophages (F) and WT or Irf1 −/− macrophages (G). Results are mean ± SEM. *p

Techniques Used: Incubation

Deleting Ifngr1 Corrects MSRN Proteins and Macrophage Cholesterol Levels in the “Obesity/IR Only” Model (A–G) WT and Ifngr1 −/− mice were fed a LFD or HFD for 9 weeks. (A) IFNγ production by splenic T cells in WT mice. (B) Metabolic parameters. (C–F) Peritoneal macrophage MSRN protein levels (C), cholesterol levels following treatment with 2% serum from WTD-fed Ldlr −/− mice (D), cholesterol metabolism gene levels (E), and IFNγ -target gene levels (F). (G) p-STAT1/STAT1 levels. Results are mean ± SEM. *p
Figure Legend Snippet: Deleting Ifngr1 Corrects MSRN Proteins and Macrophage Cholesterol Levels in the “Obesity/IR Only” Model (A–G) WT and Ifngr1 −/− mice were fed a LFD or HFD for 9 weeks. (A) IFNγ production by splenic T cells in WT mice. (B) Metabolic parameters. (C–F) Peritoneal macrophage MSRN protein levels (C), cholesterol levels following treatment with 2% serum from WTD-fed Ldlr −/− mice (D), cholesterol metabolism gene levels (E), and IFNγ -target gene levels (F). (G) p-STAT1/STAT1 levels. Results are mean ± SEM. *p

Techniques Used: Mouse Assay

Macrophage IFNGR1 Is Required for Obesity/IR to Target MSRN Proteins, Increase Macrophage Cholesterol Accumulation, and Promote Atherosclerosis (A–L) Ldlr −/− mice transplanted with WT or Ifngr1 −/− bone marrow cells were fed a chow diet, a WTD, or a HCLF diet for up to 15 weeks. (A) IFNγ production by splenic T cells in WT mice. (B) Metabolic parameters. (C–I) Peritoneal macrophage MSRN protein levels (C and D), IFNγ -target gene levels in WT mice (E), p-STAT1/STAT1 levels in WT mice (F), IFNγ -target gene levels in WT and Ifngr1 −/− mice (G), cholesterol levels (H), and cholesterol metabolism gene levels (I). (J) Aortic root lesion area and representative images. Scale bar, 200 mm. (K) Relationships between metabolic parameters and aortic root lesion area. (L) Relationships between fasting insulin or plasma cholesterol levels and aortic root lesion area. Results are mean ± SEM. *p
Figure Legend Snippet: Macrophage IFNGR1 Is Required for Obesity/IR to Target MSRN Proteins, Increase Macrophage Cholesterol Accumulation, and Promote Atherosclerosis (A–L) Ldlr −/− mice transplanted with WT or Ifngr1 −/− bone marrow cells were fed a chow diet, a WTD, or a HCLF diet for up to 15 weeks. (A) IFNγ production by splenic T cells in WT mice. (B) Metabolic parameters. (C–I) Peritoneal macrophage MSRN protein levels (C and D), IFNγ -target gene levels in WT mice (E), p-STAT1/STAT1 levels in WT mice (F), IFNγ -target gene levels in WT and Ifngr1 −/− mice (G), cholesterol levels (H), and cholesterol metabolism gene levels (I). (J) Aortic root lesion area and representative images. Scale bar, 200 mm. (K) Relationships between metabolic parameters and aortic root lesion area. (L) Relationships between fasting insulin or plasma cholesterol levels and aortic root lesion area. Results are mean ± SEM. *p

Techniques Used: Mouse Assay

17) Product Images from "DDX3 Participates in Translational Control of Inflammation Induced by Infections and Injuries"

Article Title: DDX3 Participates in Translational Control of Inflammation Induced by Infections and Injuries

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00285-18

Polysome profile analysis of STAT1, GNB2, Rac1, TAK1, and p38 MAPK translation in the DDX3 knockdown and mock-treated HEK293 cells. HEK293 cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing DDX3 shRNA (shDDX3-2). The cells were harvested for analysis at 3 days posttransduction. The cytoplasmic extracts of the cells were loaded on a linear 15% to 40% sucrose gradient and subjected to ultracentrifugation. (A) After sucrose gradient centrifugation, the polysome profiles were plotted using A 254 values. (B) After sucrose gradient centrifugation, the mRNA/ribosome complexes were separated into 12 fractions. Total RNA was extracted from each fraction for analysis. The purified RNA was resolved by 1% denaturing agarose gel electrophoresis. 18S and 28S rRNAs were visualized using ethidium bromide for staining. (C) Total RNA was extracted from each fraction for analysis. The amount of mRNA was detected by quantitative real-time RT-PCR with specific primers for STAT1, GNB2, Rac1, TAK1, MAPK14 (p38α), MAPK11 (p38β), and MAPK12 (p38γ) mRNAs. The sum of a specific mRNA from the total fractions (fractions 1 to 11) was assumed to be 1 (or 100%), and the relative mRNA level in each fraction was expressed as a percentage of the amount of total specific mRNA. (D) The translational efficiencies of STAT1, GNB2, Rac1, TAK1, MAPK14 (p38α), MAPK11 (p38β), MAPK12 (p38γ), and GAPDH mRNAs in the mock-treated (pLKO.1) and DDX3 knockdown (shDDX3-2) HEK293 cells were calculated and are represented as percentages. The detection of GAPDH mRNA served as a negative control. The bar graph shows the changes in translational efficiencies as means ± SDs from three independent experiments.*, P
Figure Legend Snippet: Polysome profile analysis of STAT1, GNB2, Rac1, TAK1, and p38 MAPK translation in the DDX3 knockdown and mock-treated HEK293 cells. HEK293 cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing DDX3 shRNA (shDDX3-2). The cells were harvested for analysis at 3 days posttransduction. The cytoplasmic extracts of the cells were loaded on a linear 15% to 40% sucrose gradient and subjected to ultracentrifugation. (A) After sucrose gradient centrifugation, the polysome profiles were plotted using A 254 values. (B) After sucrose gradient centrifugation, the mRNA/ribosome complexes were separated into 12 fractions. Total RNA was extracted from each fraction for analysis. The purified RNA was resolved by 1% denaturing agarose gel electrophoresis. 18S and 28S rRNAs were visualized using ethidium bromide for staining. (C) Total RNA was extracted from each fraction for analysis. The amount of mRNA was detected by quantitative real-time RT-PCR with specific primers for STAT1, GNB2, Rac1, TAK1, MAPK14 (p38α), MAPK11 (p38β), and MAPK12 (p38γ) mRNAs. The sum of a specific mRNA from the total fractions (fractions 1 to 11) was assumed to be 1 (or 100%), and the relative mRNA level in each fraction was expressed as a percentage of the amount of total specific mRNA. (D) The translational efficiencies of STAT1, GNB2, Rac1, TAK1, MAPK14 (p38α), MAPK11 (p38β), MAPK12 (p38γ), and GAPDH mRNAs in the mock-treated (pLKO.1) and DDX3 knockdown (shDDX3-2) HEK293 cells were calculated and are represented as percentages. The detection of GAPDH mRNA served as a negative control. The bar graph shows the changes in translational efficiencies as means ± SDs from three independent experiments.*, P

Techniques Used: Transduction, Plasmid Preparation, Expressing, shRNA, Gradient Centrifugation, Purification, Agarose Gel Electrophoresis, Staining, Quantitative RT-PCR, Negative Control

). The nucleotides are colored according to their probabilities in the structure. (C) HEK293 cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing DDX3 shRNA (shDDX3-2). After 48 h, the HEK293 cells were cotransfected with firefly luciferase (Fluc) reporters containing the 5′ UTRs of PACT, STAT1, GNB2, Rac1, TAK1, p38β MAPK, and β-actin mRNAs and the control pRL-SV40 vector encoding the Renilla luciferase (Rluc). The cells were lysed for analysis at 24 h posttransfection. For each transfectant, the Fluc activity was normalized to that of the Rluc control. The bar graph shows the relative Fluc/Rluc activities in DDX3 knockdown cells compared with those in the mock-treated controls. Data are shown as means ± SDs from three independent experiments. **, P
Figure Legend Snippet: ). The nucleotides are colored according to their probabilities in the structure. (C) HEK293 cells were transduced with the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing DDX3 shRNA (shDDX3-2). After 48 h, the HEK293 cells were cotransfected with firefly luciferase (Fluc) reporters containing the 5′ UTRs of PACT, STAT1, GNB2, Rac1, TAK1, p38β MAPK, and β-actin mRNAs and the control pRL-SV40 vector encoding the Renilla luciferase (Rluc). The cells were lysed for analysis at 24 h posttransfection. For each transfectant, the Fluc activity was normalized to that of the Rluc control. The bar graph shows the relative Fluc/Rluc activities in DDX3 knockdown cells compared with those in the mock-treated controls. Data are shown as means ± SDs from three independent experiments. **, P

Techniques Used: Transduction, Plasmid Preparation, Expressing, shRNA, Luciferase, Transfection, Activity Assay

DDX3 regulates the expression of PACT, STAT1, GNB2, Rac1, TAK1, and p38 MAPK proteins in THP-1 and HeLa cells. THP-1 and HeLa cells were transduced with either the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing the indicated shRNAs. After 24 h, puromycin was added to the culture medium for selection. The cells were harvested for analysis at 3 days posttransduction. Immunoblotting was performed using antibodies against DDX3, α-tubulin, PACT, STAT1, GNB2, Rac1, TAK1, and p38 MAPK. The detection of α-tubulin served as a loading control.
Figure Legend Snippet: DDX3 regulates the expression of PACT, STAT1, GNB2, Rac1, TAK1, and p38 MAPK proteins in THP-1 and HeLa cells. THP-1 and HeLa cells were transduced with either the empty lentiviral vector (pLKO.1) or the pLKO.1 vector expressing the indicated shRNAs. After 24 h, puromycin was added to the culture medium for selection. The cells were harvested for analysis at 3 days posttransduction. Immunoblotting was performed using antibodies against DDX3, α-tubulin, PACT, STAT1, GNB2, Rac1, TAK1, and p38 MAPK. The detection of α-tubulin served as a loading control.

Techniques Used: Expressing, Transduction, Plasmid Preparation, Selection

18) Product Images from "Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in HepG2.2.15 Cells via JAK/STAT Signaling"

Article Title: Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in HepG2.2.15 Cells via JAK/STAT Signaling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0156496

USP18 silencing increases the antiviral activity of IFN-α associated with the JAK-STAT signaling pathway. (A) Hepg2.2.15 cells were treated with shRR or shUSP18 for 48 hours and then subjected to immunofluorescence analysis using a STAT1-specific polyclonal antibody. Blue, DAPI; green, GFP; red, anti-STAT1; original magnification × 400. Hepg2.2.15 cells were treated with indicated concentration of IFN-α (0, 1, 10, 100, 1000 IU/ml) for 20 hours after either shUSP18 or shRR lentivirus transduction. (B) The protein levels of p-STAT1 and STAT1 were quantified by western blot. (C) STAT1 activation was initiated and prolonged in USP18-silenced HepG2.2.15 cells and STAT1 phosphorylation remained 24 hours after treatment compared to the control group.
Figure Legend Snippet: USP18 silencing increases the antiviral activity of IFN-α associated with the JAK-STAT signaling pathway. (A) Hepg2.2.15 cells were treated with shRR or shUSP18 for 48 hours and then subjected to immunofluorescence analysis using a STAT1-specific polyclonal antibody. Blue, DAPI; green, GFP; red, anti-STAT1; original magnification × 400. Hepg2.2.15 cells were treated with indicated concentration of IFN-α (0, 1, 10, 100, 1000 IU/ml) for 20 hours after either shUSP18 or shRR lentivirus transduction. (B) The protein levels of p-STAT1 and STAT1 were quantified by western blot. (C) STAT1 activation was initiated and prolonged in USP18-silenced HepG2.2.15 cells and STAT1 phosphorylation remained 24 hours after treatment compared to the control group.

Techniques Used: Activity Assay, Immunofluorescence, Concentration Assay, Transduction, Western Blot, Activation Assay

19) Product Images from "C-reactive protein directly suppresses T helper 1 cell differentiation and alleviates experimental autoimmune encephalomyelitis"

Article Title: C-reactive protein directly suppresses T helper 1 cell differentiation and alleviates experimental autoimmune encephalomyelitis

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

doi: 10.4049/jimmunol.1402909

Direct modulation of human Jurkat T cell and mouse T cell responses by human CRP Human Jurkat T cells ( A and B ) and mouse primary naïve T cells ( C – E ) were activated with immobilized anti-CD3 plus soluble anti-CD28 antibodies. ( A ) Jurkat cells co-incubated with human CRP for 3 days (100 μg/ml, black bars) expressed significantly more IL-4 mRNA and significantly less IFN-γ mRNA than cells not treated with CRP (vehicle, white bars; n=3). This effect of CRP was completely abrogated if CRP was denatured by boiling or if the CRP stock solution was filtered through a 10 kDa cut-off membrane. Polymyxin B (20 μg/ml) was included in all experiments to ensure there was no contribution made by residual endotoxin. ( B ) Jurkat cells transfected with a human CRP expressing vector (black bars) made significantly more IL-4 mRNA and significantly less IFN-γ mRNA than cells transfected with an empty vector (white bars; n=3). ( C and D ) Mouse naïve CD4 + CD62L + T cells co-incubated with human CRP for 3 days (100 μg/ml, black bars) expressed more IL-4 and less IFN-γ than cells not treated with CRP (white bars) at both mRNA ( C ) and protein levels ( D ) (n=3). ( E ) Mouse naïve CD4 + CD62L + T cells were activated with anti-CD3/anti-CD28 and treated with CRP, IL-4, and IL-12 alone or in combination. CRP (100 μg/ml) inhibited IL-12 induced phosphorylation of STAT1 (left blot) and STAT4 (middle blot) but enhanced IL-4 induced phosphorylation of STAT6 (right blot). * p
Figure Legend Snippet: Direct modulation of human Jurkat T cell and mouse T cell responses by human CRP Human Jurkat T cells ( A and B ) and mouse primary naïve T cells ( C – E ) were activated with immobilized anti-CD3 plus soluble anti-CD28 antibodies. ( A ) Jurkat cells co-incubated with human CRP for 3 days (100 μg/ml, black bars) expressed significantly more IL-4 mRNA and significantly less IFN-γ mRNA than cells not treated with CRP (vehicle, white bars; n=3). This effect of CRP was completely abrogated if CRP was denatured by boiling or if the CRP stock solution was filtered through a 10 kDa cut-off membrane. Polymyxin B (20 μg/ml) was included in all experiments to ensure there was no contribution made by residual endotoxin. ( B ) Jurkat cells transfected with a human CRP expressing vector (black bars) made significantly more IL-4 mRNA and significantly less IFN-γ mRNA than cells transfected with an empty vector (white bars; n=3). ( C and D ) Mouse naïve CD4 + CD62L + T cells co-incubated with human CRP for 3 days (100 μg/ml, black bars) expressed more IL-4 and less IFN-γ than cells not treated with CRP (white bars) at both mRNA ( C ) and protein levels ( D ) (n=3). ( E ) Mouse naïve CD4 + CD62L + T cells were activated with anti-CD3/anti-CD28 and treated with CRP, IL-4, and IL-12 alone or in combination. CRP (100 μg/ml) inhibited IL-12 induced phosphorylation of STAT1 (left blot) and STAT4 (middle blot) but enhanced IL-4 induced phosphorylation of STAT6 (right blot). * p

Techniques Used: Incubation, Transfection, Expressing, Plasmid Preparation

Direct modulation of Th1/Th2 differentiation of mouse primary naïve CD4 + CD62L + T cells by human CRP ( A – C ) 2 × 10 5 CD4 + CD62L + naïve T cells were cultured for 3 days with plate-bound anti-CD3 and fluid-phase anti-CD28 under Th1 polarizing conditions (10 ng/ml mIL-2, 20ng/ml mIL-12p70, 10 μg/ml anti-IL-4 mAb) or Th2 polarizing conditions (10 ng/ml mIL-2, 20 ng/ml mIL-4, 10 μg/ml anti-IL12 mAb, 10 μg/ml anti-IFN-γ mAb). Human CRP (100 μg/ml) was present continuously in the cell cultures starting from the beginning of polarization (early treatment) or starting 24 h after the beginning of polarization (late treatment). For pre-treatment, CRP was added to cells 24 h before the beginning of polarization but was absent thereafter. Intracellular staining for IL-4 and IFN-γ was assessed by flow cytometry. ( A ) CRP late treatment was most effective at inhibiting Th1 differentiation (upper panels) and promoting Th2 differentiation (lower panels) of mouse naïve T cells. ( B and C ) Th1 and Th2 signature cytokine receptors and transcription factor expression ( B ) and STAT phosphorylation levels ( C ) for mouse naïve T cells polarized towards Th1 or Th2 phenotypes with or without early CRP treatment. Following immunoblotting of GATA-3 or T-bet using the cell lysates, the blots were then stripped and re-probed for β-tubulin as the loading controls. Under Th1 polarizing conditions CRP significantly inhibited STAT1 and STAT4 phosphorylation, whereas under Th2 polarizing conditions CRP significantly increased IL-4Ra expression and STAT6 phosphorylation (n=3). * p
Figure Legend Snippet: Direct modulation of Th1/Th2 differentiation of mouse primary naïve CD4 + CD62L + T cells by human CRP ( A – C ) 2 × 10 5 CD4 + CD62L + naïve T cells were cultured for 3 days with plate-bound anti-CD3 and fluid-phase anti-CD28 under Th1 polarizing conditions (10 ng/ml mIL-2, 20ng/ml mIL-12p70, 10 μg/ml anti-IL-4 mAb) or Th2 polarizing conditions (10 ng/ml mIL-2, 20 ng/ml mIL-4, 10 μg/ml anti-IL12 mAb, 10 μg/ml anti-IFN-γ mAb). Human CRP (100 μg/ml) was present continuously in the cell cultures starting from the beginning of polarization (early treatment) or starting 24 h after the beginning of polarization (late treatment). For pre-treatment, CRP was added to cells 24 h before the beginning of polarization but was absent thereafter. Intracellular staining for IL-4 and IFN-γ was assessed by flow cytometry. ( A ) CRP late treatment was most effective at inhibiting Th1 differentiation (upper panels) and promoting Th2 differentiation (lower panels) of mouse naïve T cells. ( B and C ) Th1 and Th2 signature cytokine receptors and transcription factor expression ( B ) and STAT phosphorylation levels ( C ) for mouse naïve T cells polarized towards Th1 or Th2 phenotypes with or without early CRP treatment. Following immunoblotting of GATA-3 or T-bet using the cell lysates, the blots were then stripped and re-probed for β-tubulin as the loading controls. Under Th1 polarizing conditions CRP significantly inhibited STAT1 and STAT4 phosphorylation, whereas under Th2 polarizing conditions CRP significantly increased IL-4Ra expression and STAT6 phosphorylation (n=3). * p

Techniques Used: Cell Culture, Staining, Flow Cytometry, Cytometry, Expressing

20) Product Images from "THZ1 targeting CDK7 suppresses STAT transcriptional activity and sensitizes T-cell lymphomas to BCL2 inhibitors"

Article Title: THZ1 targeting CDK7 suppresses STAT transcriptional activity and sensitizes T-cell lymphomas to BCL2 inhibitors

Journal: Nature Communications

doi: 10.1038/ncomms14290

THZ1 inhibits STAT1 and STAT5 signalling. ( a ) Effect on STAT3 DNA binding activity of the STAT3 inhibitors cryptotanshinone (CTN) and S31-201 in STAT3 mutant cell lines OCI-Ly12 and OCI-Ly13.2. ( b ) Effect on cell viability at 48 h of the STAT3 inhibitors cryptotanshinone and S31-201 in OCI-Ly12 and OCI-Ly13.2 cells. ( c ) Activity of STAT1, STAT5a and STAT5b measured by DNA biding to a consensus sequence in nuclear extracts of OCI-Ly12 and OCI-Ly13.2 cells treated with cryptotanshinone (CTN) and S31-201. ( d ) Immunoblots of STAT5, STAT1, phospho-STAT5 and phospho-STAT1 (to actin) in PTCL cells treated with THZ1 500 nM for the indicated time points. ( e ) Activity of STAT1, STAT5a and STAT5b measured by DNA biding to a consensus sequence in nuclear extracts of OCI-Ly12 and OCI-Ly13.2 cells treated with 500 nM of THZ1 for the indicated time points. ( f ) JUND transcript (left) and protein (right) levels in OCI-Ly13.2 cells upon THZ1 500 nM treatment for the indicated time points. All data are presented as mean with 95% CI for triplicates. P -values obtained from T -test.
Figure Legend Snippet: THZ1 inhibits STAT1 and STAT5 signalling. ( a ) Effect on STAT3 DNA binding activity of the STAT3 inhibitors cryptotanshinone (CTN) and S31-201 in STAT3 mutant cell lines OCI-Ly12 and OCI-Ly13.2. ( b ) Effect on cell viability at 48 h of the STAT3 inhibitors cryptotanshinone and S31-201 in OCI-Ly12 and OCI-Ly13.2 cells. ( c ) Activity of STAT1, STAT5a and STAT5b measured by DNA biding to a consensus sequence in nuclear extracts of OCI-Ly12 and OCI-Ly13.2 cells treated with cryptotanshinone (CTN) and S31-201. ( d ) Immunoblots of STAT5, STAT1, phospho-STAT5 and phospho-STAT1 (to actin) in PTCL cells treated with THZ1 500 nM for the indicated time points. ( e ) Activity of STAT1, STAT5a and STAT5b measured by DNA biding to a consensus sequence in nuclear extracts of OCI-Ly12 and OCI-Ly13.2 cells treated with 500 nM of THZ1 for the indicated time points. ( f ) JUND transcript (left) and protein (right) levels in OCI-Ly13.2 cells upon THZ1 500 nM treatment for the indicated time points. All data are presented as mean with 95% CI for triplicates. P -values obtained from T -test.

Techniques Used: Binding Assay, Activity Assay, Mutagenesis, Sequencing, Western Blot

21) Product Images from "Adoptive transfer of bone marrow-derived dendritic cells (BMDCs) alleviates OVA-induced allergic airway inflammation in asthmatic mice"

Article Title: Adoptive transfer of bone marrow-derived dendritic cells (BMDCs) alleviates OVA-induced allergic airway inflammation in asthmatic mice

Journal: Scientific Reports

doi: 10.1038/s41598-020-70467-3

STAT signaling pathways were inhibited by adoptive transfer of BMDCs with or without the SOCS3 gene. Lung tissue samples were homogenized after BMDC adoptive transfer into OVA-sensitized mice. Mononuclear cells were isolated from homogenates. STATs and phosphorylated STATs were detected by Western blotting (WB). The right columns represent the densitometry analysis of the WB results. ( A ) STAT1 and py-STAT1 detected by WB. ( B ) STAT4 and py-STAT4 detected by WB. C. STAT6 and py-STAT6 detected by WB. Data are representative of 3 independent experiments with similar results (*P
Figure Legend Snippet: STAT signaling pathways were inhibited by adoptive transfer of BMDCs with or without the SOCS3 gene. Lung tissue samples were homogenized after BMDC adoptive transfer into OVA-sensitized mice. Mononuclear cells were isolated from homogenates. STATs and phosphorylated STATs were detected by Western blotting (WB). The right columns represent the densitometry analysis of the WB results. ( A ) STAT1 and py-STAT1 detected by WB. ( B ) STAT4 and py-STAT4 detected by WB. C. STAT6 and py-STAT6 detected by WB. Data are representative of 3 independent experiments with similar results (*P

Techniques Used: Adoptive Transfer Assay, Mouse Assay, Isolation, Western Blot

22) Product Images from "Type III Interferon Induces Distinct SOCS1 Expression Pattern that Contributes to Delayed but Prolonged Activation of Jak/STAT Signaling Pathway: Implications for Treatment Non-Response in HCV Patients"

Article Title: Type III Interferon Induces Distinct SOCS1 Expression Pattern that Contributes to Delayed but Prolonged Activation of Jak/STAT Signaling Pathway: Implications for Treatment Non-Response in HCV Patients

Journal: PLoS ONE

doi: 10.1371/journal.pone.0133800

Over-expression of SOCS1 blocked IFN-α and IFN-λ signaling pathway. Over-expression of SOCS1 repressed IFN-induced ISRE-luciferase activity (A), decreased IFN-α and IFN-λ induced ISG15 and MxA (B) mRNA and protein levels (C) and STAT1 phosphorylation (C) in Con1b replicon cell. (D) Western blot data analyzed by Quantity One are expressed as the means of ratios of targeted genes (pSTAT1, ISG15 and MxA) /β-actin. Con1b cells were cotransfected with pCR3.1 (mock) or pCR3.1/SOCS1, pISRE-luc and pRL-TK for 24 hours and then 100 IU/mL IFN-α and 50 ng/mL IFN-λ was added to the cells for 24 hours respectively. The firefly and Renilla luciferase activity was measured. Total RNA was harvested and reverse transcribed. Cells were transfected with pCR3.1 (mock) or pCR3.1/SOCS1 for 24 hours and then treated with 100 IU/mL IFN-α and 50 ng/mL IFN-λ respectively for 24 hours and the cells were collected. Cell lysates were harvested and the levels of mRNA expression of ISG15 and MxA were determined by quantitative real time PCR normalized to GAPDH. In addition, the cell were analyzed by immunoblotting with the indicated antibodies as described in Materials and Methods. The samples for Tyrosine phosphorylation of STAT1 (pSTAT1) were harvested after incubating with IFNs for 15 min. Shown is one representative Western blot out of three performed experiments. + with;—without. Data are presented as means ± SEM, n = 3. Error bars indicate standard error of mean (SEM). “*” means p values less than 0.05; “**” p values less than 0.01; “***” means p values less than 0.001.
Figure Legend Snippet: Over-expression of SOCS1 blocked IFN-α and IFN-λ signaling pathway. Over-expression of SOCS1 repressed IFN-induced ISRE-luciferase activity (A), decreased IFN-α and IFN-λ induced ISG15 and MxA (B) mRNA and protein levels (C) and STAT1 phosphorylation (C) in Con1b replicon cell. (D) Western blot data analyzed by Quantity One are expressed as the means of ratios of targeted genes (pSTAT1, ISG15 and MxA) /β-actin. Con1b cells were cotransfected with pCR3.1 (mock) or pCR3.1/SOCS1, pISRE-luc and pRL-TK for 24 hours and then 100 IU/mL IFN-α and 50 ng/mL IFN-λ was added to the cells for 24 hours respectively. The firefly and Renilla luciferase activity was measured. Total RNA was harvested and reverse transcribed. Cells were transfected with pCR3.1 (mock) or pCR3.1/SOCS1 for 24 hours and then treated with 100 IU/mL IFN-α and 50 ng/mL IFN-λ respectively for 24 hours and the cells were collected. Cell lysates were harvested and the levels of mRNA expression of ISG15 and MxA were determined by quantitative real time PCR normalized to GAPDH. In addition, the cell were analyzed by immunoblotting with the indicated antibodies as described in Materials and Methods. The samples for Tyrosine phosphorylation of STAT1 (pSTAT1) were harvested after incubating with IFNs for 15 min. Shown is one representative Western blot out of three performed experiments. + with;—without. Data are presented as means ± SEM, n = 3. Error bars indicate standard error of mean (SEM). “*” means p values less than 0.05; “**” p values less than 0.01; “***” means p values less than 0.001.

Techniques Used: Over Expression, Luciferase, Activity Assay, Western Blot, Transfection, Expressing, Real-time Polymerase Chain Reaction

23) Product Images from "Spatiotemporal control of interferon-induced JAK/STAT signalling and gene transcription by the retromer complex"

Article Title: Spatiotemporal control of interferon-induced JAK/STAT signalling and gene transcription by the retromer complex

Journal: Nature Communications

doi: 10.1038/ncomms13476

Model for the spatiotemporal control of FNAR endosomal sorting and JAK/STAT signalling by the retromer. Left: at the plasma membrane, the IFNAR complex, assembled by type-I IFNs binding to IFNAR2 and to IFNAR1, is internalized by clathrin-dependent endocytosis, which triggers activation of the JAK kinases and STAT1 phosphorylation (pSTAT1). Middle: the VPS26-29-35 cargo recognition retromer complex is recruited via Rab7 to the early endosome and controls the timely separation of IFNAR1–IFNAR2 subunits that is required for JAK/STAT signalling termination. Right: inhibiting retromer activity results in prolonged IFNAR1-IFNAR2 association at the early endosome, perturbed IFNAR trafficking and abnormally prolonged activation of the JAK/STAT pathway and gene transcription.
Figure Legend Snippet: Model for the spatiotemporal control of FNAR endosomal sorting and JAK/STAT signalling by the retromer. Left: at the plasma membrane, the IFNAR complex, assembled by type-I IFNs binding to IFNAR2 and to IFNAR1, is internalized by clathrin-dependent endocytosis, which triggers activation of the JAK kinases and STAT1 phosphorylation (pSTAT1). Middle: the VPS26-29-35 cargo recognition retromer complex is recruited via Rab7 to the early endosome and controls the timely separation of IFNAR1–IFNAR2 subunits that is required for JAK/STAT signalling termination. Right: inhibiting retromer activity results in prolonged IFNAR1-IFNAR2 association at the early endosome, perturbed IFNAR trafficking and abnormally prolonged activation of the JAK/STAT pathway and gene transcription.

Techniques Used: Binding Assay, Activation Assay, Activity Assay

VPS35 depletion results in prolonged activation of JAK/STAT signalling by type-I IFNs. ( a ) Immunoblots for tyrosine phosphorylation levels of STAT1 (pSTAT1) in CTRL and VPS35-depleted RPE1 cells stimulated with IFN-α or IFN-β for the indicated times. Representative immunoblot out of four independent experiments. ( b ) Quantification of experiments performed in a : pSTAT1 and STAT1 levels were normalized to tubulin (tub) level (loading control) and the ratio (pSTAT1/tub)/(STAT1/tub) was calculated for each condition. STAT1 activation on VPS35 depletion was normalized to CTRL as 1. ( c ) PCA using ΔCT value in CTRL and VPS35-depleted RPE1 cells with or without IFN-α or IFN-β stimulation for all replicates (biological duplicates with technical duplicates each). ( d ) Clustering analysis based on −ΔCt values for gene expression in CTRL and VPS35-depleted RPE1 cells with or without IFN-α or IFN-β stimulation for all samples replicates (biological duplicates with technical duplicates each). Each square represents a value for a given gene (row) for a specific condition (column). Genes depicted in blue are expressed at low level (low −ΔCT value); genes depicted in red are expressed at high level (high −ΔCT value). ( e ) Genes significantly and selectively upregulated by IFN-α or IFN-β stimulation in VPS35-depleted RPE1 cells. ( f ) Expression of IFN-independent genes: p21 (upper panel), and CHMP2A (lower panel) in CTRL and in VPS35-depleted RPE1 cells with or without IFN-α or IFN-β stimulation. Reproducibility of experiments: panel a shows representative data for four independent experiments, b shows quantification of data for four, four and three independent experiments for each time point, respectively. Statistical analysis with two-tailed, unpaired t -test. * P
Figure Legend Snippet: VPS35 depletion results in prolonged activation of JAK/STAT signalling by type-I IFNs. ( a ) Immunoblots for tyrosine phosphorylation levels of STAT1 (pSTAT1) in CTRL and VPS35-depleted RPE1 cells stimulated with IFN-α or IFN-β for the indicated times. Representative immunoblot out of four independent experiments. ( b ) Quantification of experiments performed in a : pSTAT1 and STAT1 levels were normalized to tubulin (tub) level (loading control) and the ratio (pSTAT1/tub)/(STAT1/tub) was calculated for each condition. STAT1 activation on VPS35 depletion was normalized to CTRL as 1. ( c ) PCA using ΔCT value in CTRL and VPS35-depleted RPE1 cells with or without IFN-α or IFN-β stimulation for all replicates (biological duplicates with technical duplicates each). ( d ) Clustering analysis based on −ΔCt values for gene expression in CTRL and VPS35-depleted RPE1 cells with or without IFN-α or IFN-β stimulation for all samples replicates (biological duplicates with technical duplicates each). Each square represents a value for a given gene (row) for a specific condition (column). Genes depicted in blue are expressed at low level (low −ΔCT value); genes depicted in red are expressed at high level (high −ΔCT value). ( e ) Genes significantly and selectively upregulated by IFN-α or IFN-β stimulation in VPS35-depleted RPE1 cells. ( f ) Expression of IFN-independent genes: p21 (upper panel), and CHMP2A (lower panel) in CTRL and in VPS35-depleted RPE1 cells with or without IFN-α or IFN-β stimulation. Reproducibility of experiments: panel a shows representative data for four independent experiments, b shows quantification of data for four, four and three independent experiments for each time point, respectively. Statistical analysis with two-tailed, unpaired t -test. * P

Techniques Used: Activation Assay, Western Blot, Expressing, Two Tailed Test

24) Product Images from "Activation of Protein Tyrosine Phosphatase Non-Receptor Type 2 by Spermidine Exerts Anti-Inflammatory Effects in Human THP-1 Monocytes and in a Mouse Model of Acute Colitis"

Article Title: Activation of Protein Tyrosine Phosphatase Non-Receptor Type 2 by Spermidine Exerts Anti-Inflammatory Effects in Human THP-1 Monocytes and in a Mouse Model of Acute Colitis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073703

Effects of spermidine treatment on the phosphorylation levels of signal transducers and activators of transcription (STATs) and p38 mitogen-activated protein kinase (MAPK) in interferon-gamma (IFN-γ)-treated THP-1 cells. Representative Western blots and densitometry (n = 3) show phosphorylation status of ( a and b ) STAT1, ( c ) STAT3 and ( d ) p38 MAPK in THP-1 cells treated with IFN-γ (1000 U/ml) and/or spermidine (100 µM) for ( a , c , and d ) 30 min or ( b ) 36 h. Blots were probed for β-actin to show equal protein loading. Significant differences compared to untreated cells (* = p
Figure Legend Snippet: Effects of spermidine treatment on the phosphorylation levels of signal transducers and activators of transcription (STATs) and p38 mitogen-activated protein kinase (MAPK) in interferon-gamma (IFN-γ)-treated THP-1 cells. Representative Western blots and densitometry (n = 3) show phosphorylation status of ( a and b ) STAT1, ( c ) STAT3 and ( d ) p38 MAPK in THP-1 cells treated with IFN-γ (1000 U/ml) and/or spermidine (100 µM) for ( a , c , and d ) 30 min or ( b ) 36 h. Blots were probed for β-actin to show equal protein loading. Significant differences compared to untreated cells (* = p

Techniques Used: Western Blot

25) Product Images from "Resistance to Alpha/Beta Interferon Is a Determinant of West Nile Virus Replication Fitness and Virulence"

Article Title: Resistance to Alpha/Beta Interferon Is a Determinant of West Nile Virus Replication Fitness and Virulence

Journal: Journal of Virology

doi: 10.1128/JVI.00768-06

MAD78 regulation of JAK-STAT signaling is attenuated. (A) A549 cells were mock infected (lanes 1 to 3) or infected (MOI = 5) with WNV (TX02, lanes 4 to 6; MAD78, lanes 7 to 9). Twenty-four h postinfection, cells were pulse treated with 1,000 U IFN-α for 0, 15, or 30 min, and whole-cell lysates were collected and analyzed by immunoblotting to determine WNV protein (NS3) abundance or the abundance of the active, tyrosine-phosphorylated (P) isoforms of Tyk2, STAT1, and STAT2. (B) A549 cells were mock infected or infected with TX02 or MAD78. Cells were then treated with 1,000 U IFN-α for the times indicated. Proteins were immunoprecipitated from whole-cell lysates by use of an antiphosphotyrosine antibody, and JAK1 immunoblot analysis was performed subsequently. (C) A549 cells were infected with TX02 or MAD78 and treated with IFN as described for panel A. Whole-cell lysates were analyzed for the presence of phospho-JAK1. (D) A549 cells were mock infected (M; lanes 1 and 12) or infected (MOI = 5) with TX02 (lanes 2 to 11) or MAD78 (lanes 13 to 22). In 5-h increments, cells were pulse treated with 1,000 U IFN-α for 30 min, and whole-cell lysates were collected and analyzed by immunoblotting to detect GAPDH, WNV, phosphotyrosine STAT isoforms, and total STAT1 or STAT2 abundance. Bars at left indicate the positions of molecular mass (kilodalton) standards. Arrows at right denote the positions of the indicated WNV proteins.
Figure Legend Snippet: MAD78 regulation of JAK-STAT signaling is attenuated. (A) A549 cells were mock infected (lanes 1 to 3) or infected (MOI = 5) with WNV (TX02, lanes 4 to 6; MAD78, lanes 7 to 9). Twenty-four h postinfection, cells were pulse treated with 1,000 U IFN-α for 0, 15, or 30 min, and whole-cell lysates were collected and analyzed by immunoblotting to determine WNV protein (NS3) abundance or the abundance of the active, tyrosine-phosphorylated (P) isoforms of Tyk2, STAT1, and STAT2. (B) A549 cells were mock infected or infected with TX02 or MAD78. Cells were then treated with 1,000 U IFN-α for the times indicated. Proteins were immunoprecipitated from whole-cell lysates by use of an antiphosphotyrosine antibody, and JAK1 immunoblot analysis was performed subsequently. (C) A549 cells were infected with TX02 or MAD78 and treated with IFN as described for panel A. Whole-cell lysates were analyzed for the presence of phospho-JAK1. (D) A549 cells were mock infected (M; lanes 1 and 12) or infected (MOI = 5) with TX02 (lanes 2 to 11) or MAD78 (lanes 13 to 22). In 5-h increments, cells were pulse treated with 1,000 U IFN-α for 30 min, and whole-cell lysates were collected and analyzed by immunoblotting to detect GAPDH, WNV, phosphotyrosine STAT isoforms, and total STAT1 or STAT2 abundance. Bars at left indicate the positions of molecular mass (kilodalton) standards. Arrows at right denote the positions of the indicated WNV proteins.

Techniques Used: Infection, Immunoprecipitation

TX02 prevents IFN-α-induced STAT1 and STAT2 nuclear translocation. (A) A549 cells were infected (MOI = 2) with TX02 or MAD78 or were left uninfected (MOCK). Twenty-four h postinfection, cells were treated with 1,000 U IFN-α for 1 h and then stained using primary antibodies directed against WNV or STAT2. Nuclei were stained with DAPI. Panels show representative confocal micrographs of images obtained (magnification, 40×). Top panels show STAT2 in mock-infected control cells. Images from infected cells show STAT2 (left column), STAT and WNV merged (middle column), and nuclei (right column). (B) A549 cells were infected (MOI = 5) with TX02 or MAD78. Twenty-four h postinfection, cells were left untreated or were treated with 1,000 U IFN-α for 1 h. Whole-cell lysates were fractionated into cytoplasmic and nuclear extracts and analyzed by immunoblotting using STAT- or phospho-STAT-specific antibodies. The fractionation of poly(ADP-ribose) polymerase (PARP) was monitored as a nuclear control protein. This shows that lanes 1 and 3 contained a residual level of nuclear material not present in lanes 5, 7, 9, and 11. P-, phosphorylated.
Figure Legend Snippet: TX02 prevents IFN-α-induced STAT1 and STAT2 nuclear translocation. (A) A549 cells were infected (MOI = 2) with TX02 or MAD78 or were left uninfected (MOCK). Twenty-four h postinfection, cells were treated with 1,000 U IFN-α for 1 h and then stained using primary antibodies directed against WNV or STAT2. Nuclei were stained with DAPI. Panels show representative confocal micrographs of images obtained (magnification, 40×). Top panels show STAT2 in mock-infected control cells. Images from infected cells show STAT2 (left column), STAT and WNV merged (middle column), and nuclei (right column). (B) A549 cells were infected (MOI = 5) with TX02 or MAD78. Twenty-four h postinfection, cells were left untreated or were treated with 1,000 U IFN-α for 1 h. Whole-cell lysates were fractionated into cytoplasmic and nuclear extracts and analyzed by immunoblotting using STAT- or phospho-STAT-specific antibodies. The fractionation of poly(ADP-ribose) polymerase (PARP) was monitored as a nuclear control protein. This shows that lanes 1 and 3 contained a residual level of nuclear material not present in lanes 5, 7, 9, and 11. P-, phosphorylated.

Techniques Used: Translocation Assay, Infection, Staining, Fractionation

26) Product Images from "Transcriptional and epigenetic characterization of early striosomes identifies Foxf2 and Olig2 as factors required for development of striatal compartmentation and neuronal phenotypic differentiation"

Article Title: Transcriptional and epigenetic characterization of early striosomes identifies Foxf2 and Olig2 as factors required for development of striatal compartmentation and neuronal phenotypic differentiation

Journal: bioRxiv

doi: 10.1101/2020.05.19.105171

STAT1 overexpression in MSNs in vitro promotes their maturation and increases the levels of Foxf2 and Olig2 mRNA. a) Network demonstrating that Stat1 may be a “master” TF in striosomes as it regulates the greatest number of TFs enriched in that compartment. b , c) Stat1 and DARPP-32 immunolabeling in DIV9 WT primary striatal neurons transduced for 96 h with ADV- STAT1 -V5 vs. non-transduced controls show that Stat1 overexpression increases the number of DARPP-32 immunopositive cells. Scale bars correspond to 50 µm. n=4 images from 4 individual cultures, t-test ***P
Figure Legend Snippet: STAT1 overexpression in MSNs in vitro promotes their maturation and increases the levels of Foxf2 and Olig2 mRNA. a) Network demonstrating that Stat1 may be a “master” TF in striosomes as it regulates the greatest number of TFs enriched in that compartment. b , c) Stat1 and DARPP-32 immunolabeling in DIV9 WT primary striatal neurons transduced for 96 h with ADV- STAT1 -V5 vs. non-transduced controls show that Stat1 overexpression increases the number of DARPP-32 immunopositive cells. Scale bars correspond to 50 µm. n=4 images from 4 individual cultures, t-test ***P

Techniques Used: Over Expression, In Vitro, Immunolabeling

Foxf2, Olig2, and Stat1, alone and in combination, promote MSN differentiation in NSCs from human HD induced pluripotent stem cells. a) HD72-NSCs transduced for 4 days with ADV- FOXF2 , ADV- OLIG2 and ADV- STAT1 were immunostained with Oprm1 (green) and DARPP-32 (red). Non-transduced cells and AD-GFP were used as control. Scale bars: 100 µm. b) RT-qPCR assay on HD72-NSCs transduced for 4 days with ADV-GFP, ADV- FOXF2 , ADV- OLIG2 and ADV- STAT1 . n=3 individual cultures. One outlier (one of the three technical data points) was excluded in Bcl11b RT-qPCR when transduced with ADV- FOXF2 and ADV -STAT1 . One-way ANOVA for multiple comparisons (Dunnett’s) *P
Figure Legend Snippet: Foxf2, Olig2, and Stat1, alone and in combination, promote MSN differentiation in NSCs from human HD induced pluripotent stem cells. a) HD72-NSCs transduced for 4 days with ADV- FOXF2 , ADV- OLIG2 and ADV- STAT1 were immunostained with Oprm1 (green) and DARPP-32 (red). Non-transduced cells and AD-GFP were used as control. Scale bars: 100 µm. b) RT-qPCR assay on HD72-NSCs transduced for 4 days with ADV-GFP, ADV- FOXF2 , ADV- OLIG2 and ADV- STAT1 . n=3 individual cultures. One outlier (one of the three technical data points) was excluded in Bcl11b RT-qPCR when transduced with ADV- FOXF2 and ADV -STAT1 . One-way ANOVA for multiple comparisons (Dunnett’s) *P

Techniques Used: Quantitative RT-PCR, Transduction

27) Product Images from "Hepatitis C Virus NS5A Disrupts STAT1 Phosphorylation and Suppresses Type I Interferon Signaling"

Article Title: Hepatitis C Virus NS5A Disrupts STAT1 Phosphorylation and Suppresses Type I Interferon Signaling

Journal: Journal of Virology

doi: 10.1128/JVI.00533-12

GT1 NS5A has stronger binding to STAT1 than GT3 NS5A. The plasmid DNA constructs encoding HCV NS5A of GT1a, GT1b, GT3a, or GT3b were transfected into Huh7.5.1 cells. Cell lysates were harvested at 48 h after transfection. STAT1 immunoprecipitation was
Figure Legend Snippet: GT1 NS5A has stronger binding to STAT1 than GT3 NS5A. The plasmid DNA constructs encoding HCV NS5A of GT1a, GT1b, GT3a, or GT3b were transfected into Huh7.5.1 cells. Cell lysates were harvested at 48 h after transfection. STAT1 immunoprecipitation was

Techniques Used: Binding Assay, Plasmid Preparation, Construct, Transfection, Immunoprecipitation

HCV NS5A reduces STAT1 phosphorylation. The inhibitory effect of NS5A on IFN-α-induced STAT1 phosphorylation was assessed in cells expressing the NS5A protein. Huh7.5.1 cells were transfected with NS5A constructs for 48 h. Huh7.5.1, JFH1-infected
Figure Legend Snippet: HCV NS5A reduces STAT1 phosphorylation. The inhibitory effect of NS5A on IFN-α-induced STAT1 phosphorylation was assessed in cells expressing the NS5A protein. Huh7.5.1 cells were transfected with NS5A constructs for 48 h. Huh7.5.1, JFH1-infected

Techniques Used: Expressing, Transfection, Construct, Infection

C-terminal NS5A displays inhibitory effects on IFN signaling through binding to STAT1. Full-length HCV NS5A (aa 1 to 447) was also classified into domain I (aa 1 to 213), domain II (aa 250 to 342), and domain III (aa 356 to 447) in a previous report (
Figure Legend Snippet: C-terminal NS5A displays inhibitory effects on IFN signaling through binding to STAT1. Full-length HCV NS5A (aa 1 to 447) was also classified into domain I (aa 1 to 213), domain II (aa 250 to 342), and domain III (aa 356 to 447) in a previous report (

Techniques Used: Binding Assay

28) Product Images from "Obesity and Insulin Resistance Promote Atherosclerosis through an IFNγ-Regulated Macrophage Protein Network"

Article Title: Obesity and Insulin Resistance Promote Atherosclerosis through an IFNγ-Regulated Macrophage Protein Network

Journal: Cell reports

doi: 10.1016/j.celrep.2018.05.010

“Metabolic Disease Appropriate” Doses of IFN γ Specifically Target MSRN Proteins (A–D) Macrophages were treated with varying levels of IFNγ. (A) APOE levels. (B) p-STAT1/STAT1 levels. (C) Irf8 levels. (D) Number of P. aeruginosa remaining after incubation with macrophages. (E) Efficiency of STAT1 knockdown in macrophages treated with control or Stat1 siRNA. STAT1 levels were quantified 30 min after IFNγ exposure. (F and G) Effects of IFNγ on APOE levels and number of P. aeruginosa remaining after incubation with siRNA control or siRNA Stat1 macrophages (F) and WT or Irf1 −/− macrophages (G). .
Figure Legend Snippet: “Metabolic Disease Appropriate” Doses of IFN γ Specifically Target MSRN Proteins (A–D) Macrophages were treated with varying levels of IFNγ. (A) APOE levels. (B) p-STAT1/STAT1 levels. (C) Irf8 levels. (D) Number of P. aeruginosa remaining after incubation with macrophages. (E) Efficiency of STAT1 knockdown in macrophages treated with control or Stat1 siRNA. STAT1 levels were quantified 30 min after IFNγ exposure. (F and G) Effects of IFNγ on APOE levels and number of P. aeruginosa remaining after incubation with siRNA control or siRNA Stat1 macrophages (F) and WT or Irf1 −/− macrophages (G). .

Techniques Used: Incubation

Deleting Ifngr1 Corrects MSRN Proteins and Macrophage Cholesterol Levels in the “Obesity/IR Only” Model (A–G) WT and Ifngr1 −/− mice were fed a LFD or HFD for 9 weeks. (A) IFNγ production by splenic T cells in WT mice. (B) Metabolic parameters. (C–F) Peritoneal macrophage MSRN protein levels (C), cholesterol levels following treatment with 2% serum from WTD-fed Ldlr −/− mice (D), cholesterol metabolism gene levels (E), and IFNγ -target gene levels (F). (G) p-STAT1/STAT1 levels. .
Figure Legend Snippet: Deleting Ifngr1 Corrects MSRN Proteins and Macrophage Cholesterol Levels in the “Obesity/IR Only” Model (A–G) WT and Ifngr1 −/− mice were fed a LFD or HFD for 9 weeks. (A) IFNγ production by splenic T cells in WT mice. (B) Metabolic parameters. (C–F) Peritoneal macrophage MSRN protein levels (C), cholesterol levels following treatment with 2% serum from WTD-fed Ldlr −/− mice (D), cholesterol metabolism gene levels (E), and IFNγ -target gene levels (F). (G) p-STAT1/STAT1 levels. .

Techniques Used: Mouse Assay

29) Product Images from "Interleukin-1β Signaling in Dendritic Cells Induces Antiviral Interferon Responses"

Article Title: Interleukin-1β Signaling in Dendritic Cells Induces Antiviral Interferon Responses

Journal: mBio

doi: 10.1128/mBio.00342-18

IL-1 signaling enhances antiviral responses. (A) WT or Il-1r −/− BMDCs were mock infected or infected with WNV at an MOI of 2.5. Expression of IFN-β and IFIT1 was measured by qRT-PCR at 24 and 48 h p.i. relative to that in matched, mock-treated controls. (B) Total cell WNV NS3, STAT1, and IFIT3 protein levels were measured by immunoblotting with GAPDH as a loading control (left). Densitometry analyses of STAT1 and IFIT3 protein abundance were compared against GAPDH abundance for each condition (right). The data are the averages of three independent experiments. Asterisks indicate values that are statistically significantly different between WT and Il - 1r −/− cells by unpaired t test (*, P
Figure Legend Snippet: IL-1 signaling enhances antiviral responses. (A) WT or Il-1r −/− BMDCs were mock infected or infected with WNV at an MOI of 2.5. Expression of IFN-β and IFIT1 was measured by qRT-PCR at 24 and 48 h p.i. relative to that in matched, mock-treated controls. (B) Total cell WNV NS3, STAT1, and IFIT3 protein levels were measured by immunoblotting with GAPDH as a loading control (left). Densitometry analyses of STAT1 and IFIT3 protein abundance were compared against GAPDH abundance for each condition (right). The data are the averages of three independent experiments. Asterisks indicate values that are statistically significantly different between WT and Il - 1r −/− cells by unpaired t test (*, P

Techniques Used: Infection, Expressing, Quantitative RT-PCR

30) Product Images from "Neuronal MHC Class I Expression Is Regulated by Activity Driven Calcium Signaling"

Article Title: Neuronal MHC Class I Expression Is Regulated by Activity Driven Calcium Signaling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0135223

PKC mediated activation of MAPK and AKT pathways by KA stimulation. (A) Western blot analysis of time-dependent activation of JAK1, STAT1, AKT, MAPK ERK1/2 and p38 after adding 100μM KA to 8 div hippocampal neurons for 30min. (B) Pretreatment of 8 div hippocampal neurons with PKC inhibitor (Staurosporine, 0.1μM) compromised the KA-induced activation of AKT, MAPK ERK1/2 and p38, but had no effect on the activation of JAK1 and STAT1. (C) Pretreatment of 8 div hippocampal neurons with PKA inhibitor (H89, 40μM) had no effect on the activation of JAK1, STAT1, AKT, MAPK ERK1/2 and p38 induced by KA. (D) Pretreatment of primary hippocampal neurons with MAPK-p38 inhibitor (Skepinone-L, 20μM) resulted in inhibition of KA-induced expression of MHC-I. (E) Pretreatment of primary hippocampal neurons with MAPK-ERK inhibitor (U0126, 10μM) blocked KA-induced expression of MHC-I. (F) Pretreatment of primary hippocampal neurons with MAPK-p38 inhibitor (Skepinone-L, 20μM) blocked KA-induced expression of p-p65, p-CREB and IRF-1. (G) Pretreatment of primary hippocampal neurons with MAPK-ERK inhibitor (U0126, 10μM) resulted in inhibition of KA-induced expression of IRF-1 and activation of NF-κB p65, but had no effect on the activation of CREB.
Figure Legend Snippet: PKC mediated activation of MAPK and AKT pathways by KA stimulation. (A) Western blot analysis of time-dependent activation of JAK1, STAT1, AKT, MAPK ERK1/2 and p38 after adding 100μM KA to 8 div hippocampal neurons for 30min. (B) Pretreatment of 8 div hippocampal neurons with PKC inhibitor (Staurosporine, 0.1μM) compromised the KA-induced activation of AKT, MAPK ERK1/2 and p38, but had no effect on the activation of JAK1 and STAT1. (C) Pretreatment of 8 div hippocampal neurons with PKA inhibitor (H89, 40μM) had no effect on the activation of JAK1, STAT1, AKT, MAPK ERK1/2 and p38 induced by KA. (D) Pretreatment of primary hippocampal neurons with MAPK-p38 inhibitor (Skepinone-L, 20μM) resulted in inhibition of KA-induced expression of MHC-I. (E) Pretreatment of primary hippocampal neurons with MAPK-ERK inhibitor (U0126, 10μM) blocked KA-induced expression of MHC-I. (F) Pretreatment of primary hippocampal neurons with MAPK-p38 inhibitor (Skepinone-L, 20μM) blocked KA-induced expression of p-p65, p-CREB and IRF-1. (G) Pretreatment of primary hippocampal neurons with MAPK-ERK inhibitor (U0126, 10μM) resulted in inhibition of KA-induced expression of IRF-1 and activation of NF-κB p65, but had no effect on the activation of CREB.

Techniques Used: Activation Assay, Western Blot, Inhibition, Expressing

Model depicting the calcium-dependent pathway mediated MHC-I expression induced by KA. Exposure of hippocampal neurons to KA leads to activation of calcium-dependent PKA and PKC, which results in the subsequent activation of the MAPK pathways and the downstream transcription factors NF-κB, CREB and IRF-1. Activation of these molecules finally leads to enhanced expression of MHC-I by binding to its promoter elements. JAK1/STAT1 and AKT pathways are also activated by KA stimulation.
Figure Legend Snippet: Model depicting the calcium-dependent pathway mediated MHC-I expression induced by KA. Exposure of hippocampal neurons to KA leads to activation of calcium-dependent PKA and PKC, which results in the subsequent activation of the MAPK pathways and the downstream transcription factors NF-κB, CREB and IRF-1. Activation of these molecules finally leads to enhanced expression of MHC-I by binding to its promoter elements. JAK1/STAT1 and AKT pathways are also activated by KA stimulation.

Techniques Used: Expressing, Activation Assay, Binding Assay

31) Product Images from "Novel High-throughput Approach for Purification of Infectious Virions"

Article Title: Novel High-throughput Approach for Purification of Infectious Virions

Journal: Scientific Reports

doi: 10.1038/srep36826

High-throughput reovirus purification using Capto Core 700 in-slurry approach. (A–D) 10 cm 2 wells of confluent reovirus-infected L929 cells were once-extracted with Vertrel XF, incubated with or without RNase/DNase then subjected to one round of in-slurry purification with 0, 50, 100, 200 or 400 μl of Capto Core 700 resin (50% slurry in virus dilution buffer) for 1 hour at room temperature. (B) Reovirus virion recovery and purity were assessed by SDS-PAGE and Coomassie blue staining. (C) L929 cells were exposed to purified reovirus samples from ( B ) at indicated dilutions. The infectivity of samples was then monitored at 18 hours post-infection by immunocytochemical staining with polyclonal anti-reovirus antibodies. (D) Plaque forming titers for samples in ( B ) were determined immediately post-purification or following 35 days of storage at 4 °C. (E–G) Four million reovirus-infected Ras-transformed NIH3T3 cells were once-extracted with Vertrel XF then subjected to 0–4 rounds of 45-minute end-over-end incubation with 50 μl of 50% Capto Core 700 resin slurry at room temperature. Results from two independent preparations are provided. (E) SDS-PAGE and Coomassie blue staining (left) or silver staining (right) of unpurified infected lysates (lysate), Vertrel-extracted samples (Vertrel), or samples exposed to 0–4 rounds Capto Core 700 resin (Capto700). (F) Infectivity was assessed by plaque titration. (G) Samples from ( E ) were subjected to western blot analysis. The same membrane was first immunoblotted with polyclonal rabbit anti-reovirus antibodies and Cy2-conjugated anti-rabbit secondary antibodies, then re-probed with mouse anti-β-actin antibodies and Cy5-conjugated anti-mouse secondary antibodies. A separate membrane was blotted with rabbit anti-STAT1 and Cy5-conjugated anti-rabbit secondary antibodies. Protein bands were visualized at respective wavelengths using an ImageQuant LAS 4010 imager (GE Healthcare).
Figure Legend Snippet: High-throughput reovirus purification using Capto Core 700 in-slurry approach. (A–D) 10 cm 2 wells of confluent reovirus-infected L929 cells were once-extracted with Vertrel XF, incubated with or without RNase/DNase then subjected to one round of in-slurry purification with 0, 50, 100, 200 or 400 μl of Capto Core 700 resin (50% slurry in virus dilution buffer) for 1 hour at room temperature. (B) Reovirus virion recovery and purity were assessed by SDS-PAGE and Coomassie blue staining. (C) L929 cells were exposed to purified reovirus samples from ( B ) at indicated dilutions. The infectivity of samples was then monitored at 18 hours post-infection by immunocytochemical staining with polyclonal anti-reovirus antibodies. (D) Plaque forming titers for samples in ( B ) were determined immediately post-purification or following 35 days of storage at 4 °C. (E–G) Four million reovirus-infected Ras-transformed NIH3T3 cells were once-extracted with Vertrel XF then subjected to 0–4 rounds of 45-minute end-over-end incubation with 50 μl of 50% Capto Core 700 resin slurry at room temperature. Results from two independent preparations are provided. (E) SDS-PAGE and Coomassie blue staining (left) or silver staining (right) of unpurified infected lysates (lysate), Vertrel-extracted samples (Vertrel), or samples exposed to 0–4 rounds Capto Core 700 resin (Capto700). (F) Infectivity was assessed by plaque titration. (G) Samples from ( E ) were subjected to western blot analysis. The same membrane was first immunoblotted with polyclonal rabbit anti-reovirus antibodies and Cy2-conjugated anti-rabbit secondary antibodies, then re-probed with mouse anti-β-actin antibodies and Cy5-conjugated anti-mouse secondary antibodies. A separate membrane was blotted with rabbit anti-STAT1 and Cy5-conjugated anti-rabbit secondary antibodies. Protein bands were visualized at respective wavelengths using an ImageQuant LAS 4010 imager (GE Healthcare).

Techniques Used: High Throughput Screening Assay, Purification, Infection, Incubation, SDS Page, Staining, Transformation Assay, Silver Staining, Titration, Western Blot

32) Product Images from "Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein"

Article Title: Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein

Journal: Proteomics

doi: 10.1002/pmic.201300001

Inhibitory effects of JEV NS5 protein on IFNβ‐induced responses. (A) To analyze ISRE promoter activity, vector control and NS5‐expressing cells were transiently cotransfected with reporter plasmid containing firefly luciferase under control of ISRE and internal control reporter pRluc‐C1. After 4‐h IFNβ treatment, firefly luciferase and renilla luciferase were measured; firefly luciferase activity normalized to renilla luciferase activity is reported. (B) To analyze ISRE‐driven gene expression, vector control and NS5‐expressing cells were treated with or without 1000 U/mL IFNβ for 8 h; relative levels of PKR and OAS mRNAs were gauged by quantitative real‐time PCR, relative fold levels of PKR or OAS mRNA presented as ratio of PKR or OAS mRNA/GAPDH mRNA. (C) To analyze STAT1 phosphorylation, Western blot of lysates from cells treated with IFNβ for 0, 30, 60, or 120 min was performed by anti‐phospho‐STAT1 (Tyr701) and anti‐β actin antibody as internal control.
Figure Legend Snippet: Inhibitory effects of JEV NS5 protein on IFNβ‐induced responses. (A) To analyze ISRE promoter activity, vector control and NS5‐expressing cells were transiently cotransfected with reporter plasmid containing firefly luciferase under control of ISRE and internal control reporter pRluc‐C1. After 4‐h IFNβ treatment, firefly luciferase and renilla luciferase were measured; firefly luciferase activity normalized to renilla luciferase activity is reported. (B) To analyze ISRE‐driven gene expression, vector control and NS5‐expressing cells were treated with or without 1000 U/mL IFNβ for 8 h; relative levels of PKR and OAS mRNAs were gauged by quantitative real‐time PCR, relative fold levels of PKR or OAS mRNA presented as ratio of PKR or OAS mRNA/GAPDH mRNA. (C) To analyze STAT1 phosphorylation, Western blot of lysates from cells treated with IFNβ for 0, 30, 60, or 120 min was performed by anti‐phospho‐STAT1 (Tyr701) and anti‐β actin antibody as internal control.

Techniques Used: Activity Assay, Plasmid Preparation, Expressing, Luciferase, Real-time Polymerase Chain Reaction, Western Blot

Functional analysis of calcineurin with siRNA‐mediated gene silencing. To analyze subcellular localization of STAT1, vector control (A) and NS5‐expresing cells (B) transfected with control or calreticulin siRNA were tested using immunfluorescent staining with anti‐STAT1 antibodies. Relative mRNA levels of PKR (C) and IL‐4 (D) were measured by quantitative PCR and normalized by GAPDH mRNA, presented as relative ratio.
Figure Legend Snippet: Functional analysis of calcineurin with siRNA‐mediated gene silencing. To analyze subcellular localization of STAT1, vector control (A) and NS5‐expresing cells (B) transfected with control or calreticulin siRNA were tested using immunfluorescent staining with anti‐STAT1 antibodies. Relative mRNA levels of PKR (C) and IL‐4 (D) were measured by quantitative PCR and normalized by GAPDH mRNA, presented as relative ratio.

Techniques Used: Functional Assay, Plasmid Preparation, Transfection, Staining, Real-time Polymerase Chain Reaction

Effect of CsA on IFNβ‐induced phosphorylation and nuclear translocation of STAT1 in NS5‐expressing cells. (A) To analyze tyrosine phosphoryl‐ated STAT1, vector control and NS5‐expressing cells were treated singly or with both IFNβ and cyclosporine A for 0, 30, 60, or 120 min. Lysates were subjected to Western blot, probed with anti‐phospho‐STAT1 (Tyr701). For subcellular location of STAT1, vector control (B) and NS5‐expressing (C) cells were treated singly or with both CsA and IFNβ for 24 h, then washed, fixed, and reacted with anti‐STAT1 and FITC‐conjugated anti‐mouse IgG antibodies. Finally, cells were stained with DAPI for 10 min, imaging analyzed by immunofluorescent microscopy.
Figure Legend Snippet: Effect of CsA on IFNβ‐induced phosphorylation and nuclear translocation of STAT1 in NS5‐expressing cells. (A) To analyze tyrosine phosphoryl‐ated STAT1, vector control and NS5‐expressing cells were treated singly or with both IFNβ and cyclosporine A for 0, 30, 60, or 120 min. Lysates were subjected to Western blot, probed with anti‐phospho‐STAT1 (Tyr701). For subcellular location of STAT1, vector control (B) and NS5‐expressing (C) cells were treated singly or with both CsA and IFNβ for 24 h, then washed, fixed, and reacted with anti‐STAT1 and FITC‐conjugated anti‐mouse IgG antibodies. Finally, cells were stained with DAPI for 10 min, imaging analyzed by immunofluorescent microscopy.

Techniques Used: Translocation Assay, Expressing, Plasmid Preparation, Western Blot, Staining, Imaging, Microscopy

Functional characterization of calreticulin with siRNA‐mediated gene silencing. (A) To detect intracellular Ca 2+ , cells transfected with control or calreticulin siRNA were harvested 4 h posttreatment with or without IFNβ, stained with FLUO3/AM and analyzed by flow cytometry. To analyze subcellular localization of STAT1 (B, C) and NFAT‐1 (D, E), vector control (B, D) and NS5‐expressing cells (C, E) transfected with control or calreticulin siRNA were tested by immunfluorescent staining with anti‐STAT1 or anti‐NFAT‐1 antibodies.
Figure Legend Snippet: Functional characterization of calreticulin with siRNA‐mediated gene silencing. (A) To detect intracellular Ca 2+ , cells transfected with control or calreticulin siRNA were harvested 4 h posttreatment with or without IFNβ, stained with FLUO3/AM and analyzed by flow cytometry. To analyze subcellular localization of STAT1 (B, C) and NFAT‐1 (D, E), vector control (B, D) and NS5‐expressing cells (C, E) transfected with control or calreticulin siRNA were tested by immunfluorescent staining with anti‐STAT1 or anti‐NFAT‐1 antibodies.

Techniques Used: Functional Assay, Transfection, Staining, Flow Cytometry, Plasmid Preparation, Expressing

33) Product Images from "Cucurbitacin I inhibits STAT3, but enhances STAT1 signaling in human cancer cells in vitro through disrupting actin filaments"

Article Title: Cucurbitacin I inhibits STAT3, but enhances STAT1 signaling in human cancer cells in vitro through disrupting actin filaments

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2017.99

Disrupting actin cytoskeleton inhibited STAT3 phosphorylation and p-STAT1 dephosphorylation. (A and B) A549 cells in suspension were treated with CytoD (A) or Jasp (B) at indicated concentrations for 2 h, followed by IL-6 (10 ng/mL) stimulation for 5 min. (C and D) Following IFN-γ (10 ng/mL) stimulation for 30 min, A549 cells were washed and incubated with fresh medium containing DMSO, CytoD (1 μmol/L) (C) or Jasp (0.5 μmol/L) (D) for indicated time periods.
Figure Legend Snippet: Disrupting actin cytoskeleton inhibited STAT3 phosphorylation and p-STAT1 dephosphorylation. (A and B) A549 cells in suspension were treated with CytoD (A) or Jasp (B) at indicated concentrations for 2 h, followed by IL-6 (10 ng/mL) stimulation for 5 min. (C and D) Following IFN-γ (10 ng/mL) stimulation for 30 min, A549 cells were washed and incubated with fresh medium containing DMSO, CytoD (1 μmol/L) (C) or Jasp (0.5 μmol/L) (D) for indicated time periods.

Techniques Used: De-Phosphorylation Assay, Incubation

STAT3 and STAT1 signaling proteins were physically associated with actin filaments. (A–C) A549 cells were lysed, and actin filaments were pulled-down using biotinylated-phalloidin, followed by elution with 10 mmol/L biotin. For drug treatments in B and C, A549 cells were treated with DMSO, 0.5 μmol/L CuI or 1 μmol/L CytoD for 2 h. input, 3% of the total cell lysates compared with 100% of the elution; bio, biotin; bio-pha, biotinylated-phalloidin. (D–H) Confocal micrographs of A549 cells staining with rhodamine-phalloidin (for actin, red), DAPI (for nucleus, blue), and antibodies against the indicated proteins (green). Arrowheads indicated colocalization of STATs signaling proteins with actin. For drug treatment, A549 cells were treated with DMSO, 0.5 μmol/L CuI or 1 μmol/L CytoD for 2 h prior to immunochemical staining. Scale bars, 10 μm.
Figure Legend Snippet: STAT3 and STAT1 signaling proteins were physically associated with actin filaments. (A–C) A549 cells were lysed, and actin filaments were pulled-down using biotinylated-phalloidin, followed by elution with 10 mmol/L biotin. For drug treatments in B and C, A549 cells were treated with DMSO, 0.5 μmol/L CuI or 1 μmol/L CytoD for 2 h. input, 3% of the total cell lysates compared with 100% of the elution; bio, biotin; bio-pha, biotinylated-phalloidin. (D–H) Confocal micrographs of A549 cells staining with rhodamine-phalloidin (for actin, red), DAPI (for nucleus, blue), and antibodies against the indicated proteins (green). Arrowheads indicated colocalization of STATs signaling proteins with actin. For drug treatment, A549 cells were treated with DMSO, 0.5 μmol/L CuI or 1 μmol/L CytoD for 2 h prior to immunochemical staining. Scale bars, 10 μm.

Techniques Used: Staining

Cucurbitacin I inhibited phosphorylation of STAT3 but dephosphorylation of p-STAT1. (A and B) A549 cells were treated with CuI at indicated concentrations for 2 h, followed by IL-6 (10 ng/mL, A) or IFN-γ (10 ng/mL, B) stimulation for 15 min. (C–E) Following IFN-γ (C and D) or IL-6 (E) stimulation for 30 min, A549 cells were washed with fresh medium for 3 times and then incubated with or without CuI at indicated concentrations (C) or for indicated time periods (D and E). Whole cell lysates were processed for Western blot analyses and probed with indicated antibodies.
Figure Legend Snippet: Cucurbitacin I inhibited phosphorylation of STAT3 but dephosphorylation of p-STAT1. (A and B) A549 cells were treated with CuI at indicated concentrations for 2 h, followed by IL-6 (10 ng/mL, A) or IFN-γ (10 ng/mL, B) stimulation for 15 min. (C–E) Following IFN-γ (C and D) or IL-6 (E) stimulation for 30 min, A549 cells were washed with fresh medium for 3 times and then incubated with or without CuI at indicated concentrations (C) or for indicated time periods (D and E). Whole cell lysates were processed for Western blot analyses and probed with indicated antibodies.

Techniques Used: De-Phosphorylation Assay, Incubation, Western Blot

Actin filaments regulated p-STAT1 dephosphorylation through modulating p-STAT1 proteins, rather than the activity of protein tyrosine phosphatases (PTPs). (A and B) A549 cells were treated with IFN-γ (10 ng/mL) for 30 min, and then were lysed and incubated in vitro at 36 °C in the presence or absence of 1% Triton X-100 (A) or DNase I (B) for indicated time periods. Following incubation, the lysates were mixed with laemmli 2×sample buffer and processed for Western blot analyses with indicated antibodies. (C) Two dishes of A549 cells were stimulated with or without IFN-γ (10 ng/mL) respectively for 30 min and lysed. The IFN-γ-stimulated lysate was divided into two parts, one of which was heat-denatured while the other undenatured, and then were mixed with unstimulated cell lysates respectively and incubated at 36 °C for indicated time periods.
Figure Legend Snippet: Actin filaments regulated p-STAT1 dephosphorylation through modulating p-STAT1 proteins, rather than the activity of protein tyrosine phosphatases (PTPs). (A and B) A549 cells were treated with IFN-γ (10 ng/mL) for 30 min, and then were lysed and incubated in vitro at 36 °C in the presence or absence of 1% Triton X-100 (A) or DNase I (B) for indicated time periods. Following incubation, the lysates were mixed with laemmli 2×sample buffer and processed for Western blot analyses with indicated antibodies. (C) Two dishes of A549 cells were stimulated with or without IFN-γ (10 ng/mL) respectively for 30 min and lysed. The IFN-γ-stimulated lysate was divided into two parts, one of which was heat-denatured while the other undenatured, and then were mixed with unstimulated cell lysates respectively and incubated at 36 °C for indicated time periods.

Techniques Used: De-Phosphorylation Assay, Activity Assay, Incubation, In Vitro, Western Blot

Cucurbitacin I did not inhibit the STAT kinases or phosphatases directly. (A) A549 cells in suspension were stimulated with IL-6 (10 ng/mL), IFN-γ (10 ng/mL) or IFN-α (1000 IU/mL) for 5 min. The p-JAK2 and p-TYK2 antibodies non-specifically recognized other phosphorylated JAK family members as indicated by arrows. (B and C) A549 cells in adhesion (B) or suspension (C) were treated with CuI at various concentrations for 2 h, followed by stimulation with or without 10 ng/mL IL-6 for 5 min. (D and E) JAK2, and SHP2 or TCPTP proteins immunoprecipitated from A549 cells was subjected to in vitro kinase (D) or phosphatase (E) assays respectively in the presence of indicated concentrations of CuI. 2-MS, a JAK2 inhibitor identified by our lab, and vanadate, a protein tyrosine phosphatase inhibitor, were served as positive controls. (F) Effect of Cul or vanadate on p-STAT1 dephosphorylation in A549 cells. Data are represented as mean±SD of three experiments. CuI, 2-MS and vanadate groups were compared with vehicle group by Student's t -test. ns, not significant; * P
Figure Legend Snippet: Cucurbitacin I did not inhibit the STAT kinases or phosphatases directly. (A) A549 cells in suspension were stimulated with IL-6 (10 ng/mL), IFN-γ (10 ng/mL) or IFN-α (1000 IU/mL) for 5 min. The p-JAK2 and p-TYK2 antibodies non-specifically recognized other phosphorylated JAK family members as indicated by arrows. (B and C) A549 cells in adhesion (B) or suspension (C) were treated with CuI at various concentrations for 2 h, followed by stimulation with or without 10 ng/mL IL-6 for 5 min. (D and E) JAK2, and SHP2 or TCPTP proteins immunoprecipitated from A549 cells was subjected to in vitro kinase (D) or phosphatase (E) assays respectively in the presence of indicated concentrations of CuI. 2-MS, a JAK2 inhibitor identified by our lab, and vanadate, a protein tyrosine phosphatase inhibitor, were served as positive controls. (F) Effect of Cul or vanadate on p-STAT1 dephosphorylation in A549 cells. Data are represented as mean±SD of three experiments. CuI, 2-MS and vanadate groups were compared with vehicle group by Student's t -test. ns, not significant; * P

Techniques Used: Immunoprecipitation, In Vitro, Mass Spectrometry, De-Phosphorylation Assay

Cucurbitacin I inhibited STAT3 phosphorylation but enhanced STAT1 phosphorylation. Lung adenocarcinoma cell line A549 (A), cervical adenocarcinoma cell line Hela (B), neuroglioma cell line H4 (C), prostate cancer cell line PC3 (D) and mammary adenocarcinoma MDA-MB-468 (E) were treated with CuI for 2 h, and whole cell lysates were processed for Western blot analyses with indicated antibodies. The levels of pY705-STAT3 and pY701-STAT1 in CuI treatments were normalized by α-tubulin and quantified to DMSO treatment, and were graphed below the corresponding blots.
Figure Legend Snippet: Cucurbitacin I inhibited STAT3 phosphorylation but enhanced STAT1 phosphorylation. Lung adenocarcinoma cell line A549 (A), cervical adenocarcinoma cell line Hela (B), neuroglioma cell line H4 (C), prostate cancer cell line PC3 (D) and mammary adenocarcinoma MDA-MB-468 (E) were treated with CuI for 2 h, and whole cell lysates were processed for Western blot analyses with indicated antibodies. The levels of pY705-STAT3 and pY701-STAT1 in CuI treatments were normalized by α-tubulin and quantified to DMSO treatment, and were graphed below the corresponding blots.

Techniques Used: Multiple Displacement Amplification, Western Blot

34) Product Images from "E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts"

Article Title: E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-018-1713-x

STAT1/MyD88 complexes mediate E2F2 regulation of inflammatory cytokines. a – d STAT1/MyD88 complex is found in RASFs and MEFs. E2F2-silenced RASFs and E2f2 −/− MEFs were cultured with or without lipopolysaccharide (LPS; 10 μg/mL). Co-IP ( a , b ) was performed to test the binding. Confocal immunofluorescence ( c , d ) was performed to confirm the result (magnification 10 × 40, MyD88 (green) and STAT1 (red)). e – g Effect of STAT1/MyD88 complexes on the expression of cytokines. qRT-PCR was performed to detect expression of interleukin (IL)-1α ( e ), IL-1β ( f ), and tumor necrosis factor (TNF)-α ( g ) in STAT1/MyD88 knockdown RASFs with or without E2F2 overexpression in the presence of LPS (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. ** P
Figure Legend Snippet: STAT1/MyD88 complexes mediate E2F2 regulation of inflammatory cytokines. a – d STAT1/MyD88 complex is found in RASFs and MEFs. E2F2-silenced RASFs and E2f2 −/− MEFs were cultured with or without lipopolysaccharide (LPS; 10 μg/mL). Co-IP ( a , b ) was performed to test the binding. Confocal immunofluorescence ( c , d ) was performed to confirm the result (magnification 10 × 40, MyD88 (green) and STAT1 (red)). e – g Effect of STAT1/MyD88 complexes on the expression of cytokines. qRT-PCR was performed to detect expression of interleukin (IL)-1α ( e ), IL-1β ( f ), and tumor necrosis factor (TNF)-α ( g ) in STAT1/MyD88 knockdown RASFs with or without E2F2 overexpression in the presence of LPS (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. ** P

Techniques Used: Cell Culture, Co-Immunoprecipitation Assay, Binding Assay, Immunofluorescence, Expressing, Quantitative RT-PCR, Over Expression

STAT1 mediates E2F2 regulation of interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α expression. a – d Effect of E2F2 on STAT1. E2F2 was overexpressed by adenovirus infection or inhibited by small interfering RNA (siRNA) with or without lipopolysaccharide (LPS; 10 μg/mL). qRT-PCR ( a , b ) and Western blot ( c , d ) were performed to detect expression of STAT1. e Schematic representation of STAT1 promoters, primers for the ChIP assay, and the E2F2 binding motif in the STAT1 promoter. ChIP ( f ) and luciferase (Luc) reporter assays ( g ) were performed to show that E2F2 was recruited to the STAT1 gene promoter in RASFs in the presence of LPS. Nuclear and cytoplasmic proteins were fractionally extracted from E2F2 knocked-down RASFs ( h ) and E2f2 −/− MEFs ( i ). Effects of E2F2 on nuclear translocation of STAT1 were determined by Western blot. (Lamin A/C as a reference for nuclear extraction (N); Tubulin as a reference for cytoplasmic extraction (C).) Effect of E2F2 on nuclear translocation of STAT1 was observed using confocal fluorescence microscopy both in E2F2-silenced RASFs ( j ) and E2f2 −/− MEFs ( k ). STAT1 (green) was detected using anti-STAT1 antibody. Nuclei were stained with DAPI (blue). l In E2F2-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing STAT1 in the presence of LPS stimulation (10 μg/mL). m In STAT1-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing E2F2 in the presence of LPS stimulation (10 μg/mL). n In E2f2 −/− MEFs, expression of IL-1α, IL-1β, and TNF-α was detected using qRT-PCR after STAT1 overexpression in the presence of LPS stimulation (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. * P
Figure Legend Snippet: STAT1 mediates E2F2 regulation of interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α expression. a – d Effect of E2F2 on STAT1. E2F2 was overexpressed by adenovirus infection or inhibited by small interfering RNA (siRNA) with or without lipopolysaccharide (LPS; 10 μg/mL). qRT-PCR ( a , b ) and Western blot ( c , d ) were performed to detect expression of STAT1. e Schematic representation of STAT1 promoters, primers for the ChIP assay, and the E2F2 binding motif in the STAT1 promoter. ChIP ( f ) and luciferase (Luc) reporter assays ( g ) were performed to show that E2F2 was recruited to the STAT1 gene promoter in RASFs in the presence of LPS. Nuclear and cytoplasmic proteins were fractionally extracted from E2F2 knocked-down RASFs ( h ) and E2f2 −/− MEFs ( i ). Effects of E2F2 on nuclear translocation of STAT1 were determined by Western blot. (Lamin A/C as a reference for nuclear extraction (N); Tubulin as a reference for cytoplasmic extraction (C).) Effect of E2F2 on nuclear translocation of STAT1 was observed using confocal fluorescence microscopy both in E2F2-silenced RASFs ( j ) and E2f2 −/− MEFs ( k ). STAT1 (green) was detected using anti-STAT1 antibody. Nuclei were stained with DAPI (blue). l In E2F2-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing STAT1 in the presence of LPS stimulation (10 μg/mL). m In STAT1-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing E2F2 in the presence of LPS stimulation (10 μg/mL). n In E2f2 −/− MEFs, expression of IL-1α, IL-1β, and TNF-α was detected using qRT-PCR after STAT1 overexpression in the presence of LPS stimulation (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. * P

Techniques Used: Expressing, Infection, Small Interfering RNA, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Luciferase, Translocation Assay, Fluorescence, Microscopy, Staining, Over Expression

E2F2 participates in RA inflammation through STAT1 and PI3K/AKT/NF-κB pathways. RNA-seq was performed to screen target genes downstream of E2F2 in RASFs. a , b Heat maps indicate the most differentially expressed genes in RASFs with E2F2 knocked-down. Colored bands represent the change in gene expression: red, downregulation; blue, upregulation. c – e In-vitro verification of genes related to inflammation in RA was performed using qRT-PCR. mRNA levels of STAT1 ( c ), AKT1 ( d ), and AKT2 ( e ). f Western blot was performed to test inhibitory effects of siE2F2 on expression of E2F2, STAT1, AKT1, AKT2, p-AKT, and the p65 subunit of NF-κB. All results are presented as the mean ± SEM of three independent experiments performed in triplicate. NC knockdown scramble control, si small interfering
Figure Legend Snippet: E2F2 participates in RA inflammation through STAT1 and PI3K/AKT/NF-κB pathways. RNA-seq was performed to screen target genes downstream of E2F2 in RASFs. a , b Heat maps indicate the most differentially expressed genes in RASFs with E2F2 knocked-down. Colored bands represent the change in gene expression: red, downregulation; blue, upregulation. c – e In-vitro verification of genes related to inflammation in RA was performed using qRT-PCR. mRNA levels of STAT1 ( c ), AKT1 ( d ), and AKT2 ( e ). f Western blot was performed to test inhibitory effects of siE2F2 on expression of E2F2, STAT1, AKT1, AKT2, p-AKT, and the p65 subunit of NF-κB. All results are presented as the mean ± SEM of three independent experiments performed in triplicate. NC knockdown scramble control, si small interfering

Techniques Used: RNA Sequencing Assay, Expressing, In Vitro, Quantitative RT-PCR, Western Blot

35) Product Images from "Protective Immunity against Pulmonary Cryptococcosis Is Associated with STAT1-Mediated Classical Macrophage Activation"

Article Title: Protective Immunity against Pulmonary Cryptococcosis Is Associated with STAT1-Mediated Classical Macrophage Activation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1103455

STAT1 signaling cascade is upregulated in macrophages from infected H99γ-immunized mice. BALB/c mice were intranasally immunized with 1 × 10 4 CFU C. neoformans strain H99γ or HK Cn in 50 μl sterile PBS. Thirty-five days
Figure Legend Snippet: STAT1 signaling cascade is upregulated in macrophages from infected H99γ-immunized mice. BALB/c mice were intranasally immunized with 1 × 10 4 CFU C. neoformans strain H99γ or HK Cn in 50 μl sterile PBS. Thirty-five days

Techniques Used: Infection, Mouse Assay

STAT1 phosphorylation is increased in macrophages from H99γ-immunized mice during cryptococcal rechallenge. BALB/c mice were intranasally immunized with 1 × 10 4 CFU C. neoformans strain H99γ or HK Cn in 50 μl sterile PBS.
Figure Legend Snippet: STAT1 phosphorylation is increased in macrophages from H99γ-immunized mice during cryptococcal rechallenge. BALB/c mice were intranasally immunized with 1 × 10 4 CFU C. neoformans strain H99γ or HK Cn in 50 μl sterile PBS.

Techniques Used: Mouse Assay

36) Product Images from "Cucurbitacin I inhibits STAT3, but enhances STAT1 signaling in human cancer cells in vitro through disrupting actin filaments"

Article Title: Cucurbitacin I inhibits STAT3, but enhances STAT1 signaling in human cancer cells in vitro through disrupting actin filaments

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2017.99

Disrupting actin cytoskeleton inhibited STAT3 phosphorylation and p-STAT1 dephosphorylation. (A and B) A549 cells in suspension were treated with CytoD (A) or Jasp (B) at indicated concentrations for 2 h, followed by IL-6 (10 ng/mL) stimulation for 5 min. (C and D) Following IFN-γ (10 ng/mL) stimulation for 30 min, A549 cells were washed and incubated with fresh medium containing DMSO, CytoD (1 μmol/L) (C) or Jasp (0.5 μmol/L) (D) for indicated time periods.
Figure Legend Snippet: Disrupting actin cytoskeleton inhibited STAT3 phosphorylation and p-STAT1 dephosphorylation. (A and B) A549 cells in suspension were treated with CytoD (A) or Jasp (B) at indicated concentrations for 2 h, followed by IL-6 (10 ng/mL) stimulation for 5 min. (C and D) Following IFN-γ (10 ng/mL) stimulation for 30 min, A549 cells were washed and incubated with fresh medium containing DMSO, CytoD (1 μmol/L) (C) or Jasp (0.5 μmol/L) (D) for indicated time periods.

Techniques Used: De-Phosphorylation Assay, Incubation

STAT3 and STAT1 signaling proteins were physically associated with actin filaments. (A–C) A549 cells were lysed, and actin filaments were pulled-down using biotinylated-phalloidin, followed by elution with 10 mmol/L biotin. For drug treatments in B and C, A549 cells were treated with DMSO, 0.5 μmol/L CuI or 1 μmol/L CytoD for 2 h. input, 3% of the total cell lysates compared with 100% of the elution; bio, biotin; bio-pha, biotinylated-phalloidin. (D–H) Confocal micrographs of A549 cells staining with rhodamine-phalloidin (for actin, red), DAPI (for nucleus, blue), and antibodies against the indicated proteins (green). Arrowheads indicated colocalization of STATs signaling proteins with actin. For drug treatment, A549 cells were treated with DMSO, 0.5 μmol/L CuI or 1 μmol/L CytoD for 2 h prior to immunochemical staining. Scale bars, 10 μm.
Figure Legend Snippet: STAT3 and STAT1 signaling proteins were physically associated with actin filaments. (A–C) A549 cells were lysed, and actin filaments were pulled-down using biotinylated-phalloidin, followed by elution with 10 mmol/L biotin. For drug treatments in B and C, A549 cells were treated with DMSO, 0.5 μmol/L CuI or 1 μmol/L CytoD for 2 h. input, 3% of the total cell lysates compared with 100% of the elution; bio, biotin; bio-pha, biotinylated-phalloidin. (D–H) Confocal micrographs of A549 cells staining with rhodamine-phalloidin (for actin, red), DAPI (for nucleus, blue), and antibodies against the indicated proteins (green). Arrowheads indicated colocalization of STATs signaling proteins with actin. For drug treatment, A549 cells were treated with DMSO, 0.5 μmol/L CuI or 1 μmol/L CytoD for 2 h prior to immunochemical staining. Scale bars, 10 μm.

Techniques Used: Staining

Cucurbitacin I inhibited phosphorylation of STAT3 but dephosphorylation of p-STAT1. (A and B) A549 cells were treated with CuI at indicated concentrations for 2 h, followed by IL-6 (10 ng/mL, A) or IFN-γ (10 ng/mL, B) stimulation for 15 min. (C–E) Following IFN-γ (C and D) or IL-6 (E) stimulation for 30 min, A549 cells were washed with fresh medium for 3 times and then incubated with or without CuI at indicated concentrations (C) or for indicated time periods (D and E). Whole cell lysates were processed for Western blot analyses and probed with indicated antibodies.
Figure Legend Snippet: Cucurbitacin I inhibited phosphorylation of STAT3 but dephosphorylation of p-STAT1. (A and B) A549 cells were treated with CuI at indicated concentrations for 2 h, followed by IL-6 (10 ng/mL, A) or IFN-γ (10 ng/mL, B) stimulation for 15 min. (C–E) Following IFN-γ (C and D) or IL-6 (E) stimulation for 30 min, A549 cells were washed with fresh medium for 3 times and then incubated with or without CuI at indicated concentrations (C) or for indicated time periods (D and E). Whole cell lysates were processed for Western blot analyses and probed with indicated antibodies.

Techniques Used: De-Phosphorylation Assay, Incubation, Western Blot

Actin filaments regulated p-STAT1 dephosphorylation through modulating p-STAT1 proteins, rather than the activity of protein tyrosine phosphatases (PTPs). (A and B) A549 cells were treated with IFN-γ (10 ng/mL) for 30 min, and then were lysed and incubated in vitro at 36 °C in the presence or absence of 1% Triton X-100 (A) or DNase I (B) for indicated time periods. Following incubation, the lysates were mixed with laemmli 2×sample buffer and processed for Western blot analyses with indicated antibodies. (C) Two dishes of A549 cells were stimulated with or without IFN-γ (10 ng/mL) respectively for 30 min and lysed. The IFN-γ-stimulated lysate was divided into two parts, one of which was heat-denatured while the other undenatured, and then were mixed with unstimulated cell lysates respectively and incubated at 36 °C for indicated time periods.
Figure Legend Snippet: Actin filaments regulated p-STAT1 dephosphorylation through modulating p-STAT1 proteins, rather than the activity of protein tyrosine phosphatases (PTPs). (A and B) A549 cells were treated with IFN-γ (10 ng/mL) for 30 min, and then were lysed and incubated in vitro at 36 °C in the presence or absence of 1% Triton X-100 (A) or DNase I (B) for indicated time periods. Following incubation, the lysates were mixed with laemmli 2×sample buffer and processed for Western blot analyses with indicated antibodies. (C) Two dishes of A549 cells were stimulated with or without IFN-γ (10 ng/mL) respectively for 30 min and lysed. The IFN-γ-stimulated lysate was divided into two parts, one of which was heat-denatured while the other undenatured, and then were mixed with unstimulated cell lysates respectively and incubated at 36 °C for indicated time periods.

Techniques Used: De-Phosphorylation Assay, Activity Assay, Incubation, In Vitro, Western Blot

Cucurbitacin I did not inhibit the STAT kinases or phosphatases directly. (A) A549 cells in suspension were stimulated with IL-6 (10 ng/mL), IFN-γ (10 ng/mL) or IFN-α (1000 IU/mL) for 5 min. The p-JAK2 and p-TYK2 antibodies non-specifically recognized other phosphorylated JAK family members as indicated by arrows. (B and C) A549 cells in adhesion (B) or suspension (C) were treated with CuI at various concentrations for 2 h, followed by stimulation with or without 10 ng/mL IL-6 for 5 min. (D and E) JAK2, and SHP2 or TCPTP proteins immunoprecipitated from A549 cells was subjected to in vitro kinase (D) or phosphatase (E) assays respectively in the presence of indicated concentrations of CuI. 2-MS, a JAK2 inhibitor identified by our lab, and vanadate, a protein tyrosine phosphatase inhibitor, were served as positive controls. (F) Effect of Cul or vanadate on p-STAT1 dephosphorylation in A549 cells. Data are represented as mean±SD of three experiments. CuI, 2-MS and vanadate groups were compared with vehicle group by Student's t -test. ns, not significant; * P
Figure Legend Snippet: Cucurbitacin I did not inhibit the STAT kinases or phosphatases directly. (A) A549 cells in suspension were stimulated with IL-6 (10 ng/mL), IFN-γ (10 ng/mL) or IFN-α (1000 IU/mL) for 5 min. The p-JAK2 and p-TYK2 antibodies non-specifically recognized other phosphorylated JAK family members as indicated by arrows. (B and C) A549 cells in adhesion (B) or suspension (C) were treated with CuI at various concentrations for 2 h, followed by stimulation with or without 10 ng/mL IL-6 for 5 min. (D and E) JAK2, and SHP2 or TCPTP proteins immunoprecipitated from A549 cells was subjected to in vitro kinase (D) or phosphatase (E) assays respectively in the presence of indicated concentrations of CuI. 2-MS, a JAK2 inhibitor identified by our lab, and vanadate, a protein tyrosine phosphatase inhibitor, were served as positive controls. (F) Effect of Cul or vanadate on p-STAT1 dephosphorylation in A549 cells. Data are represented as mean±SD of three experiments. CuI, 2-MS and vanadate groups were compared with vehicle group by Student's t -test. ns, not significant; * P

Techniques Used: Immunoprecipitation, In Vitro, Mass Spectrometry, De-Phosphorylation Assay

Cucurbitacin I inhibited STAT3 phosphorylation but enhanced STAT1 phosphorylation. Lung adenocarcinoma cell line A549 (A), cervical adenocarcinoma cell line Hela (B), neuroglioma cell line H4 (C), prostate cancer cell line PC3 (D) and mammary adenocarcinoma MDA-MB-468 (E) were treated with CuI for 2 h, and whole cell lysates were processed for Western blot analyses with indicated antibodies. The levels of pY705-STAT3 and pY701-STAT1 in CuI treatments were normalized by α-tubulin and quantified to DMSO treatment, and were graphed below the corresponding blots.
Figure Legend Snippet: Cucurbitacin I inhibited STAT3 phosphorylation but enhanced STAT1 phosphorylation. Lung adenocarcinoma cell line A549 (A), cervical adenocarcinoma cell line Hela (B), neuroglioma cell line H4 (C), prostate cancer cell line PC3 (D) and mammary adenocarcinoma MDA-MB-468 (E) were treated with CuI for 2 h, and whole cell lysates were processed for Western blot analyses with indicated antibodies. The levels of pY705-STAT3 and pY701-STAT1 in CuI treatments were normalized by α-tubulin and quantified to DMSO treatment, and were graphed below the corresponding blots.

Techniques Used: Multiple Displacement Amplification, Western Blot

37) Product Images from "The USP18 cysteine protease promotes HBV production independent of its protease activity"

Article Title: The USP18 cysteine protease promotes HBV production independent of its protease activity

Journal: Virology Journal

doi: 10.1186/s12985-020-01304-2

USP18 upregulation inhibited IFN-induced Jak/STAT signaling pathway. HepAD38 cells and HepG2 cells were seeded in 6-well plate without any treatment, respectively. Forty-eight hours later, supernatant IFNα ( a, left ) and IFNβ ( a, right ) and intracellular mRNA expression of ISGs ( b ) were analyzed by ELISA assay and real-time PCR, respectively. To investigate the effects of USP18 on STAT phosphorylation, HepAD38 cells were transfected with WT-USP18, USP18-C64S or MOCK for 48 h and treated with 500 IU/ml IFNα for 30 min before harvested. Western blot was used to analyze the expression of STAT1 and p-STAT1 ( c, left ). The interferon stimulated response element (ISRE) activity was quantified by dual luciferase reporter gene assay. Briefly, HepAD38 cells were co-transfected with WT-USP18, USP18-C64S or MOCK and ISRE-luc reporter plasmid /pRL-TK reporter plasmid for 24 h, and then left untreated or treated with 100 IU/ml or 1000 IU/ml IFNα for 24 h before the cells were lysed ( c, middle ). HepAD38 cells were transfected with WT-USP18, USP18-C64S or MOCK for 48 h and treated with 500 IU/ml IFNα for additional 24 h. Expression of ISGs mRNA including MxA and OAS2 were detected by real-time PCR ( c, right ). WT-USP18, wide type USP18; MOCK, empty plasmid. Results are presented as means ± SD (n ≥ 3). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
Figure Legend Snippet: USP18 upregulation inhibited IFN-induced Jak/STAT signaling pathway. HepAD38 cells and HepG2 cells were seeded in 6-well plate without any treatment, respectively. Forty-eight hours later, supernatant IFNα ( a, left ) and IFNβ ( a, right ) and intracellular mRNA expression of ISGs ( b ) were analyzed by ELISA assay and real-time PCR, respectively. To investigate the effects of USP18 on STAT phosphorylation, HepAD38 cells were transfected with WT-USP18, USP18-C64S or MOCK for 48 h and treated with 500 IU/ml IFNα for 30 min before harvested. Western blot was used to analyze the expression of STAT1 and p-STAT1 ( c, left ). The interferon stimulated response element (ISRE) activity was quantified by dual luciferase reporter gene assay. Briefly, HepAD38 cells were co-transfected with WT-USP18, USP18-C64S or MOCK and ISRE-luc reporter plasmid /pRL-TK reporter plasmid for 24 h, and then left untreated or treated with 100 IU/ml or 1000 IU/ml IFNα for 24 h before the cells were lysed ( c, middle ). HepAD38 cells were transfected with WT-USP18, USP18-C64S or MOCK for 48 h and treated with 500 IU/ml IFNα for additional 24 h. Expression of ISGs mRNA including MxA and OAS2 were detected by real-time PCR ( c, right ). WT-USP18, wide type USP18; MOCK, empty plasmid. Results are presented as means ± SD (n ≥ 3). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Activity Assay, Luciferase, Reporter Gene Assay, Plasmid Preparation

38) Product Images from "Intrinsically photosensitive retinal ganglion cells are resistant to N-methyl-D-aspartic acid excitotoxicity"

Article Title: Intrinsically photosensitive retinal ganglion cells are resistant to N-methyl-D-aspartic acid excitotoxicity

Journal: Molecular Vision

doi:

Intravitreal N-methyl-D-aspartic acid injection activates endogenous rescue and stress pathways. A : Shown is the relative expression of Lif, Clc, Edn2, Fgf2, Stat1, Stat3, Gfap, Casp1, and Mcp-1 in retinas of 129S6 wild type mice at 6 h, 24 h, 48 h, and 6 days after injection of N-methyl-D-aspartic acid (NMDA, black bars) or phosphate buffered saline (PBS, grey bars). Expression after NMDA injection was expressed relative to expression after PBS injection, which was set to 1 for each time point. Shown are means±SD of n=4-6. *: p
Figure Legend Snippet: Intravitreal N-methyl-D-aspartic acid injection activates endogenous rescue and stress pathways. A : Shown is the relative expression of Lif, Clc, Edn2, Fgf2, Stat1, Stat3, Gfap, Casp1, and Mcp-1 in retinas of 129S6 wild type mice at 6 h, 24 h, 48 h, and 6 days after injection of N-methyl-D-aspartic acid (NMDA, black bars) or phosphate buffered saline (PBS, grey bars). Expression after NMDA injection was expressed relative to expression after PBS injection, which was set to 1 for each time point. Shown are means±SD of n=4-6. *: p

Techniques Used: Injection, Expressing, Mouse Assay

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Immunoprecipitation:

Article Title: Nitroalkenes Suppress Lipopolysaccharide-Induced Signal Transducer and Activator of Transcription Signaling in Macrophages: A Critical Role of Mitogen-Activated Protein Kinase Phosphatase 1
Article Snippet: .. The cell lysates were subjected to immunoprecipitation and immunoblot as previously described ( ) using antibodies of phospho-STAT1 (pSTAT1) (Tyr-701), pSTAT3 (Tyr-705), STAT1, and STAT3 (Cell Signaling Technology, Inc., Danvers, MA), MKP-1 and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and iNOS, (BD Biosciences, San Jose, CA). .. For quantitation of the changes in STAT1 phosphorylation, the intensities of the bands representing STAT1 and pSTAT1 (Tyr-701) were measured by densitometry using an image scanner (EPSON GT-8000) and National Institutes of Health Image software.

other:

Article Title: MiR-155 Induction by Microbes/Microbial Ligands Requires NF-?B-Dependent de novo Protein Synthesis
Article Snippet: Antibodies Antibodies against phospho-Stat1, c-Fos, and c-Jun were purchased from Cell Signaling (Beverly, Massachusetts).

Article Title: Characterization and allergic role of IL-33-induced neutrophil polarization
Article Snippet: The primary antibodies against p-JNK (Thr183/Tyr185), JNK, p-p38 (Thr180/Tyr182), p38, p-STAT1 (Ser727), STAT1, p-ERK (Thr202/Tyr204), ERK, p-p65 (Ser536), p65 and H3 were purchased from Cell Signaling Technology.

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    Cell Signaling Technology Inc anti stat1
    DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 <t>(STAT1),</t> and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P
    Anti Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Activation <t>STAT1</t> and STAT3 by IFN-γ in MCs. MCs were treated with 0–20 ng/ml IFN-γ for 8 hours. After treatment, STAT1 ((a) N =3 replicates per condition from three independent experiments) and STAT3 phosphorylation ((b) N =3 replicates per condition from three independent experiments) were evaluated by western blot analysis. These phosphorylation levels were normalised based on β-actin levels. Whether IFN-γ induces translocalisation of STAT1 and STAT3 into nucleus was evaluated. MCs were treated with 5 ng/mL IFN-γ for 8 hours. Thereafter, localisations of STAT1 and STAT3 were determined by immunofluorescence staining ((c) and (d)). Data are expressed as mean ± SE. Asterisk indicates a significant difference compared with the control group at each time point ( P
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    Species-specific activity of rec Mg IFN-γ and mouse IFN-γ on different rodent cell lines. NIH/3T3, BVK168, FMN-R and AAL-R cells were tested and either left untreated as control (lower panel), stimulated for 1 h with rec Mg IFN-γ (200 ng/ml, upper panel) or mouse IFN-γ (200 U/ml, central panel). In each panel the following stainings are shown from left to right: <t>phospho-Tyr701-STAT1</t> staining (green), DAPI staining (white) and overlay of the two signals. Activated phospho-STAT1 is indicated by nuclear translocation. Of note, minor background signal in the cytoplasma was observed in some control treated cells. A representative experiment of three replicates is shown. The scale bar represents 50 μm.
    Rabbit Anti Phospho Stat1 Tyr701 Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti phospho stat1 ser 727
    Suppression of type I IFN signaling pathways is not associated with early temperature effects. (A, C, and D) Immunoblot assay of <t>STAT1</t> phosphorylated at Tyr-701 (A and D) or <t>Ser-727</t> (C) from Vero cells treated with 1,000 IU/ml IFN-α/β at 30, 37, or 39°C and corresponding densitometry quantification with STAT1-p bands normalized to β-actin. Data are presented as mean STAT1-p/actin ratio ± SD. (B) Vero cells treated with 1,000 IU/ml IFN-α/β for 30 min at 30, 37, or 39°C were subjected to immunocytochemistry staining for STAT1-p (Y701). Confocal imaging shows phosphorylated STAT1 nuclear translocation. Graph displays average nuclear STAT1-p signal intensity per imaged nuclear area at each temperature. At least 199 cells/nucleus were analyzed in each temperature group. Statistics for panel A: **, P
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    DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 (STAT1), and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Dengue Virus Infects Primary Human Hair Follicle Dermal Papilla Cells

    doi: 10.3389/fcimb.2018.00268

    Figure Lengend Snippet: DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 (STAT1), and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P

    Article Snippet: The following primary antibodies were used: anti-caspase 1 (GTX111630, GeneTex, Irvine, CA), anti-caspase 3 (#9665, Cell Signaling Technology, Danvers, MA), anti-caspase 7 (GTX1002337, GeneTex), anti-caspase 8 (#4790, Cell Signaling Technology), anti-bone morphogenetic protein 4 (BMP-4; GTX100875, GeneTex), anti-phospho-STAT1 (phospho-Tyr701, #9167 Cell signaling), anti-STAT1 (#14994, Cell Signaling), anti-phospho-STAT2 (phospho-Tyr690, GTX50721, GeneTex), anti-STAT2 (#14994, Cell Signaling), anti-DENV NS3 (GTX124252, GeneTex), and anti-GAPDH (#60004-1-Ig, Proteintech Group, Rosemont, IL).

    Techniques: Expressing, Quantitative RT-PCR, Infection

    Activation STAT1 and STAT3 by IFN-γ in MCs. MCs were treated with 0–20 ng/ml IFN-γ for 8 hours. After treatment, STAT1 ((a) N =3 replicates per condition from three independent experiments) and STAT3 phosphorylation ((b) N =3 replicates per condition from three independent experiments) were evaluated by western blot analysis. These phosphorylation levels were normalised based on β-actin levels. Whether IFN-γ induces translocalisation of STAT1 and STAT3 into nucleus was evaluated. MCs were treated with 5 ng/mL IFN-γ for 8 hours. Thereafter, localisations of STAT1 and STAT3 were determined by immunofluorescence staining ((c) and (d)). Data are expressed as mean ± SE. Asterisk indicates a significant difference compared with the control group at each time point ( P

    Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

    Article Title: STAT1 regulates interferon-γ-induced angiotensinogen and MCP-1 expression in a bidirectional manner in primary cultured mesangial cells

    doi: 10.1177/1470320320946527

    Figure Lengend Snippet: Activation STAT1 and STAT3 by IFN-γ in MCs. MCs were treated with 0–20 ng/ml IFN-γ for 8 hours. After treatment, STAT1 ((a) N =3 replicates per condition from three independent experiments) and STAT3 phosphorylation ((b) N =3 replicates per condition from three independent experiments) were evaluated by western blot analysis. These phosphorylation levels were normalised based on β-actin levels. Whether IFN-γ induces translocalisation of STAT1 and STAT3 into nucleus was evaluated. MCs were treated with 5 ng/mL IFN-γ for 8 hours. Thereafter, localisations of STAT1 and STAT3 were determined by immunofluorescence staining ((c) and (d)). Data are expressed as mean ± SE. Asterisk indicates a significant difference compared with the control group at each time point ( P

    Article Snippet: A mouse anti-phospho-STAT1 (Tyr 701) antibody, a rabbit anti-phospho-STAT3 (Tyr 705) antibody, a rabbit anti-STAT1 antibody and a rabbit anti-STAT3 antibody were obtained from Cell Signaling Technology.

    Techniques: Activation Assay, Western Blot, Immunofluorescence, Staining

    Contribution of STAT1 to the IFN-γ-induced AGT and MCP-1 augmentations. To elucidate further the mechanisms underlying the regulation of AGT and MCP-1 by IFN-γ, basal STAT1 expression was suppressed using STAT1-siRNA ((a) N =3 replicates per condition from three independent experiments). Thereafter, the cells were treated with 20 ng/ml IFN-γ for 8 hours. AGT mRNA ((b) N =3 replicates per condition from three independent experiments) and MCP-1 mRNA ((c) N =3 replicates per condition from three independent experiments) levels were measured by qRT–PCR. AGT and MCP-1 mRNA levels were normalised based on β-actin mRNA levels. Data are expressed as relative values compared with each control group and represent the mean ± SE. Asterisk ( P

    Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

    Article Title: STAT1 regulates interferon-γ-induced angiotensinogen and MCP-1 expression in a bidirectional manner in primary cultured mesangial cells

    doi: 10.1177/1470320320946527

    Figure Lengend Snippet: Contribution of STAT1 to the IFN-γ-induced AGT and MCP-1 augmentations. To elucidate further the mechanisms underlying the regulation of AGT and MCP-1 by IFN-γ, basal STAT1 expression was suppressed using STAT1-siRNA ((a) N =3 replicates per condition from three independent experiments). Thereafter, the cells were treated with 20 ng/ml IFN-γ for 8 hours. AGT mRNA ((b) N =3 replicates per condition from three independent experiments) and MCP-1 mRNA ((c) N =3 replicates per condition from three independent experiments) levels were measured by qRT–PCR. AGT and MCP-1 mRNA levels were normalised based on β-actin mRNA levels. Data are expressed as relative values compared with each control group and represent the mean ± SE. Asterisk ( P

    Article Snippet: A mouse anti-phospho-STAT1 (Tyr 701) antibody, a rabbit anti-phospho-STAT3 (Tyr 705) antibody, a rabbit anti-STAT1 antibody and a rabbit anti-STAT3 antibody were obtained from Cell Signaling Technology.

    Techniques: Expressing, Quantitative RT-PCR

    Schematic summary of proposed MCP-1, AGT regulation by IFN-γ-STAT1 in rat MCs. IFN-γ induces MCP-1 and SOCS1 expression directly via STAT1 in rat MCs. Conversely, STAT1-induced expression of SOCS1 attenuates IFN-γ-STAT3-mediated expression of AGT, providing feedback regulation of IFN-γ-AGT signaling in rat MCs. These results demonstrate that while IFN-γ increases both AGT and MCP-1 expression, STAT1 plays an opposing role in the regulation of each factor in MCs. MCP-1: monocyte chemoattractant protein 1; AGT: angiotensinogen; IFN-γ: interferon-γ; SOCS1: suppressor of cytokine signaling 1; MCs: mesangial cells.

    Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

    Article Title: STAT1 regulates interferon-γ-induced angiotensinogen and MCP-1 expression in a bidirectional manner in primary cultured mesangial cells

    doi: 10.1177/1470320320946527

    Figure Lengend Snippet: Schematic summary of proposed MCP-1, AGT regulation by IFN-γ-STAT1 in rat MCs. IFN-γ induces MCP-1 and SOCS1 expression directly via STAT1 in rat MCs. Conversely, STAT1-induced expression of SOCS1 attenuates IFN-γ-STAT3-mediated expression of AGT, providing feedback regulation of IFN-γ-AGT signaling in rat MCs. These results demonstrate that while IFN-γ increases both AGT and MCP-1 expression, STAT1 plays an opposing role in the regulation of each factor in MCs. MCP-1: monocyte chemoattractant protein 1; AGT: angiotensinogen; IFN-γ: interferon-γ; SOCS1: suppressor of cytokine signaling 1; MCs: mesangial cells.

    Article Snippet: A mouse anti-phospho-STAT1 (Tyr 701) antibody, a rabbit anti-phospho-STAT3 (Tyr 705) antibody, a rabbit anti-STAT1 antibody and a rabbit anti-STAT3 antibody were obtained from Cell Signaling Technology.

    Techniques: Expressing

    Participation of SOCS1 in IFN-γ-induced AGT augmentations. Involvement of STAT1 in SOCS1 regulation was tested using STAT1-siRNA as shown in Figure 4 (a) . Furthermore, MCs were treated with SOCS1-siRNA ((b) N =3 replicates per condition from three independent experiments). Thereafter, the cells were treated with 20 ng/ml IFN-γ for 24 hours. Then, AGT mRNA levels were measured by qRT–PCR (c). AGT mRNA levels were normalised based on β-actin mRNA levels. Data are expressed as relative values compared with each control group and represent the mean ± SE. Asterisk ( P

    Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

    Article Title: STAT1 regulates interferon-γ-induced angiotensinogen and MCP-1 expression in a bidirectional manner in primary cultured mesangial cells

    doi: 10.1177/1470320320946527

    Figure Lengend Snippet: Participation of SOCS1 in IFN-γ-induced AGT augmentations. Involvement of STAT1 in SOCS1 regulation was tested using STAT1-siRNA as shown in Figure 4 (a) . Furthermore, MCs were treated with SOCS1-siRNA ((b) N =3 replicates per condition from three independent experiments). Thereafter, the cells were treated with 20 ng/ml IFN-γ for 24 hours. Then, AGT mRNA levels were measured by qRT–PCR (c). AGT mRNA levels were normalised based on β-actin mRNA levels. Data are expressed as relative values compared with each control group and represent the mean ± SE. Asterisk ( P

    Article Snippet: A mouse anti-phospho-STAT1 (Tyr 701) antibody, a rabbit anti-phospho-STAT3 (Tyr 705) antibody, a rabbit anti-STAT1 antibody and a rabbit anti-STAT3 antibody were obtained from Cell Signaling Technology.

    Techniques: Quantitative RT-PCR

    Species-specific activity of rec Mg IFN-γ and mouse IFN-γ on different rodent cell lines. NIH/3T3, BVK168, FMN-R and AAL-R cells were tested and either left untreated as control (lower panel), stimulated for 1 h with rec Mg IFN-γ (200 ng/ml, upper panel) or mouse IFN-γ (200 U/ml, central panel). In each panel the following stainings are shown from left to right: phospho-Tyr701-STAT1 staining (green), DAPI staining (white) and overlay of the two signals. Activated phospho-STAT1 is indicated by nuclear translocation. Of note, minor background signal in the cytoplasma was observed in some control treated cells. A representative experiment of three replicates is shown. The scale bar represents 50 μm.

    Journal: Scientific Reports

    Article Title: Recombinant IFN-γ from the bank vole Myodes glareolus: a novel tool for research on rodent reservoirs of zoonotic pathogens

    doi: 10.1038/s41598-018-21143-0

    Figure Lengend Snippet: Species-specific activity of rec Mg IFN-γ and mouse IFN-γ on different rodent cell lines. NIH/3T3, BVK168, FMN-R and AAL-R cells were tested and either left untreated as control (lower panel), stimulated for 1 h with rec Mg IFN-γ (200 ng/ml, upper panel) or mouse IFN-γ (200 U/ml, central panel). In each panel the following stainings are shown from left to right: phospho-Tyr701-STAT1 staining (green), DAPI staining (white) and overlay of the two signals. Activated phospho-STAT1 is indicated by nuclear translocation. Of note, minor background signal in the cytoplasma was observed in some control treated cells. A representative experiment of three replicates is shown. The scale bar represents 50 μm.

    Article Snippet: For the immunofluorescent analysis a rabbit anti-phospho-STAT1 (Tyr701) monoclonal antibody (dilution 1:50) or rabbit polyclonal anti-phospho-STAT1 (Ser727) antibody (dilution 1:100; both Cell Signaling Technology) and an AlexaFluor 488-conjugated goat anti-rabbit secondary antibody (dilution 1:1,000; Abcam) were used.

    Techniques: Activity Assay, Staining, Translocation Assay

    Suppression of type I IFN signaling pathways is not associated with early temperature effects. (A, C, and D) Immunoblot assay of STAT1 phosphorylated at Tyr-701 (A and D) or Ser-727 (C) from Vero cells treated with 1,000 IU/ml IFN-α/β at 30, 37, or 39°C and corresponding densitometry quantification with STAT1-p bands normalized to β-actin. Data are presented as mean STAT1-p/actin ratio ± SD. (B) Vero cells treated with 1,000 IU/ml IFN-α/β for 30 min at 30, 37, or 39°C were subjected to immunocytochemistry staining for STAT1-p (Y701). Confocal imaging shows phosphorylated STAT1 nuclear translocation. Graph displays average nuclear STAT1-p signal intensity per imaged nuclear area at each temperature. At least 199 cells/nucleus were analyzed in each temperature group. Statistics for panel A: **, P

    Journal: mBio

    Article Title: The Efficacy of the Interferon Alpha/Beta Response versus Arboviruses Is Temperature Dependent

    doi: 10.1128/mBio.00535-18

    Figure Lengend Snippet: Suppression of type I IFN signaling pathways is not associated with early temperature effects. (A, C, and D) Immunoblot assay of STAT1 phosphorylated at Tyr-701 (A and D) or Ser-727 (C) from Vero cells treated with 1,000 IU/ml IFN-α/β at 30, 37, or 39°C and corresponding densitometry quantification with STAT1-p bands normalized to β-actin. Data are presented as mean STAT1-p/actin ratio ± SD. (B) Vero cells treated with 1,000 IU/ml IFN-α/β for 30 min at 30, 37, or 39°C were subjected to immunocytochemistry staining for STAT1-p (Y701). Confocal imaging shows phosphorylated STAT1 nuclear translocation. Graph displays average nuclear STAT1-p signal intensity per imaged nuclear area at each temperature. At least 199 cells/nucleus were analyzed in each temperature group. Statistics for panel A: **, P

    Article Snippet: The following antibodies were used for Western blotting and/or immunostaining: mouse anti-β-actin (BA3R; Invitrogen; RRID AB_10979409 ), rabbit anti-STAT1 (M-22; Santa Cruz; RRID AB_632434 ), rabbit anti-ISG15 (H-150; Santa Cruz; RRID AB_2126309 ), rabbit anti-IFIT1 (Invitrogen catalog no. PA5-27907 for human/nonhuman primate samples; RRID AB_2545383 ), rabbit anti-IFIT1 (Invitrogen catalog no. PA3-846 for murine samples; RRID AB_1958734 ), rabbit anti-phospho-STAT1 (Tyr-701) (catalog no. 9171; Cell Signaling Technology, Inc.; used for Western blotting assays; RRID AB_561284 ), rabbit anti-phospho-STAT1 (Tyr-701) (D4A7; Cell Signaling Technology, Inc.; used for immunofluorescence; RRID AB_10950970 ), rabbit anti-phospho-STAT1 (Ser-727) (catalog no. 9177; Cell Signaling Technology, Inc.; RRID AB_2197983 ), and rabbit anti-phospho-IRF3 (Ser-396) (catalog no. 29047; Cell Signaling Technology, Inc.).

    Techniques: Immunocytochemistry, Staining, Imaging, Translocation Assay