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  • 99
    Thermo Fisher gene exp stat1 hs01013996 m1
    Gene Exp Stat1 Hs01013996 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc stat1
    VPA treatment elevates <t>STAT1</t> expression in the lesioned spinal cord 7 days after SCI. a STAT1 expressions were higher in the SCI than in the sham group. The STAT1 protein levels were upregulated following the VPA treatment ( P
    Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology stat1
    7kchol-induced apoptosis is dependent on an autocrine action of IFN-β. (A) Neutralizing antibody to IFN-β decreases <t>Stat1</t> phosphorylation at Ser 727 . Cell extracts were prepared at the times indicated after treatment with 7kchol (15 μg/ml). One set of cells was pretreated with 0.1-μg/ml neutralizing antibody to IFN-β. Western transfers were probed with an antibody to the Ser 727 phosphorylated form of Stat1. Cells treated with IFN-γ were used as positive controls. (B) Neutralizing antibody to IFN-β decreases 7kchol-induced apoptosis in 2fTGH cells. Apoptosis induced by 7kchol (15 μg/ml) was quantified by staining with acridine orange and ethidium bromide in cells treated or untreated with neutralizing antibody to IFN-β. (C) Cells deficient in IFN receptor have a blunted apoptosis response to 7kchol. Apoptosis induced by 7kchol (15 μg/ml) was quantified as described above for 2fTGH and U5A (IFNAR-2-deficient) cells. Data are the means of three separate experiments; error bars represent the standard error of the means.
    Stat1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2779 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti stat1
    Blockade of ERBB Induces MHC Class I Gene Expression (A) DFTD tumor cell line T1 was treated with recombinant interferon-γ (rIFN-γ) and/or 1 μM sapitinib. Control cells were treated with solvents. Forty-eight hours after treatment, expression of B2M , SAHA-UC , <t>STAT1</t> , and STAT3 were measured by real-time PCR (n = 3 replicates). (B) Reciprocal co-immunoprecipitation of STAT3 and STAT1 followed by western blots for STAT3 and STAT1 in DFTD tumor cell line (T1), fibroblasts, and human HT29 colon cancer cells as control. (C and D) Tumor volume (C) and tumor weight (D) of DFTD tumor cell line T1 transplanted into NSG mice and treated with either vehicle or 50 mg/kg sapitinib once daily (bilateral tumors, n = 5 mice per group). One out of two representative experiments is shown. (E) H E and IHC analyses for total STAT3, pS-STAT3, pY-STAT3, Ki67, and Cleaved Caspase 3 of tumor tissues. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Quantification of total STAT3, pS-STAT3, pY-STAT3, Ki67, and Cleaved Caspase 3. Scale bars, 200 and 25 μm. (F) Western blots for total STAT3, pY-STAT3, pS-STAT3, and STAT1 from representative xenograft tumors. (G) Expression of B2M and STAT3 by real-time PCR from xenograft tumor tissue. .
    Anti Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1486 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc phospho stat1
    IDO expression in IFN-γ-primed MSCs via a <t>JAK-STAT1</t> signaling pathway. MSCs derived from four different tissues (BM-, AT-, CB-, and WJ-MSC) were used. (a) MSCs were incubated with 200 IU/mL IFN-γ for the indicated amounts of time. The expression levels of phospho-JAK1/2, phospho-STAT1, STAT1, and IRF-1 in these MSCs were detected by immunoblotting. (b) To inhibit the activity of JAK, an intracellular domain of the IFN-γ receptor, MSCs were incubated with 1 μM AG490 (a JAK inhibitor) for 24 h before IFN-γ priming. The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in AG490-treated MSCs were detected by immunoblotting. AG490 treatment induced the down-regulation of STAT1 activity and IDO expression. (c) To down-regulate STAT1 activity, MSCs were transfected with a scrambled siRNA or with an siRNA targeting STAT1 . The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in these transfected MSCs were detected by immunoblotting. Down-regulation of STAT1 activity effectively induced a decrease in IDO expression in IFN-γ-primed MSCs. (d) MSCs were treated with 200 IU/mL IFN-γ or 100 μg/mL poly I:C for 24 h. The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in these MSCs were detected by immunoblotting. β-Actin was used as a loading control for all western blots.
    Phospho Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 933 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology anti stat1
    Influence of a neutralizing IFN receptor antibody on <t>STAT1</t> phosphorylation in response to poly(I:C). (A) Following pretreatment with the anti-IFNAR neutralizing antibody (2.5 μg/well) for 1 h, A549 cells were transfected with poly(I:C) (200 ng)
    Anti Stat1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 745 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson stat1
    <t>STAT1</t> regulates Bcl-xL, Fas and Bad expression A-C . CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight, and analyzed for the expression level of genes as indicated by semi-quantitative RT-PCR. Cell surface Fas protein levels were measured by staining cells with Fas-specific mAb and analyzed by flow cytometry. Fas protein level is quantified by mean fluorescence intensity and presented in bottom of panel C. * p =0.05. D . Bcl-xL and Bad form a protein complex in sarcoma cells. Cell lysates were prepared from CMS4 cells, immunoprecipitated with Bcl-2- and Bcl-xL-specific mAbs, respectively, and then analyzed for Bcl-2, Bcl-xL and Bad protein association by Western blotting analysis.
    Stat1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 583 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    stat1  (Abcam)
    95
    Abcam stat1
    <t>STAT1</t> regulates Bcl-xL, Fas and Bad expression A-C . CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight, and analyzed for the expression level of genes as indicated by semi-quantitative RT-PCR. Cell surface Fas protein levels were measured by staining cells with Fas-specific mAb and analyzed by flow cytometry. Fas protein level is quantified by mean fluorescence intensity and presented in bottom of panel C. * p =0.05. D . Bcl-xL and Bad form a protein complex in sarcoma cells. Cell lysates were prepared from CMS4 cells, immunoprecipitated with Bcl-2- and Bcl-xL-specific mAbs, respectively, and then analyzed for Bcl-2, Bcl-xL and Bad protein association by Western blotting analysis.
    Stat1, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 456 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho stat1 tyr701
    <t>STAT1</t> regulates Bcl-xL, Fas and Bad expression A-C . CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight, and analyzed for the expression level of genes as indicated by semi-quantitative RT-PCR. Cell surface Fas protein levels were measured by staining cells with Fas-specific mAb and analyzed by flow cytometry. Fas protein level is quantified by mean fluorescence intensity and presented in bottom of panel C. * p =0.05. D . Bcl-xL and Bad form a protein complex in sarcoma cells. Cell lysates were prepared from CMS4 cells, immunoprecipitated with Bcl-2- and Bcl-xL-specific mAbs, respectively, and then analyzed for Bcl-2, Bcl-xL and Bad protein association by Western blotting analysis.
    Phospho Stat1 Tyr701, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher stat1
    Interferon response-like signalling is upregulated in amoeboid cells. ( a ) Ten most significant GO Biological Process terms enriched in the genes affected concordantly by both iRhoA and dasatinib (DAS) treatment. Only transcripts with adjusted p -value ≤ 0.25 and fold change > 1.5 in either direction were considered differentially expressed. Network plot was generated using ShinyGO v0.61 online tool [ 30 ]. ( b ) Gene expression (log2 fold change) of <t>STAT1,</t> STAT2, STAT3 and IRF9 in cells after mesenchymal–amoeboid transition (MAT) in 3D collagen (48 h) determined by RT-qPCR, normalized to control cells without MAT induction. ( c ) Immunoblotting detection of Stat transcription factors Stat1, Stat2 and Stat3 in HT1080 cells before and after MAT. p -values: ** p
    Stat1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc stat1
    DN forms of CaMKII inhibit <t>Stat1</t> S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
    Stat1, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Taconic Biosciences stat1
    Replication of ICP0 - and ICP4 - viruses in cell culture and immunodeficient mice . Vero cells were A . untreated or B . treated with 200 U per ml of IFN-β and were inoculated with 2.5 pfu per cell of wild-type HSV-1 (KOS), an ICP0 - virus (0 - -GFP), or an ICP4 - virus (n12). The mean ± sem of the logarithm of viral titers recovered from Vero cells is plotted over time (n = 4 per time point). C . Rag2 -/- <t>stat1</t> -/- mice and D . rag2 -/- mice were inoculated with 2 × 10 5 pfu per eye of the ICP0 - virus 0 - -GFP or the ICP4 - virus n12 (n = 4 mice per group). The mean ± sem of the logarithm of viral titers recovered from mouse eyes is plotted over time (open black symbols). Dashed lines indicate the lower limit of detection of each plaque assay. The survival of 0 - -GFP-infected mice and ICP4 - virus-infected mice is plotted over time (open red symbols).
    Stat1, supplied by Taconic Biosciences, used in various techniques. Bioz Stars score: 96/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc phosphorylated stat1
    Effects of 4AAQB on the LPS-induced activation of MAP kinases, IkBα, NFκB p65 and <t>STAT1</t> in RAW 264.7 macrophages and peritoneal macrophages. RAW264.7 cells were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Cells were harvested and total cell extracts were prepared. a Phosphorylated-ERK, phosphorylated-JNK, phosphorylated-p38, or b Phosphorylated-IκBα and NFκB p65 subunit and c Phosphorylated-STAT1 were detected by Western blot analysis. Total ERK, JNK, p38 and α-tubulin were used as internal standard. d Peritoneal macrophages were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Phosphorylated-ERK and total ERK were detected by Western blot analysis
    Phosphorylated Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 289 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology p stat1
    Co-Immunoprecipitation and ubiquitination of <t>P-STAT1</t> in CLP Cells were lysed and cleared by centrifugation. Aliquots were precleared by addition of normal IgG and 20 µl of appropriate agarose conjugate. After centrifugation, the supernatant was collected and incubated with 2 µg of P-Stat1 Ab. After washing, immunoblotting was performed using Ub AB. Blot is representative of four experiments.
    P Stat1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 267 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti stat1
    Time response of JAK/STAT pathway activation in RT4-D6P2T after BDNF administration. BDNF (100 pM) was administered to treat rat Schwann cells RT4-D6P2T at 0 min, 10 min, 30 min, 60 min, 120 min and 24 hr. <t>STAT3/STAT1</t> were activated at 10 min after the treatment with BDNF (lane 2), and the phosphorylation level peaked at 30 min (lane 3) (*P
    Anti Stat1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti stat1
    <t>STAT1</t> 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
    Rabbit Anti Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    VPA treatment elevates STAT1 expression in the lesioned spinal cord 7 days after SCI. a STAT1 expressions were higher in the SCI than in the sham group. The STAT1 protein levels were upregulated following the VPA treatment ( P

    Journal: Journal of Neuroinflammation

    Article Title: Valproic acid attenuates traumatic spinal cord injury-induced inflammation via STAT1 and NF-κB pathway dependent of HDAC3

    doi: 10.1186/s12974-018-1193-6

    Figure Lengend Snippet: VPA treatment elevates STAT1 expression in the lesioned spinal cord 7 days after SCI. a STAT1 expressions were higher in the SCI than in the sham group. The STAT1 protein levels were upregulated following the VPA treatment ( P

    Article Snippet: The sections were incubated with a STAT1 (1:100; Cell Signaling Technology, Danvers, MA, USA) antibody, washed, and incubated with secondary antibody for 1 h at room temperature.

    Techniques: Expressing

    VPA treatment suppressed the NF-κB pathway via elevating STAT1 expression in the lesioned spinal cord 7 days after SCI. a Co-IP analysis showed a greater elevation in the STAT1 after the VPA treatment than in the SCI group ( P

    Journal: Journal of Neuroinflammation

    Article Title: Valproic acid attenuates traumatic spinal cord injury-induced inflammation via STAT1 and NF-κB pathway dependent of HDAC3

    doi: 10.1186/s12974-018-1193-6

    Figure Lengend Snippet: VPA treatment suppressed the NF-κB pathway via elevating STAT1 expression in the lesioned spinal cord 7 days after SCI. a Co-IP analysis showed a greater elevation in the STAT1 after the VPA treatment than in the SCI group ( P

    Article Snippet: The sections were incubated with a STAT1 (1:100; Cell Signaling Technology, Danvers, MA, USA) antibody, washed, and incubated with secondary antibody for 1 h at room temperature.

    Techniques: Expressing, Co-Immunoprecipitation Assay

    Increased STAT3 phosphorylation is maintained 24 h post-stimulation. A , STAT3, STAT1, and STAT5 phosphorylation levels in NIH3T3 cells transduced with shRNA directed against mLIFR (mOSMR signaling) 24 h after cytokine stimulation. B , relative quantification of receptor activation by each cytokine; P-STAT band intensities were normalized against total STAT levels, followed by data transformation relative to the basal ( Ctrl ) level, which was set to 0; in the case of STAT5, the stronger basal signal was set to 1. Values are presented as mean ± S.E. ( error bars ), n = 3 independent cultures. C , STAT3, STAT1, and STAT5 phosphorylation levels in MH-S cells (mLIFR signaling) 24 h after cytokine stimulation. D , relative quantification of receptor activation by each cytokine; P-STAT band intensities were normalized and transformed as described above. Values are presented as mean ± S.E., n = 3 independent cultures; not significant ( n.s. ), p > 0.05; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The AB loop of oncostatin M (OSM) determines species-specific signaling in humans and mice

    doi: 10.1074/jbc.RA118.004375

    Figure Lengend Snippet: Increased STAT3 phosphorylation is maintained 24 h post-stimulation. A , STAT3, STAT1, and STAT5 phosphorylation levels in NIH3T3 cells transduced with shRNA directed against mLIFR (mOSMR signaling) 24 h after cytokine stimulation. B , relative quantification of receptor activation by each cytokine; P-STAT band intensities were normalized against total STAT levels, followed by data transformation relative to the basal ( Ctrl ) level, which was set to 0; in the case of STAT5, the stronger basal signal was set to 1. Values are presented as mean ± S.E. ( error bars ), n = 3 independent cultures. C , STAT3, STAT1, and STAT5 phosphorylation levels in MH-S cells (mLIFR signaling) 24 h after cytokine stimulation. D , relative quantification of receptor activation by each cytokine; P-STAT band intensities were normalized and transformed as described above. Values are presented as mean ± S.E., n = 3 independent cultures; not significant ( n.s. ), p > 0.05; *, p

    Article Snippet: These were probed with antibodies against phospho-STAT3 (Tyr-705) (catalog no. 9131, Cell Signaling Technology), phospho-STAT1 (Tyr-701) (catalog no. 7649, Cell Signaling Technology), phospho-STAT5 (Tyr-694) (catalog no. 9351, Cell Signaling Technology), phospho-SAP/JNK (Thr-183/Tyr-185) (catalog no. 9251, Cell Signaling Technology), phospho-ERK1/2 (Thr-202/Tyr-204) (catalog no. 9101, Cell Signaling Technology), phospho-AKT (Thr-308) (catalog no. 9275, Cell Signaling Technology), phospho-AKT (Ser-473) (catalog no. 9271, Cell Signaling Technology), phospho-SHP2 (Tyr-580) (catalog no. 3754, Cell Signaling Technology), Phospho-p38 (Thr-180/Tyr-182) (catalog. no. 9211, Cell Signaling Technology), pan-actin (catalog no. 4968, Cell Signaling Technology), human TIMP1 (catalog no. 8946, Cell Signaling Technology), mouse TIMP1 (catalog no. MAB9801, R & D Systems), HIF1α (catalog no. A300-286A, Bethyl Laboratories), SOD2 (catalog no. MAB3419, R & D Systems), VEGF 164 (catalog no. AF-493-NA, R & D Systems), total STAT3 (catalog no. 9139, Cell Signaling Technology), total STAT1 (catalog no. 9172, Cell Signaling Technology), total STAT5 (catalog no. 9310, Cell Signaling Technology), total SAP/JNK (catalog no. 9258, Cell Signaling Technology), total ERK1/2 (catalog no. 4695, Cell Signaling Technology), total AKT (catalog no. 9272, Cell Signaling Technology), total SHP2 (catalog no. 3752, Cell Signaling Technology), and total p38 (catalog no. 9212, Cell Signaling Technology).

    Techniques: Transduction, shRNA, Activation Assay, Transformation Assay

    Short-term changes in receptor activation are not restricted to STAT3 signaling. A , STAT3, STAT1, and STAT5 phosphorylation levels in NIH3T3 cells transduced with shRNA directed against mLIFR (mOSMR signaling) 10 min after cytokine stimulation. B , relative quantification of receptor activation by each cytokine; P-STAT band intensities were normalized against total STAT levels, followed by data transformation relative to the basal ( Ctrl ) level, which was set to 0. Values are presented as mean ± S.E. ( error bars ), n = 5 independent cultures. C , STAT3, STAT1, and STAT5 phosphorylation levels in MH-S cells (mLIFR signaling) 10 min after cytokine stimulation. D , relative quantification of receptor activation by each cytokine; P-STAT band intensities were normalized and transformed as described above. Values are presented as mean ± S.E., n = 3 independent cultures; not significant ( n.s. ), p > 0.05; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The AB loop of oncostatin M (OSM) determines species-specific signaling in humans and mice

    doi: 10.1074/jbc.RA118.004375

    Figure Lengend Snippet: Short-term changes in receptor activation are not restricted to STAT3 signaling. A , STAT3, STAT1, and STAT5 phosphorylation levels in NIH3T3 cells transduced with shRNA directed against mLIFR (mOSMR signaling) 10 min after cytokine stimulation. B , relative quantification of receptor activation by each cytokine; P-STAT band intensities were normalized against total STAT levels, followed by data transformation relative to the basal ( Ctrl ) level, which was set to 0. Values are presented as mean ± S.E. ( error bars ), n = 5 independent cultures. C , STAT3, STAT1, and STAT5 phosphorylation levels in MH-S cells (mLIFR signaling) 10 min after cytokine stimulation. D , relative quantification of receptor activation by each cytokine; P-STAT band intensities were normalized and transformed as described above. Values are presented as mean ± S.E., n = 3 independent cultures; not significant ( n.s. ), p > 0.05; *, p

    Article Snippet: These were probed with antibodies against phospho-STAT3 (Tyr-705) (catalog no. 9131, Cell Signaling Technology), phospho-STAT1 (Tyr-701) (catalog no. 7649, Cell Signaling Technology), phospho-STAT5 (Tyr-694) (catalog no. 9351, Cell Signaling Technology), phospho-SAP/JNK (Thr-183/Tyr-185) (catalog no. 9251, Cell Signaling Technology), phospho-ERK1/2 (Thr-202/Tyr-204) (catalog no. 9101, Cell Signaling Technology), phospho-AKT (Thr-308) (catalog no. 9275, Cell Signaling Technology), phospho-AKT (Ser-473) (catalog no. 9271, Cell Signaling Technology), phospho-SHP2 (Tyr-580) (catalog no. 3754, Cell Signaling Technology), Phospho-p38 (Thr-180/Tyr-182) (catalog. no. 9211, Cell Signaling Technology), pan-actin (catalog no. 4968, Cell Signaling Technology), human TIMP1 (catalog no. 8946, Cell Signaling Technology), mouse TIMP1 (catalog no. MAB9801, R & D Systems), HIF1α (catalog no. A300-286A, Bethyl Laboratories), SOD2 (catalog no. MAB3419, R & D Systems), VEGF 164 (catalog no. AF-493-NA, R & D Systems), total STAT3 (catalog no. 9139, Cell Signaling Technology), total STAT1 (catalog no. 9172, Cell Signaling Technology), total STAT5 (catalog no. 9310, Cell Signaling Technology), total SAP/JNK (catalog no. 9258, Cell Signaling Technology), total ERK1/2 (catalog no. 4695, Cell Signaling Technology), total AKT (catalog no. 9272, Cell Signaling Technology), total SHP2 (catalog no. 3752, Cell Signaling Technology), and total p38 (catalog no. 9212, Cell Signaling Technology).

    Techniques: Activation Assay, Transduction, shRNA, Transformation Assay

    7kchol-induced apoptosis is dependent on an autocrine action of IFN-β. (A) Neutralizing antibody to IFN-β decreases Stat1 phosphorylation at Ser 727 . Cell extracts were prepared at the times indicated after treatment with 7kchol (15 μg/ml). One set of cells was pretreated with 0.1-μg/ml neutralizing antibody to IFN-β. Western transfers were probed with an antibody to the Ser 727 phosphorylated form of Stat1. Cells treated with IFN-γ were used as positive controls. (B) Neutralizing antibody to IFN-β decreases 7kchol-induced apoptosis in 2fTGH cells. Apoptosis induced by 7kchol (15 μg/ml) was quantified by staining with acridine orange and ethidium bromide in cells treated or untreated with neutralizing antibody to IFN-β. (C) Cells deficient in IFN receptor have a blunted apoptosis response to 7kchol. Apoptosis induced by 7kchol (15 μg/ml) was quantified as described above for 2fTGH and U5A (IFNAR-2-deficient) cells. Data are the means of three separate experiments; error bars represent the standard error of the means.

    Journal: Molecular and Cellular Biology

    Article Title: Stat1-Dependent, p53-Independent Expression of p21waf1 Modulates Oxysterol-Induced Apoptosis

    doi: 10.1128/MCB.22.7.1981-1992.2002

    Figure Lengend Snippet: 7kchol-induced apoptosis is dependent on an autocrine action of IFN-β. (A) Neutralizing antibody to IFN-β decreases Stat1 phosphorylation at Ser 727 . Cell extracts were prepared at the times indicated after treatment with 7kchol (15 μg/ml). One set of cells was pretreated with 0.1-μg/ml neutralizing antibody to IFN-β. Western transfers were probed with an antibody to the Ser 727 phosphorylated form of Stat1. Cells treated with IFN-γ were used as positive controls. (B) Neutralizing antibody to IFN-β decreases 7kchol-induced apoptosis in 2fTGH cells. Apoptosis induced by 7kchol (15 μg/ml) was quantified by staining with acridine orange and ethidium bromide in cells treated or untreated with neutralizing antibody to IFN-β. (C) Cells deficient in IFN receptor have a blunted apoptosis response to 7kchol. Apoptosis induced by 7kchol (15 μg/ml) was quantified as described above for 2fTGH and U5A (IFNAR-2-deficient) cells. Data are the means of three separate experiments; error bars represent the standard error of the means.

    Article Snippet: However, our observations that fibroblasts lacking Stat1 express reduced p21waf1 protein and that the lack of either Stat1 or p21waf1 leads to markedly reduced apoptosis in response to 7kchol establish a connection between Stat1 and p21waf1 and also place cytochrome c release and the activation of caspase 3 downstream of the regulatory roles played by Stat1 and p21waf1 .

    Techniques: Western Blot, Staining

    Apoptosis in response to 7kchol is impaired in cells expressing Stat1 mutants with substituted Ser 727 or a deleted C terminus. Apoptosis was quantified after 18 h treatment with 7kchol (15 μg/ml) in 2fTGH control cells, U3A cells, and U3A cells stably transfected to express various mutants of Stat1 as described in the text. The graph displays the means of data from four different experiments, and the error bars reflect standard errors of the means.

    Journal: Molecular and Cellular Biology

    Article Title: Stat1-Dependent, p53-Independent Expression of p21waf1 Modulates Oxysterol-Induced Apoptosis

    doi: 10.1128/MCB.22.7.1981-1992.2002

    Figure Lengend Snippet: Apoptosis in response to 7kchol is impaired in cells expressing Stat1 mutants with substituted Ser 727 or a deleted C terminus. Apoptosis was quantified after 18 h treatment with 7kchol (15 μg/ml) in 2fTGH control cells, U3A cells, and U3A cells stably transfected to express various mutants of Stat1 as described in the text. The graph displays the means of data from four different experiments, and the error bars reflect standard errors of the means.

    Article Snippet: However, our observations that fibroblasts lacking Stat1 express reduced p21waf1 protein and that the lack of either Stat1 or p21waf1 leads to markedly reduced apoptosis in response to 7kchol establish a connection between Stat1 and p21waf1 and also place cytochrome c release and the activation of caspase 3 downstream of the regulatory roles played by Stat1 and p21waf1 .

    Techniques: Expressing, Stable Transfection, Transfection

    p21 waf1 deficiency in Stat1 −/− cells. The data shown in panels A and B were from Stat1 −/− mice developed in two independent laboratories. (A) p21 waf1 is decreased in Stat1 −/− MEF compared to wild-type cells; Stat1 is not decreased in p21 waf1−/− MEF. The levels of the proteins were determined by Western analysis with combined antibodies to p21 waf1 , p53, and Stat1. (B) p21 waf1 is decreased in Stat1 −/− mouse fibroblasts. Western analysis was performed with an antibody to p21 waf1 showing the basal levels of p21 waf1 in Stat1 −/− and wild-type mouse fibroblasts. (C) p21 waf1 is decreased in Stat1 −/− primary cultures of MEF compared to wild-type primary cultures of MEF. Western analysis was performed with an antibody to p21 waf1 and shows the relative basal levels of p21 waf1 present in primary Stat1 −/− and wild-type MEF. (D) Restoration of p21 waf1 to Stat1 −/− MEF restores apoptosis rates in response to 7kchol. Stat1 −/− MEF were infected with Ad-p21 carrying p21 waf1 cDNA at an MOI of 100 FFU per cell or with Ad-CMV (control virus) at an MOI of 125 FFU per cell for 48 h. Cells were treated with 7kchol (15 μg/ml) for 18 h, and apoptosis was quantified by staining with acridine orange and ethidium bromide.

    Journal: Molecular and Cellular Biology

    Article Title: Stat1-Dependent, p53-Independent Expression of p21waf1 Modulates Oxysterol-Induced Apoptosis

    doi: 10.1128/MCB.22.7.1981-1992.2002

    Figure Lengend Snippet: p21 waf1 deficiency in Stat1 −/− cells. The data shown in panels A and B were from Stat1 −/− mice developed in two independent laboratories. (A) p21 waf1 is decreased in Stat1 −/− MEF compared to wild-type cells; Stat1 is not decreased in p21 waf1−/− MEF. The levels of the proteins were determined by Western analysis with combined antibodies to p21 waf1 , p53, and Stat1. (B) p21 waf1 is decreased in Stat1 −/− mouse fibroblasts. Western analysis was performed with an antibody to p21 waf1 showing the basal levels of p21 waf1 in Stat1 −/− and wild-type mouse fibroblasts. (C) p21 waf1 is decreased in Stat1 −/− primary cultures of MEF compared to wild-type primary cultures of MEF. Western analysis was performed with an antibody to p21 waf1 and shows the relative basal levels of p21 waf1 present in primary Stat1 −/− and wild-type MEF. (D) Restoration of p21 waf1 to Stat1 −/− MEF restores apoptosis rates in response to 7kchol. Stat1 −/− MEF were infected with Ad-p21 carrying p21 waf1 cDNA at an MOI of 100 FFU per cell or with Ad-CMV (control virus) at an MOI of 125 FFU per cell for 48 h. Cells were treated with 7kchol (15 μg/ml) for 18 h, and apoptosis was quantified by staining with acridine orange and ethidium bromide.

    Article Snippet: However, our observations that fibroblasts lacking Stat1 express reduced p21waf1 protein and that the lack of either Stat1 or p21waf1 leads to markedly reduced apoptosis in response to 7kchol establish a connection between Stat1 and p21waf1 and also place cytochrome c release and the activation of caspase 3 downstream of the regulatory roles played by Stat1 and p21waf1 .

    Techniques: Mouse Assay, Western Blot, Infection, Staining

    7kchol-induced apoptosis involves cytochrome c release from mitochondria and activation of caspase 3. (A) Apoptosis induced by 7kchol in MEF was inhibited by caspase 3 inhibitors. Cells were pretreated for 2 h with DEVD-FMK (100 μM) and DEVD-CHO (150 μM) and then incubated with 7kchol (15 g/ml) for 18 h. Apoptotic cells were counted after being stained with acridine orange and ethidium bromide. (B) Caspase 3 activity in response to 7kchol is reduced in Stat1 −/− and p21 waf1−/− MEF. Caspase 3 activity was assayed by using DEVD-AMC, a fluorogenic substrate, in MEF. Wild-type, Stat1 −/− , and p21 −/− MEF were treated with 7kchol (15 μg/ml) for 15 h. (C) Procaspase 3 is not deficient in Stat1 −/− or p21waf1 −/− MEF. Western analysis was performed with procaspase 3 antibody in MEF. (D) Cytochrome c is increased in cytosolic fractions of wild-type MEF, but not p21 waf1−/− or Stat1 −/− MEF, treated with 7kchol. Western analysis was performed for cytochrome c in cytosolic fractions at the times shown after addition of 7kchol (15 μg/ml).

    Journal: Molecular and Cellular Biology

    Article Title: Stat1-Dependent, p53-Independent Expression of p21waf1 Modulates Oxysterol-Induced Apoptosis

    doi: 10.1128/MCB.22.7.1981-1992.2002

    Figure Lengend Snippet: 7kchol-induced apoptosis involves cytochrome c release from mitochondria and activation of caspase 3. (A) Apoptosis induced by 7kchol in MEF was inhibited by caspase 3 inhibitors. Cells were pretreated for 2 h with DEVD-FMK (100 μM) and DEVD-CHO (150 μM) and then incubated with 7kchol (15 g/ml) for 18 h. Apoptotic cells were counted after being stained with acridine orange and ethidium bromide. (B) Caspase 3 activity in response to 7kchol is reduced in Stat1 −/− and p21 waf1−/− MEF. Caspase 3 activity was assayed by using DEVD-AMC, a fluorogenic substrate, in MEF. Wild-type, Stat1 −/− , and p21 −/− MEF were treated with 7kchol (15 μg/ml) for 15 h. (C) Procaspase 3 is not deficient in Stat1 −/− or p21waf1 −/− MEF. Western analysis was performed with procaspase 3 antibody in MEF. (D) Cytochrome c is increased in cytosolic fractions of wild-type MEF, but not p21 waf1−/− or Stat1 −/− MEF, treated with 7kchol. Western analysis was performed for cytochrome c in cytosolic fractions at the times shown after addition of 7kchol (15 μg/ml).

    Article Snippet: However, our observations that fibroblasts lacking Stat1 express reduced p21waf1 protein and that the lack of either Stat1 or p21waf1 leads to markedly reduced apoptosis in response to 7kchol establish a connection between Stat1 and p21waf1 and also place cytochrome c release and the activation of caspase 3 downstream of the regulatory roles played by Stat1 and p21waf1 .

    Techniques: Activation Assay, Incubation, Staining, Activity Assay, Western Blot

    Cytochrome c release from mitochondria is impaired in Stat1-deficient U3A cells compared to control cells. Western analysis was done to evaluate the levels of cytochrome c and cytochrome c peroxidase in cytosolic fractions. Cytosolic extracts were prepared at the indicated times after treatment with 7kchol (15 μg/ml). Western analysis for cytochrome c peroxidase 2 served as an indicator of mitochondrial contamination in cytosol (none was detected). The mitochondrial fraction from untreated cells was used as a positive control for cytochrome c and cytochrome c peroxidase 2 (rightmost lane).

    Journal: Molecular and Cellular Biology

    Article Title: Stat1-Dependent, p53-Independent Expression of p21waf1 Modulates Oxysterol-Induced Apoptosis

    doi: 10.1128/MCB.22.7.1981-1992.2002

    Figure Lengend Snippet: Cytochrome c release from mitochondria is impaired in Stat1-deficient U3A cells compared to control cells. Western analysis was done to evaluate the levels of cytochrome c and cytochrome c peroxidase in cytosolic fractions. Cytosolic extracts were prepared at the indicated times after treatment with 7kchol (15 μg/ml). Western analysis for cytochrome c peroxidase 2 served as an indicator of mitochondrial contamination in cytosol (none was detected). The mitochondrial fraction from untreated cells was used as a positive control for cytochrome c and cytochrome c peroxidase 2 (rightmost lane).

    Article Snippet: However, our observations that fibroblasts lacking Stat1 express reduced p21waf1 protein and that the lack of either Stat1 or p21waf1 leads to markedly reduced apoptosis in response to 7kchol establish a connection between Stat1 and p21waf1 and also place cytochrome c release and the activation of caspase 3 downstream of the regulatory roles played by Stat1 and p21waf1 .

    Techniques: Western Blot, Positive Control

    7kchol-induced apoptosis depends on p21 waf1 and Stat1. (A) Apoptosis in MEF. Subconfluent cells were treated with 7kchol (15 μg/ml) for 18 h, and apoptosis was quantified by staining with acridine orange and ethidium bromide. (B) Apoptosis in mouse fibroblasts. Mouse fibroblasts were treated with 7kchol (15 μg/ml) for 18 h, and apoptotic cells were counted as described above. (C) Quantification of fragmented DNA in p21 −/− and Stat1 −/− cells. Cells were incubated with 3 H-labeled thymidine for 18 h and treated with 15 μg of 7kchol/ml for 18 h, and DNA fragmentation was quantified. (D) Colony-forming efficiency of p21 −/− MEF. Cells were treated with 7kchol (10 to 30 μg/ml) for 18 h, and cells were then seeded in equal numbers (1,000 cells/100-mm dish) in 10% FBS and DMEM. Colonies were counted after 15 days. (E) Colony-forming efficiency of Stat1 −/− mouse fibroblasts. Cells were treated with 7kchol as described for panel D. A total of 2,000 cells were then seeded per 100-mm dish, and colonies were counted after 15 days. The data in the graphs in panels D and E depict the colonies formed after 7kchol treatment expressed as a percentage of the colonies formed from cells not treated with 7kchol. Shown in panels D and E are the means and standard deviations of data combined from two experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Stat1-Dependent, p53-Independent Expression of p21waf1 Modulates Oxysterol-Induced Apoptosis

    doi: 10.1128/MCB.22.7.1981-1992.2002

    Figure Lengend Snippet: 7kchol-induced apoptosis depends on p21 waf1 and Stat1. (A) Apoptosis in MEF. Subconfluent cells were treated with 7kchol (15 μg/ml) for 18 h, and apoptosis was quantified by staining with acridine orange and ethidium bromide. (B) Apoptosis in mouse fibroblasts. Mouse fibroblasts were treated with 7kchol (15 μg/ml) for 18 h, and apoptotic cells were counted as described above. (C) Quantification of fragmented DNA in p21 −/− and Stat1 −/− cells. Cells were incubated with 3 H-labeled thymidine for 18 h and treated with 15 μg of 7kchol/ml for 18 h, and DNA fragmentation was quantified. (D) Colony-forming efficiency of p21 −/− MEF. Cells were treated with 7kchol (10 to 30 μg/ml) for 18 h, and cells were then seeded in equal numbers (1,000 cells/100-mm dish) in 10% FBS and DMEM. Colonies were counted after 15 days. (E) Colony-forming efficiency of Stat1 −/− mouse fibroblasts. Cells were treated with 7kchol as described for panel D. A total of 2,000 cells were then seeded per 100-mm dish, and colonies were counted after 15 days. The data in the graphs in panels D and E depict the colonies formed after 7kchol treatment expressed as a percentage of the colonies formed from cells not treated with 7kchol. Shown in panels D and E are the means and standard deviations of data combined from two experiments.

    Article Snippet: However, our observations that fibroblasts lacking Stat1 express reduced p21waf1 protein and that the lack of either Stat1 or p21waf1 leads to markedly reduced apoptosis in response to 7kchol establish a connection between Stat1 and p21waf1 and also place cytochrome c release and the activation of caspase 3 downstream of the regulatory roles played by Stat1 and p21waf1 .

    Techniques: Staining, Incubation, Labeling

    p21 waf1 expression and apoptosis in response to 7kchol are decreased in Stat1-deficient U3A cells compared to 2fTGH control cells; enhanced p21 waf1 expression restores apoptosis. (A) Western analysis for p21 waf1 reveals decreased protein in Stat1-null U3A cells compared to control 2fTGH cells. The membrane was reprobed with antibody to actin as a control. (B) Northern analysis for p21 waf1 mRNA reveals decreased expression in Stat1-null U3A cells compared tocontrol cells. GAPDH mRNA was probed as a control. (C) Increased p21 waf1 expression restores apoptosis in Stat1-deficient U3A cells in response to 7kchol. U3A cells were infected with Ad-p21 at an MOI of 100 FFU per cell or with Ad-CMV at an MOI of 125 FFU per cell for 48 h. 2fTGH control cells and Ad-infected U3A cells were treated with 7kchol (15 μg/ml) for 18 h, and apoptosis was quantified by staining with acridine orange and ethidium bromide.

    Journal: Molecular and Cellular Biology

    Article Title: Stat1-Dependent, p53-Independent Expression of p21waf1 Modulates Oxysterol-Induced Apoptosis

    doi: 10.1128/MCB.22.7.1981-1992.2002

    Figure Lengend Snippet: p21 waf1 expression and apoptosis in response to 7kchol are decreased in Stat1-deficient U3A cells compared to 2fTGH control cells; enhanced p21 waf1 expression restores apoptosis. (A) Western analysis for p21 waf1 reveals decreased protein in Stat1-null U3A cells compared to control 2fTGH cells. The membrane was reprobed with antibody to actin as a control. (B) Northern analysis for p21 waf1 mRNA reveals decreased expression in Stat1-null U3A cells compared tocontrol cells. GAPDH mRNA was probed as a control. (C) Increased p21 waf1 expression restores apoptosis in Stat1-deficient U3A cells in response to 7kchol. U3A cells were infected with Ad-p21 at an MOI of 100 FFU per cell or with Ad-CMV at an MOI of 125 FFU per cell for 48 h. 2fTGH control cells and Ad-infected U3A cells were treated with 7kchol (15 μg/ml) for 18 h, and apoptosis was quantified by staining with acridine orange and ethidium bromide.

    Article Snippet: However, our observations that fibroblasts lacking Stat1 express reduced p21waf1 protein and that the lack of either Stat1 or p21waf1 leads to markedly reduced apoptosis in response to 7kchol establish a connection between Stat1 and p21waf1 and also place cytochrome c release and the activation of caspase 3 downstream of the regulatory roles played by Stat1 and p21waf1 .

    Techniques: Expressing, Western Blot, Northern Blot, Infection, Staining

    Deficiencies in p21 waf1 and Stat1 in human cells is associated with impaired apoptosis after treatment with 7kchol. (A) Induction of apoptosis in LF-1 (control) human fibroblasts and p21-null H07.2-1 cells. Cells were incubated with 7kchol (15 μg/ml) for 18 h, and apoptosis was quantified by staining with acridine orange and ethidium bromide. (B) 7kchol-induced apoptosis (18 h, 15 μg/ml) in control 2fTGH cells is decreased in Stat1-null U3A cells and restored in Stat1-reconstituted U3AR cells. Apoptosis was quantified by staining with acridine orange and ethidium bromide. Shown are the means and standard deviations of data from three separate experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Stat1-Dependent, p53-Independent Expression of p21waf1 Modulates Oxysterol-Induced Apoptosis

    doi: 10.1128/MCB.22.7.1981-1992.2002

    Figure Lengend Snippet: Deficiencies in p21 waf1 and Stat1 in human cells is associated with impaired apoptosis after treatment with 7kchol. (A) Induction of apoptosis in LF-1 (control) human fibroblasts and p21-null H07.2-1 cells. Cells were incubated with 7kchol (15 μg/ml) for 18 h, and apoptosis was quantified by staining with acridine orange and ethidium bromide. (B) 7kchol-induced apoptosis (18 h, 15 μg/ml) in control 2fTGH cells is decreased in Stat1-null U3A cells and restored in Stat1-reconstituted U3AR cells. Apoptosis was quantified by staining with acridine orange and ethidium bromide. Shown are the means and standard deviations of data from three separate experiments.

    Article Snippet: However, our observations that fibroblasts lacking Stat1 express reduced p21waf1 protein and that the lack of either Stat1 or p21waf1 leads to markedly reduced apoptosis in response to 7kchol establish a connection between Stat1 and p21waf1 and also place cytochrome c release and the activation of caspase 3 downstream of the regulatory roles played by Stat1 and p21waf1 .

    Techniques: Incubation, Staining

    Blockade of ERBB Induces MHC Class I Gene Expression (A) DFTD tumor cell line T1 was treated with recombinant interferon-γ (rIFN-γ) and/or 1 μM sapitinib. Control cells were treated with solvents. Forty-eight hours after treatment, expression of B2M , SAHA-UC , STAT1 , and STAT3 were measured by real-time PCR (n = 3 replicates). (B) Reciprocal co-immunoprecipitation of STAT3 and STAT1 followed by western blots for STAT3 and STAT1 in DFTD tumor cell line (T1), fibroblasts, and human HT29 colon cancer cells as control. (C and D) Tumor volume (C) and tumor weight (D) of DFTD tumor cell line T1 transplanted into NSG mice and treated with either vehicle or 50 mg/kg sapitinib once daily (bilateral tumors, n = 5 mice per group). One out of two representative experiments is shown. (E) H E and IHC analyses for total STAT3, pS-STAT3, pY-STAT3, Ki67, and Cleaved Caspase 3 of tumor tissues. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Quantification of total STAT3, pS-STAT3, pY-STAT3, Ki67, and Cleaved Caspase 3. Scale bars, 200 and 25 μm. (F) Western blots for total STAT3, pY-STAT3, pS-STAT3, and STAT1 from representative xenograft tumors. (G) Expression of B2M and STAT3 by real-time PCR from xenograft tumor tissue. .

    Journal: Cancer Cell

    Article Title: The ERBB-STAT3 Axis Drives Tasmanian Devil Facial Tumor Disease

    doi: 10.1016/j.ccell.2018.11.018

    Figure Lengend Snippet: Blockade of ERBB Induces MHC Class I Gene Expression (A) DFTD tumor cell line T1 was treated with recombinant interferon-γ (rIFN-γ) and/or 1 μM sapitinib. Control cells were treated with solvents. Forty-eight hours after treatment, expression of B2M , SAHA-UC , STAT1 , and STAT3 were measured by real-time PCR (n = 3 replicates). (B) Reciprocal co-immunoprecipitation of STAT3 and STAT1 followed by western blots for STAT3 and STAT1 in DFTD tumor cell line (T1), fibroblasts, and human HT29 colon cancer cells as control. (C and D) Tumor volume (C) and tumor weight (D) of DFTD tumor cell line T1 transplanted into NSG mice and treated with either vehicle or 50 mg/kg sapitinib once daily (bilateral tumors, n = 5 mice per group). One out of two representative experiments is shown. (E) H E and IHC analyses for total STAT3, pS-STAT3, pY-STAT3, Ki67, and Cleaved Caspase 3 of tumor tissues. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Quantification of total STAT3, pS-STAT3, pY-STAT3, Ki67, and Cleaved Caspase 3. Scale bars, 200 and 25 μm. (F) Western blots for total STAT3, pY-STAT3, pS-STAT3, and STAT1 from representative xenograft tumors. (G) Expression of B2M and STAT3 by real-time PCR from xenograft tumor tissue. .

    Article Snippet: For immunoprecipitation, 1 mg protein lysate was incubated with 2 μg of anti-STAT3 (9139; Cell Signaling) or anti-STAT1 (9172; Cell Signaling) at 4°C overnight and immunoprecipitated with 25 μl Dynabeads Protein G (10004D; Thermo Fisher Scientific, Waltham, MA, USA) for 2 hr at 4°C.

    Techniques: Expressing, Recombinant, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Mouse Assay, Immunohistochemistry

    Xenograft Model with STAT3 Inhibitor DR-1-55 (A and B) Tumor volume (A) and tumor weight (B) of NSG mice transplanted with DFTD tumor cell line T1 and, 22 days after transplantation, treated with either vehicle or 10 mg/kg DR-1-55 each day (bilateral tumors, n = 5 mice per group). (C) Serum concentration of alanine aminotransferase, aspartate aminotransferase, and blood urea nitrogen. (D) Tumor tissue immunohistochemically stained and quantified for total STAT3, pS-STAT3, pY-STAT3, Ki67, Cleaved Caspase 3, and MMP2. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200 and 25 μm. (E) Western blots for total STAT3, pY-STAT3, pS-STAT3, and STAT1 from representative xenograft tumors. (F) Expression of B2M and STAT3 by real-time PCR from xenograft tumor tissue. .

    Journal: Cancer Cell

    Article Title: The ERBB-STAT3 Axis Drives Tasmanian Devil Facial Tumor Disease

    doi: 10.1016/j.ccell.2018.11.018

    Figure Lengend Snippet: Xenograft Model with STAT3 Inhibitor DR-1-55 (A and B) Tumor volume (A) and tumor weight (B) of NSG mice transplanted with DFTD tumor cell line T1 and, 22 days after transplantation, treated with either vehicle or 10 mg/kg DR-1-55 each day (bilateral tumors, n = 5 mice per group). (C) Serum concentration of alanine aminotransferase, aspartate aminotransferase, and blood urea nitrogen. (D) Tumor tissue immunohistochemically stained and quantified for total STAT3, pS-STAT3, pY-STAT3, Ki67, Cleaved Caspase 3, and MMP2. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200 and 25 μm. (E) Western blots for total STAT3, pY-STAT3, pS-STAT3, and STAT1 from representative xenograft tumors. (F) Expression of B2M and STAT3 by real-time PCR from xenograft tumor tissue. .

    Article Snippet: For immunoprecipitation, 1 mg protein lysate was incubated with 2 μg of anti-STAT3 (9139; Cell Signaling) or anti-STAT1 (9172; Cell Signaling) at 4°C overnight and immunoprecipitated with 25 μl Dynabeads Protein G (10004D; Thermo Fisher Scientific, Waltham, MA, USA) for 2 hr at 4°C.

    Techniques: Mouse Assay, Transplantation Assay, Concentration Assay, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction

    Working Model for the Impact of the ERBB-STAT3 Axis in DFTD (A) Aggressive social interactions in the highly inbred population of Tasmanian devils enabled the rapid spread of DFTD with fatal consequences. The hyperactive ERBB-STAT3 axis induces the expression of downstream metastasis-related genes (i.e., MMP2 ) while suppressing the expression of MHC class I genes (i.e., B2M ). We hypothesize that highly abundant phosphorylated STAT3 protein traps unphosphorylated STAT1 proteins in heterodimers, thereby preventing the transcriptional regulation of STAT1 downstream target genes such as B2M . This may contribute to immune evasion and the known lack of tumor rejection upon horizontal transmission. (B) Interference with the ERBB-STAT3 axis by using either ERBB inhibitors or STAT3 inhibitors results in killing of DFTD tumor cells.

    Journal: Cancer Cell

    Article Title: The ERBB-STAT3 Axis Drives Tasmanian Devil Facial Tumor Disease

    doi: 10.1016/j.ccell.2018.11.018

    Figure Lengend Snippet: Working Model for the Impact of the ERBB-STAT3 Axis in DFTD (A) Aggressive social interactions in the highly inbred population of Tasmanian devils enabled the rapid spread of DFTD with fatal consequences. The hyperactive ERBB-STAT3 axis induces the expression of downstream metastasis-related genes (i.e., MMP2 ) while suppressing the expression of MHC class I genes (i.e., B2M ). We hypothesize that highly abundant phosphorylated STAT3 protein traps unphosphorylated STAT1 proteins in heterodimers, thereby preventing the transcriptional regulation of STAT1 downstream target genes such as B2M . This may contribute to immune evasion and the known lack of tumor rejection upon horizontal transmission. (B) Interference with the ERBB-STAT3 axis by using either ERBB inhibitors or STAT3 inhibitors results in killing of DFTD tumor cells.

    Article Snippet: For immunoprecipitation, 1 mg protein lysate was incubated with 2 μg of anti-STAT3 (9139; Cell Signaling) or anti-STAT1 (9172; Cell Signaling) at 4°C overnight and immunoprecipitated with 25 μl Dynabeads Protein G (10004D; Thermo Fisher Scientific, Waltham, MA, USA) for 2 hr at 4°C.

    Techniques: Expressing, Transmission Assay

    Treatment with the STAT1 inhibitor fludarabine reduces renal inflammation and ameliorates glomerular lesions in Habu GN. (A) Western blots showing P-STAT1, STAT1, and MHC class II expression levels in glomeruli of Habu GN, which were treated with the STAT1 inhibitor fludarabine, on days 3 and 7; representative images and a summary of the normalized quantification are shown. (B) PAS-stained kidney tissues were examined in Habu GN treated with fludarabine on days 3 and 7. (C) Mesangiolysis index and glomerulosclerosis index in the fludarabine-treated Habu GN group compared with those in the control group on days 3 and 7. (D) CD4+ T cell infiltration was suppressed in the fludarabine-treated group compared with that in the control group on days 3 and 7. (E) Relative IFN- γ, TNF- α, IL-12A, IL-12B, IL-6, IL-17A. and IL-23A mRNA levels in the fludarabine-treated Habu GN glomeruli as determined by RT-PCR. Data are presented as the mean ± SEM. (F) Effects of fludarabine on plasma creatinine and blood urea nitrogen (BUN) levels measured in Habu GN on D3 and D7. Data are presented as the mean ± SEM ( n = 10 per group). The results are representative of three independent experiments. * P

    Journal: Frontiers in Immunology

    Article Title: Suppressor of Cytokine Signaling-1/STAT1 Regulates Renal Inflammation in Mesangial Proliferative Glomerulonephritis Models

    doi: 10.3389/fimmu.2018.01982

    Figure Lengend Snippet: Treatment with the STAT1 inhibitor fludarabine reduces renal inflammation and ameliorates glomerular lesions in Habu GN. (A) Western blots showing P-STAT1, STAT1, and MHC class II expression levels in glomeruli of Habu GN, which were treated with the STAT1 inhibitor fludarabine, on days 3 and 7; representative images and a summary of the normalized quantification are shown. (B) PAS-stained kidney tissues were examined in Habu GN treated with fludarabine on days 3 and 7. (C) Mesangiolysis index and glomerulosclerosis index in the fludarabine-treated Habu GN group compared with those in the control group on days 3 and 7. (D) CD4+ T cell infiltration was suppressed in the fludarabine-treated group compared with that in the control group on days 3 and 7. (E) Relative IFN- γ, TNF- α, IL-12A, IL-12B, IL-6, IL-17A. and IL-23A mRNA levels in the fludarabine-treated Habu GN glomeruli as determined by RT-PCR. Data are presented as the mean ± SEM. (F) Effects of fludarabine on plasma creatinine and blood urea nitrogen (BUN) levels measured in Habu GN on D3 and D7. Data are presented as the mean ± SEM ( n = 10 per group). The results are representative of three independent experiments. * P

    Article Snippet: Sections were used for immunohistochemical analysis with the following antibodies: proliferating cell nuclear antigen (PCNA; Cell Signaling Technology), anti-CD4 (Abcam), P-STAT1 (Cell Signaling Technology), and rabbit IgG polyclonal-Isotype Control (Abcam).

    Techniques: Western Blot, Expressing, Staining, Reverse Transcription Polymerase Chain Reaction

    SOCS-1 inhibits IFN-γ-induced CIITA promoter IV activity and gene expression via STAT1 regulation. (A) Quantitative RT-PCR analysis of CIITA mRNA expression in MMCs transfected with SOCS1 plasmids or treated with fludarabine for 48 h incubated in the absence or presence of IFN-γ for 48 h. (B) MMCs were cotransfected with the CIITA promoter and SOCS1 plasmid or treated with fludarabine for 48 h in the absence or presence of IFN-γ for 48 h prior to analyze for luciferase activity as indicated in the Materials and Methods. Statistical significance was determined relative to the negative control. Data are presented as the mean ± SEM ( n = 3) of at least three experiments. * P

    Journal: Frontiers in Immunology

    Article Title: Suppressor of Cytokine Signaling-1/STAT1 Regulates Renal Inflammation in Mesangial Proliferative Glomerulonephritis Models

    doi: 10.3389/fimmu.2018.01982

    Figure Lengend Snippet: SOCS-1 inhibits IFN-γ-induced CIITA promoter IV activity and gene expression via STAT1 regulation. (A) Quantitative RT-PCR analysis of CIITA mRNA expression in MMCs transfected with SOCS1 plasmids or treated with fludarabine for 48 h incubated in the absence or presence of IFN-γ for 48 h. (B) MMCs were cotransfected with the CIITA promoter and SOCS1 plasmid or treated with fludarabine for 48 h in the absence or presence of IFN-γ for 48 h prior to analyze for luciferase activity as indicated in the Materials and Methods. Statistical significance was determined relative to the negative control. Data are presented as the mean ± SEM ( n = 3) of at least three experiments. * P

    Article Snippet: Sections were used for immunohistochemical analysis with the following antibodies: proliferating cell nuclear antigen (PCNA; Cell Signaling Technology), anti-CD4 (Abcam), P-STAT1 (Cell Signaling Technology), and rabbit IgG polyclonal-Isotype Control (Abcam).

    Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Transfection, Incubation, Plasmid Preparation, Luciferase, Negative Control

    Fludarabine inhibits IFN-γ-induced MHC class II expression and chemokine production. (A) Western blot analysis of P-STAT1 and STAT1 in IFN-γ-treated MMCs in the absence or presence of fludarabine for 48 h. (B) Quantitative RT-PCR analysis of MHC class II mRNA expression in IFN-γ-stimulated MMCs in the absence or presence of fludarabine for 48 h. (C) Representative Western blots showing the levels of MHC class II in IFN-γ-stimulated mesangial cells in the absence or presence of fludarabine for 48 h. (D) Relative levels of IP-10 and Mig mRNA in IFN-γ-treated MMCs in the absence or presence of fludarabine for 48 h as determined by RT-PCR. Data are presented as the mean ± SEM ( n = 3). The results are representative of three independent experiments. * P

    Journal: Frontiers in Immunology

    Article Title: Suppressor of Cytokine Signaling-1/STAT1 Regulates Renal Inflammation in Mesangial Proliferative Glomerulonephritis Models

    doi: 10.3389/fimmu.2018.01982

    Figure Lengend Snippet: Fludarabine inhibits IFN-γ-induced MHC class II expression and chemokine production. (A) Western blot analysis of P-STAT1 and STAT1 in IFN-γ-treated MMCs in the absence or presence of fludarabine for 48 h. (B) Quantitative RT-PCR analysis of MHC class II mRNA expression in IFN-γ-stimulated MMCs in the absence or presence of fludarabine for 48 h. (C) Representative Western blots showing the levels of MHC class II in IFN-γ-stimulated mesangial cells in the absence or presence of fludarabine for 48 h. (D) Relative levels of IP-10 and Mig mRNA in IFN-γ-treated MMCs in the absence or presence of fludarabine for 48 h as determined by RT-PCR. Data are presented as the mean ± SEM ( n = 3). The results are representative of three independent experiments. * P

    Article Snippet: Sections were used for immunohistochemical analysis with the following antibodies: proliferating cell nuclear antigen (PCNA; Cell Signaling Technology), anti-CD4 (Abcam), P-STAT1 (Cell Signaling Technology), and rabbit IgG polyclonal-Isotype Control (Abcam).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    SOCS1 and STAT1 expression in MsGN models. (A) Immunofluorescence analysis was performed to determine SOCS1 expression in Thy1.1 and Habu GN glomeruli (Thy1.1: magnification × 200; Habu: magnification × 400). (B) SOCS1 protein expression in Thy1.1 and Habu GN glomeruli. Quantitative analysis of SOCS1 protein expression in glomeruli of MsGN models. (C) Quantitative RT-PCR analysis of SOCS1 mRNA expression in glomeruli of MsGN models. (D) Immunohistochemistry analysis of P-STAT1 in Thy1.1 and Habu GN kidney sections. Representative micrographs and quantification of positive cells in glomeruli are shown (original magnification, × 400). (E) Western blot analysis of P-STAT1 and STAT1 expression in Thy1.1 and Habu GN glomeruli; representative images and the summary of the normalized quantification are shown. Data are presented as the mean ± SEM ( n = 10 per group). The results are representative of three independent experiments. * P

    Journal: Frontiers in Immunology

    Article Title: Suppressor of Cytokine Signaling-1/STAT1 Regulates Renal Inflammation in Mesangial Proliferative Glomerulonephritis Models

    doi: 10.3389/fimmu.2018.01982

    Figure Lengend Snippet: SOCS1 and STAT1 expression in MsGN models. (A) Immunofluorescence analysis was performed to determine SOCS1 expression in Thy1.1 and Habu GN glomeruli (Thy1.1: magnification × 200; Habu: magnification × 400). (B) SOCS1 protein expression in Thy1.1 and Habu GN glomeruli. Quantitative analysis of SOCS1 protein expression in glomeruli of MsGN models. (C) Quantitative RT-PCR analysis of SOCS1 mRNA expression in glomeruli of MsGN models. (D) Immunohistochemistry analysis of P-STAT1 in Thy1.1 and Habu GN kidney sections. Representative micrographs and quantification of positive cells in glomeruli are shown (original magnification, × 400). (E) Western blot analysis of P-STAT1 and STAT1 expression in Thy1.1 and Habu GN glomeruli; representative images and the summary of the normalized quantification are shown. Data are presented as the mean ± SEM ( n = 10 per group). The results are representative of three independent experiments. * P

    Article Snippet: Sections were used for immunohistochemical analysis with the following antibodies: proliferating cell nuclear antigen (PCNA; Cell Signaling Technology), anti-CD4 (Abcam), P-STAT1 (Cell Signaling Technology), and rabbit IgG polyclonal-Isotype Control (Abcam).

    Techniques: Expressing, Immunofluorescence, Quantitative RT-PCR, Immunohistochemistry, Western Blot

    SOCS1 expression inhibits IFN-γ-induced MHC class II expression and chemokine production. (A) Flow cytometric analysis of MHC class II expression in IFN-γ-stimulated mesangial cells. (B) Quantitative RT-PCR analysis of MHC class II mRNA expression in IFN-γ-stimulated MMCs transfected with SOCS1 plasmids. (C) MMCs transfected with SOCS1 plasmids were incubated in the absence or presence of IFN-γ for 48 h. Representative Western blots show MHC class II levels in IFN-γ-stimulated mesangial cells. (D) Relative IP-10 and Mig mRNA levels in IFN-γ-treated MMCs transfected with SOCS1 plasmids as determined by RT-PCR. (E) Western blot analysis of P-STAT1 and STAT1 expression in SOCS1 plasmid-transfected MMCs incubated in the absence or presence of IFN-γ for 48 h; representative images and the summary of the normalized quantification are shown. Data are presented as the mean ± SEM ( n = 3). The results are representative of three independent experiments. * P

    Journal: Frontiers in Immunology

    Article Title: Suppressor of Cytokine Signaling-1/STAT1 Regulates Renal Inflammation in Mesangial Proliferative Glomerulonephritis Models

    doi: 10.3389/fimmu.2018.01982

    Figure Lengend Snippet: SOCS1 expression inhibits IFN-γ-induced MHC class II expression and chemokine production. (A) Flow cytometric analysis of MHC class II expression in IFN-γ-stimulated mesangial cells. (B) Quantitative RT-PCR analysis of MHC class II mRNA expression in IFN-γ-stimulated MMCs transfected with SOCS1 plasmids. (C) MMCs transfected with SOCS1 plasmids were incubated in the absence or presence of IFN-γ for 48 h. Representative Western blots show MHC class II levels in IFN-γ-stimulated mesangial cells. (D) Relative IP-10 and Mig mRNA levels in IFN-γ-treated MMCs transfected with SOCS1 plasmids as determined by RT-PCR. (E) Western blot analysis of P-STAT1 and STAT1 expression in SOCS1 plasmid-transfected MMCs incubated in the absence or presence of IFN-γ for 48 h; representative images and the summary of the normalized quantification are shown. Data are presented as the mean ± SEM ( n = 3). The results are representative of three independent experiments. * P

    Article Snippet: Sections were used for immunohistochemical analysis with the following antibodies: proliferating cell nuclear antigen (PCNA; Cell Signaling Technology), anti-CD4 (Abcam), P-STAT1 (Cell Signaling Technology), and rabbit IgG polyclonal-Isotype Control (Abcam).

    Techniques: Expressing, Flow Cytometry, Quantitative RT-PCR, Transfection, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

    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

    The key component of ISGF3 complex is necessary to restrict RV replication. ( A ) STAT1 knockdown by lentiviral shRNA vectors. Western blot analysis confirms a successful knockdown of total STAT1 protein. ( B ) Correspondingly, knockdown of STAT1 led to a significant increase of RV replication (n = 3 independent experiments with 3–4 replicates each). ( C ) Knockdwon of STAT2 by lentiviral shRNA vectors. Western blot analysis shows a potent decrease of total STAT2 protein level. ( D ) Similarly, silencing of STAT2 resulted in a prominent increase of RV RNA level (n = 3 independent experiments with 3–4 replicates each). ( E ) IRF9 knockdwon by lentiviral shRNA vectors. Western blot analysis shows a potent decrease of IRF9 protein level. ( F ) Knockdown of IRF9 led to a notable increase of RV replication (n = 3 independent experiments with 3–4 replicates each). Data were presented as means ± SEM., ** P

    Journal: Scientific Reports

    Article Title: Basal interferon signaling and therapeutic use of interferons in controlling rotavirus infection in human intestinal cells and organoids

    doi: 10.1038/s41598-018-26784-9

    Figure Lengend Snippet: The key component of ISGF3 complex is necessary to restrict RV replication. ( A ) STAT1 knockdown by lentiviral shRNA vectors. Western blot analysis confirms a successful knockdown of total STAT1 protein. ( B ) Correspondingly, knockdown of STAT1 led to a significant increase of RV replication (n = 3 independent experiments with 3–4 replicates each). ( C ) Knockdwon of STAT2 by lentiviral shRNA vectors. Western blot analysis shows a potent decrease of total STAT2 protein level. ( D ) Similarly, silencing of STAT2 resulted in a prominent increase of RV RNA level (n = 3 independent experiments with 3–4 replicates each). ( E ) IRF9 knockdwon by lentiviral shRNA vectors. Western blot analysis shows a potent decrease of IRF9 protein level. ( F ) Knockdown of IRF9 led to a notable increase of RV replication (n = 3 independent experiments with 3–4 replicates each). Data were presented as means ± SEM., ** P

    Article Snippet: Anti-STAT1 antibody (#9172) was purchased from Cell Signaling Technology.

    Techniques: shRNA, Western Blot

    Expression of Ifng increases in the kidneys of the Ifng GOF mouse. A. Schematic depicting the Ifng GOF mouse line. Murine Ifng is ectopically expressed in the Pax3-expressing domain after Cre-mediated excision of a LoxP-CAT-Stop cassette. B. The amount of Ifng in kidneys increases about 13 fold in mutant vs. normal kidneys, when Ifng expression is targeted to the MM. The amount of Ifng was measured by ELISA. C. Ifng expression (red) is elevated in the MM-derived areas. The UB is marked with Hoxb7/myr-venus (green). Scale bar = 20 μm. D. Ifng mRNA is elevated in kidneys from Ifng GOF mice. mRNA is measured by semi-quantitative RT-PCR. Ifngr1 and Ifngr2 are also detected in mouse embryonic kidneys. E. Stat1 protein is activated in mutant kidneys, as determined by phosphorylation of Y701 and S727 in immunoblots. As a loading control, β-Actin was used. All these experiments used E14.5 kidneys. nor—normal, mu—mutant.

    Journal: PLoS ONE

    Article Title: Constitutive metanephric mesenchyme-specific expression of interferon-gamma causes renal dysplasia by regulating Sall1 expression

    doi: 10.1371/journal.pone.0197356

    Figure Lengend Snippet: Expression of Ifng increases in the kidneys of the Ifng GOF mouse. A. Schematic depicting the Ifng GOF mouse line. Murine Ifng is ectopically expressed in the Pax3-expressing domain after Cre-mediated excision of a LoxP-CAT-Stop cassette. B. The amount of Ifng in kidneys increases about 13 fold in mutant vs. normal kidneys, when Ifng expression is targeted to the MM. The amount of Ifng was measured by ELISA. C. Ifng expression (red) is elevated in the MM-derived areas. The UB is marked with Hoxb7/myr-venus (green). Scale bar = 20 μm. D. Ifng mRNA is elevated in kidneys from Ifng GOF mice. mRNA is measured by semi-quantitative RT-PCR. Ifngr1 and Ifngr2 are also detected in mouse embryonic kidneys. E. Stat1 protein is activated in mutant kidneys, as determined by phosphorylation of Y701 and S727 in immunoblots. As a loading control, β-Actin was used. All these experiments used E14.5 kidneys. nor—normal, mu—mutant.

    Article Snippet: Proteins were detected with specific antibodies against Stat1 (Cell Signaling), phospho-Stat1 (Cell Signaling), β-Actin (Sigma) and active β-Catenin (Millipore).

    Techniques: Expressing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Derivative Assay, Mouse Assay, Quantitative RT-PCR, Western Blot

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

    Journal: EBioMedicine

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

    doi: 10.1016/j.ebiom.2018.01.002

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

    Article Snippet: Primary antibodies specific for phospho-JAK1/2 (#3771), STAT1 (#9172), phospho-STAT1 (#9171), and β-Actin (#4967) were obtained from Cell Signaling Technology (Danvers, MA).

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

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

    Journal: EBioMedicine

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

    doi: 10.1016/j.ebiom.2018.01.002

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

    Article Snippet: Primary antibodies specific for phospho-JAK1/2 (#3771), STAT1 (#9172), phospho-STAT1 (#9171), and β-Actin (#4967) were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Incubation, Activity Assay

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.01881-12

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

    Article Snippet: The primary antibodies were anti-STAT1 and anti-phospho-STAT1 (Tyr 701) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-MDA-5 antibody (Immuno-Biological Laboratories, Japan), anti-RIG-I and anti-MAVS antibodies (Enzo Life Sciences, Miami, FL), an anti-DYKDDDDK tag antibody (Wako, Japan), and an anti-β-actin antibody (Sigma-Aldrich).

    Techniques: Transfection

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.01881-12

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

    Article Snippet: The primary antibodies were anti-STAT1 and anti-phospho-STAT1 (Tyr 701) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-MDA-5 antibody (Immuno-Biological Laboratories, Japan), anti-RIG-I and anti-MAVS antibodies (Enzo Life Sciences, Miami, FL), an anti-DYKDDDDK tag antibody (Wako, Japan), and an anti-β-actin antibody (Sigma-Aldrich).

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

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.01881-12

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

    Article Snippet: The primary antibodies were anti-STAT1 and anti-phospho-STAT1 (Tyr 701) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-MDA-5 antibody (Immuno-Biological Laboratories, Japan), anti-RIG-I and anti-MAVS antibodies (Enzo Life Sciences, Miami, FL), an anti-DYKDDDDK tag antibody (Wako, Japan), and an anti-β-actin antibody (Sigma-Aldrich).

    Techniques: Multiple Displacement Amplification

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.01881-12

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

    Article Snippet: The primary antibodies were anti-STAT1 and anti-phospho-STAT1 (Tyr 701) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-MDA-5 antibody (Immuno-Biological Laboratories, Japan), anti-RIG-I and anti-MAVS antibodies (Enzo Life Sciences, Miami, FL), an anti-DYKDDDDK tag antibody (Wako, Japan), and an anti-β-actin antibody (Sigma-Aldrich).

    Techniques: Multiple Displacement Amplification, Transfection, Incubation

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.01881-12

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

    Article Snippet: The primary antibodies were anti-STAT1 and anti-phospho-STAT1 (Tyr 701) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-MDA-5 antibody (Immuno-Biological Laboratories, Japan), anti-RIG-I and anti-MAVS antibodies (Enzo Life Sciences, Miami, FL), an anti-DYKDDDDK tag antibody (Wako, Japan), and an anti-β-actin antibody (Sigma-Aldrich).

    Techniques: Transfection

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.01881-12

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

    Article Snippet: The primary antibodies were anti-STAT1 and anti-phospho-STAT1 (Tyr 701) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-MDA-5 antibody (Immuno-Biological Laboratories, Japan), anti-RIG-I and anti-MAVS antibodies (Enzo Life Sciences, Miami, FL), an anti-DYKDDDDK tag antibody (Wako, Japan), and an anti-β-actin antibody (Sigma-Aldrich).

    Techniques: Transfection, Real-time Polymerase Chain Reaction

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.01881-12

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

    Article Snippet: The primary antibodies were anti-STAT1 and anti-phospho-STAT1 (Tyr 701) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-MDA-5 antibody (Immuno-Biological Laboratories, Japan), anti-RIG-I and anti-MAVS antibodies (Enzo Life Sciences, Miami, FL), an anti-DYKDDDDK tag antibody (Wako, Japan), and an anti-β-actin antibody (Sigma-Aldrich).

    Techniques: Transfection, Plasmid Preparation

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.01881-12

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

    Article Snippet: The primary antibodies were anti-STAT1 and anti-phospho-STAT1 (Tyr 701) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-MDA-5 antibody (Immuno-Biological Laboratories, Japan), anti-RIG-I and anti-MAVS antibodies (Enzo Life Sciences, Miami, FL), an anti-DYKDDDDK tag antibody (Wako, Japan), and an anti-β-actin antibody (Sigma-Aldrich).

    Techniques:

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.01881-12

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

    Article Snippet: The primary antibodies were anti-STAT1 and anti-phospho-STAT1 (Tyr 701) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-MDA-5 antibody (Immuno-Biological Laboratories, Japan), anti-RIG-I and anti-MAVS antibodies (Enzo Life Sciences, Miami, FL), an anti-DYKDDDDK tag antibody (Wako, Japan), and an anti-β-actin antibody (Sigma-Aldrich).

    Techniques: Transfection, Expressing, Construct, Plasmid Preparation

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.01881-12

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

    Article Snippet: The primary antibodies were anti-STAT1 and anti-phospho-STAT1 (Tyr 701) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-MDA-5 antibody (Immuno-Biological Laboratories, Japan), anti-RIG-I and anti-MAVS antibodies (Enzo Life Sciences, Miami, FL), an anti-DYKDDDDK tag antibody (Wako, Japan), and an anti-β-actin antibody (Sigma-Aldrich).

    Techniques: Stable Transfection, Transfection, Plasmid Preparation

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.01097-14

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

    Article Snippet: For input control, 50 μl of the extract was mixed with 25 μl of SDS buffer and was boiled for 10 min. For immunoprecipitation of STAT1 or STAT2, 250 μl of whole-cell extract was mixed with 4 μg of an anti-STAT1 antibody (catalog no. sc-346; Santa Cruz) or anti-STAT2 (catalog no. 07-140; Upstate) antibody, respectively, and incubated on a rotating wheel at 4°C overnight.

    Techniques: De-Phosphorylation Assay, Inhibition

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.01097-14

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

    Article Snippet: For input control, 50 μl of the extract was mixed with 25 μl of SDS buffer and was boiled for 10 min. For immunoprecipitation of STAT1 or STAT2, 250 μl of whole-cell extract was mixed with 4 μg of an anti-STAT1 antibody (catalog no. sc-346; Santa Cruz) or anti-STAT2 (catalog no. 07-140; Upstate) antibody, respectively, and incubated on a rotating wheel at 4°C overnight.

    Techniques: Inhibition, De-Phosphorylation Assay

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.01097-14

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

    Article Snippet: For input control, 50 μl of the extract was mixed with 25 μl of SDS buffer and was boiled for 10 min. For immunoprecipitation of STAT1 or STAT2, 250 μl of whole-cell extract was mixed with 4 μg of an anti-STAT1 antibody (catalog no. sc-346; Santa Cruz) or anti-STAT2 (catalog no. 07-140; Upstate) antibody, respectively, and incubated on a rotating wheel at 4°C overnight.

    Techniques: Expressing

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.01097-14

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

    Article Snippet: For input control, 50 μl of the extract was mixed with 25 μl of SDS buffer and was boiled for 10 min. For immunoprecipitation of STAT1 or STAT2, 250 μl of whole-cell extract was mixed with 4 μg of an anti-STAT1 antibody (catalog no. sc-346; Santa Cruz) or anti-STAT2 (catalog no. 07-140; Upstate) antibody, respectively, and incubated on a rotating wheel at 4°C overnight.

    Techniques: Inhibition

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.01097-14

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

    Article Snippet: For input control, 50 μl of the extract was mixed with 25 μl of SDS buffer and was boiled for 10 min. For immunoprecipitation of STAT1 or STAT2, 250 μl of whole-cell extract was mixed with 4 μg of an anti-STAT1 antibody (catalog no. sc-346; Santa Cruz) or anti-STAT2 (catalog no. 07-140; Upstate) antibody, respectively, and incubated on a rotating wheel at 4°C overnight.

    Techniques: De-Phosphorylation Assay

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.01097-14

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

    Article Snippet: For input control, 50 μl of the extract was mixed with 25 μl of SDS buffer and was boiled for 10 min. For immunoprecipitation of STAT1 or STAT2, 250 μl of whole-cell extract was mixed with 4 μg of an anti-STAT1 antibody (catalog no. sc-346; Santa Cruz) or anti-STAT2 (catalog no. 07-140; Upstate) antibody, respectively, and incubated on a rotating wheel at 4°C overnight.

    Techniques: De-Phosphorylation Assay

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.01097-14

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

    Article Snippet: For input control, 50 μl of the extract was mixed with 25 μl of SDS buffer and was boiled for 10 min. For immunoprecipitation of STAT1 or STAT2, 250 μl of whole-cell extract was mixed with 4 μg of an anti-STAT1 antibody (catalog no. sc-346; Santa Cruz) or anti-STAT2 (catalog no. 07-140; Upstate) antibody, respectively, and incubated on a rotating wheel at 4°C overnight.

    Techniques: Inhibition

    STAT1 regulates Bcl-xL, Fas and Bad expression A-C . CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight, and analyzed for the expression level of genes as indicated by semi-quantitative RT-PCR. Cell surface Fas protein levels were measured by staining cells with Fas-specific mAb and analyzed by flow cytometry. Fas protein level is quantified by mean fluorescence intensity and presented in bottom of panel C. * p =0.05. D . Bcl-xL and Bad form a protein complex in sarcoma cells. Cell lysates were prepared from CMS4 cells, immunoprecipitated with Bcl-2- and Bcl-xL-specific mAbs, respectively, and then analyzed for Bcl-2, Bcl-xL and Bad protein association by Western blotting analysis.

    Journal: Cancer research

    Article Title: Unphosphorylated STAT1 promotes sarcoma development through repressing expression of Fas and Bad and conferring apoptotic resistance

    doi: 10.1158/0008-5472.CAN-12-1347

    Figure Lengend Snippet: STAT1 regulates Bcl-xL, Fas and Bad expression A-C . CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight, and analyzed for the expression level of genes as indicated by semi-quantitative RT-PCR. Cell surface Fas protein levels were measured by staining cells with Fas-specific mAb and analyzed by flow cytometry. Fas protein level is quantified by mean fluorescence intensity and presented in bottom of panel C. * p =0.05. D . Bcl-xL and Bad form a protein complex in sarcoma cells. Cell lysates were prepared from CMS4 cells, immunoprecipitated with Bcl-2- and Bcl-xL-specific mAbs, respectively, and then analyzed for Bcl-2, Bcl-xL and Bad protein association by Western blotting analysis.

    Article Snippet: The blot was probed with pSTAT1-, STAT1-, Bcl-2-, Bcl-xL-, Bad- and Jak1-specific antibodies (BD biosciences).

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry, Fluorescence, Immunoprecipitation, Western Blot

    uSTAT1 represses Jak1 expression to inhibit IFN-γ-induced STAT1 phosphorylation A . Disruption of IRF8 function results in diminished pSTAT1 and Jak1 level in sarcoma cells. The indicated 4 cell sublines were cultured in the presence and absence of IFN-γ for 24 h and analyzed for pSTAT1 and Jak1 protein levels by Western blotting analysis. B . Disruption of IRF8 function results in diminished pSTAT1 activity in sarcoma cells. CMS4 cells were cultured in the presence or absence of IFN-γ for 4 h. Nuclear extracts were prepared from the cells and incubated with a GAS element-containing DNA probe. The protein-DNA interactions were then analyzed by EMSA. C . STAT1 is a repressor of Jak1. CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight and analyzed for STAT1 and Jak1 expression level by semi-quantitative RT-PCR. D . IFN-γ-induced STAT1 activation (pSTAT1) and expression (uSTAT1) kinetics in sarcoma cells. CMS4 cells were cultured in the presence of IFN-γ for the indicated time and analyzed for pSTAT1 and total STAT1 protein level by Western blotting analysis.

    Journal: Cancer research

    Article Title: Unphosphorylated STAT1 promotes sarcoma development through repressing expression of Fas and Bad and conferring apoptotic resistance

    doi: 10.1158/0008-5472.CAN-12-1347

    Figure Lengend Snippet: uSTAT1 represses Jak1 expression to inhibit IFN-γ-induced STAT1 phosphorylation A . Disruption of IRF8 function results in diminished pSTAT1 and Jak1 level in sarcoma cells. The indicated 4 cell sublines were cultured in the presence and absence of IFN-γ for 24 h and analyzed for pSTAT1 and Jak1 protein levels by Western blotting analysis. B . Disruption of IRF8 function results in diminished pSTAT1 activity in sarcoma cells. CMS4 cells were cultured in the presence or absence of IFN-γ for 4 h. Nuclear extracts were prepared from the cells and incubated with a GAS element-containing DNA probe. The protein-DNA interactions were then analyzed by EMSA. C . STAT1 is a repressor of Jak1. CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight and analyzed for STAT1 and Jak1 expression level by semi-quantitative RT-PCR. D . IFN-γ-induced STAT1 activation (pSTAT1) and expression (uSTAT1) kinetics in sarcoma cells. CMS4 cells were cultured in the presence of IFN-γ for the indicated time and analyzed for pSTAT1 and total STAT1 protein level by Western blotting analysis.

    Article Snippet: The blot was probed with pSTAT1-, STAT1-, Bcl-2-, Bcl-xL-, Bad- and Jak1-specific antibodies (BD biosciences).

    Techniques: Expressing, Cell Culture, Western Blot, Activity Assay, Incubation, Transfection, Quantitative RT-PCR, Activation Assay

    STAT1 immunohistochemistry on human sarcoma Human STS TMA slides were stained with STAT1 mAb as described in the Materials and Methods. Shown are examples of low (A, C, E, G I: 40X) and high (B, D J: 200X; F H: 400x) power STAT1 staining of strong (A,B) and weak (C,D) cytoplasmic staining, strong (E,F) and weak (G,H) nuclear staining, and negative (I,J) staiing in tumor cells. Infiltrating T-cell lymphocytes serve as a positive internal control (highlighted in panel I J) and there identity was confirmed using CD3 immunohistochemistry.

    Journal: Cancer research

    Article Title: Unphosphorylated STAT1 promotes sarcoma development through repressing expression of Fas and Bad and conferring apoptotic resistance

    doi: 10.1158/0008-5472.CAN-12-1347

    Figure Lengend Snippet: STAT1 immunohistochemistry on human sarcoma Human STS TMA slides were stained with STAT1 mAb as described in the Materials and Methods. Shown are examples of low (A, C, E, G I: 40X) and high (B, D J: 200X; F H: 400x) power STAT1 staining of strong (A,B) and weak (C,D) cytoplasmic staining, strong (E,F) and weak (G,H) nuclear staining, and negative (I,J) staiing in tumor cells. Infiltrating T-cell lymphocytes serve as a positive internal control (highlighted in panel I J) and there identity was confirmed using CD3 immunohistochemistry.

    Article Snippet: The blot was probed with pSTAT1-, STAT1-, Bcl-2-, Bcl-xL-, Bad- and Jak1-specific antibodies (BD biosciences).

    Techniques: Immunohistochemistry, Staining

    Correlation analysis between STAT1 protein level in tumor cells and disease-specific survival A. Kaplan-Meier survival curve for disease-specific survival (DSS) between nSTAT1-positive and negative tumors. STS specimens from 123 STS patients were stained with STAT1-specific mAb and examined for nSTAT1 protein in the tumor cells. The nSTAT1-positive and negative groups were then analyzed for correlation with DSS. B . Kaplan-Meier survival curve for DSS and correlation with cSTAT1 protein level in the tumor cells. STS specimens were analyzed as in A and cSTAT1 high protein level group was compared to the cSTAT1 low protein level group for correlation with DSS. C . The IRF8 promoter DNA is hypermethylated in human STS. Genomic DNA was isolated from dissected human STS cells and treated with bisulfite. The modified DNA was then analyzed with MS-PCR primers specific for the human IRF8 promoter. U: unmethylation; M: methylation.

    Journal: Cancer research

    Article Title: Unphosphorylated STAT1 promotes sarcoma development through repressing expression of Fas and Bad and conferring apoptotic resistance

    doi: 10.1158/0008-5472.CAN-12-1347

    Figure Lengend Snippet: Correlation analysis between STAT1 protein level in tumor cells and disease-specific survival A. Kaplan-Meier survival curve for disease-specific survival (DSS) between nSTAT1-positive and negative tumors. STS specimens from 123 STS patients were stained with STAT1-specific mAb and examined for nSTAT1 protein in the tumor cells. The nSTAT1-positive and negative groups were then analyzed for correlation with DSS. B . Kaplan-Meier survival curve for DSS and correlation with cSTAT1 protein level in the tumor cells. STS specimens were analyzed as in A and cSTAT1 high protein level group was compared to the cSTAT1 low protein level group for correlation with DSS. C . The IRF8 promoter DNA is hypermethylated in human STS. Genomic DNA was isolated from dissected human STS cells and treated with bisulfite. The modified DNA was then analyzed with MS-PCR primers specific for the human IRF8 promoter. U: unmethylation; M: methylation.

    Article Snippet: The blot was probed with pSTAT1-, STAT1-, Bcl-2-, Bcl-xL-, Bad- and Jak1-specific antibodies (BD biosciences).

    Techniques: Staining, Isolation, Modification, Mass Spectrometry, Polymerase Chain Reaction, Methylation

    Silencing STAT1 expression increases sarcoma cell sensitivity to Fas-mediated apoptosis in vitro A . CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight, and analyzed for STAT1 expression level by semi-quantitative RT-PCR (left panel) and real-time RT-PCR (right panel). B . The STAT1 siRNA-transfected cells were incubated in the absence or presence of FasL (250 ng/ml) overnight and then stained with PI and Annexin V. The stained cells were analyzed with flow cytometry. The number in each plot indicated the PI and Annexin V double positive cells. Apoptotic cell death was quantified by the formula: percentage of PI + and Annexin V + cells in the presence of FasL - percentage of PI + and Annexin V + cells without FasL treatment, and presented at the right panel.

    Journal: Cancer research

    Article Title: Unphosphorylated STAT1 promotes sarcoma development through repressing expression of Fas and Bad and conferring apoptotic resistance

    doi: 10.1158/0008-5472.CAN-12-1347

    Figure Lengend Snippet: Silencing STAT1 expression increases sarcoma cell sensitivity to Fas-mediated apoptosis in vitro A . CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight, and analyzed for STAT1 expression level by semi-quantitative RT-PCR (left panel) and real-time RT-PCR (right panel). B . The STAT1 siRNA-transfected cells were incubated in the absence or presence of FasL (250 ng/ml) overnight and then stained with PI and Annexin V. The stained cells were analyzed with flow cytometry. The number in each plot indicated the PI and Annexin V double positive cells. Apoptotic cell death was quantified by the formula: percentage of PI + and Annexin V + cells in the presence of FasL - percentage of PI + and Annexin V + cells without FasL treatment, and presented at the right panel.

    Article Snippet: The blot was probed with pSTAT1-, STAT1-, Bcl-2-, Bcl-xL-, Bad- and Jak1-specific antibodies (BD biosciences).

    Techniques: Expressing, In Vitro, Transfection, Quantitative RT-PCR, Incubation, Staining, Flow Cytometry, Cytometry

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

    Journal: Cancers

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

    doi: 10.3390/cancers12092450

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

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

    Techniques: Generated, Expressing, Quantitative RT-PCR

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

    Journal: Cancers

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

    doi: 10.3390/cancers12092450

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

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

    Techniques: Inhibition, Cell Culture, Activation Assay

    DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

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

    Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

    doi: 10.1073/pnas.052159099

    Figure Lengend Snippet: DN forms of CaMKII inhibit Stat1 S727 phosphorylation and IFN-γ-induced transcription activation. ( A ) NIH 3T3 cells were transfected with the indicated mutant CaMKIIs and selected in G418-containing medium for 6 days. G418-resistant cells were pooled and treated with 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. CMV, RcCMV vector; ( A ) a fragment containing the association region of CaMKII; DN, CaMKII containing a K43M mutation. ( B ) U3A cells were transfected with Stat1 and the WT or DN CaMKII. Twenty-four hours after transfection, cells were treated with 5 ng/ml human IFN-γ for 3 h, and total RNA was analyzed by reverse transcription–PCR with [ 32 P]dCTP and primer pairs for the indicated genes. ( C ) The gels of B were quantitated and analyzed by a PhosphorImager. The relative intensities were ratios of IRF-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

    Article Snippet: The specific interaction between Stat1 and CaMKII also suggests the possibility that CaMKII may phosphorylate sites in chromatin to participate in chromatin remodeling near the Stat1 binding sites, which may occur in addition to the acetylation of histones by CBP/p300, also recruited by Stat1 ( , ).

    Techniques: Activation Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Polymerase Chain Reaction

    Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

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

    Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

    doi: 10.1073/pnas.052159099

    Figure Lengend Snippet: Requirement of Ca 2+ and CaMKII for IFN-γ-induced phosphorylation of Stat1 S727 and gene activation. ( A ) Whole-cell extracts were prepared from NIH 3T3 cells with the indicated treatment and analyzed by Western blotting. Pretreatments with DMSO (D) or 50 μM BAPTA-AM (B) were for 30 min followed by 200 units/ml mouse IFN-γ for the length of time indicated. PS, phospho-S727; PY, phospho-Y701. ( B ) Total RNAs were prepared from NIH 3T3 cells with the indicated treatment as described for A and analyzed by Northern blotting. ( C ) NIH 3T3 cells were pretreated with the indicated reagents for 30 min followed by 200 units/ml mouse IFN-γ for 30 min, and whole-cell extracts were analyzed by Western blotting. 93 , KN93 (5 μM); BIS, bisindolylmaleimide 1 (20 μM); SB, SB203580 (10 μM); P-PKC, activated phospho-PKC.

    Article Snippet: The specific interaction between Stat1 and CaMKII also suggests the possibility that CaMKII may phosphorylate sites in chromatin to participate in chromatin remodeling near the Stat1 binding sites, which may occur in addition to the acetylation of histones by CBP/p300, also recruited by Stat1 ( , ).

    Techniques: Activation Assay, Western Blot, Northern Blot

    CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

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

    Article Title: Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-?

    doi: 10.1073/pnas.052159099

    Figure Lengend Snippet: CaMKII phosphorylates Stat1 S727 in vitro . ( A Upper ) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of 32 P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. ( Lower ) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. ( B ) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of 32 P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. ( C ) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of 32 P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.

    Article Snippet: The specific interaction between Stat1 and CaMKII also suggests the possibility that CaMKII may phosphorylate sites in chromatin to participate in chromatin remodeling near the Stat1 binding sites, which may occur in addition to the acetylation of histones by CBP/p300, also recruited by Stat1 ( , ).

    Techniques: In Vitro, Purification, Western Blot, Affinity Column, Chick Chorioallantoic Membrane Assay, Mutagenesis, Autoradiography, SDS Page

    Replication of ICP0 - and ICP4 - viruses in cell culture and immunodeficient mice . Vero cells were A . untreated or B . treated with 200 U per ml of IFN-β and were inoculated with 2.5 pfu per cell of wild-type HSV-1 (KOS), an ICP0 - virus (0 - -GFP), or an ICP4 - virus (n12). The mean ± sem of the logarithm of viral titers recovered from Vero cells is plotted over time (n = 4 per time point). C . Rag2 -/- stat1 -/- mice and D . rag2 -/- mice were inoculated with 2 × 10 5 pfu per eye of the ICP0 - virus 0 - -GFP or the ICP4 - virus n12 (n = 4 mice per group). The mean ± sem of the logarithm of viral titers recovered from mouse eyes is plotted over time (open black symbols). Dashed lines indicate the lower limit of detection of each plaque assay. The survival of 0 - -GFP-infected mice and ICP4 - virus-infected mice is plotted over time (open red symbols).

    Journal: Virology Journal

    Article Title: ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency

    doi: 10.1186/1743-422X-3-44

    Figure Lengend Snippet: Replication of ICP0 - and ICP4 - viruses in cell culture and immunodeficient mice . Vero cells were A . untreated or B . treated with 200 U per ml of IFN-β and were inoculated with 2.5 pfu per cell of wild-type HSV-1 (KOS), an ICP0 - virus (0 - -GFP), or an ICP4 - virus (n12). The mean ± sem of the logarithm of viral titers recovered from Vero cells is plotted over time (n = 4 per time point). C . Rag2 -/- stat1 -/- mice and D . rag2 -/- mice were inoculated with 2 × 10 5 pfu per eye of the ICP0 - virus 0 - -GFP or the ICP4 - virus n12 (n = 4 mice per group). The mean ± sem of the logarithm of viral titers recovered from mouse eyes is plotted over time (open black symbols). Dashed lines indicate the lower limit of detection of each plaque assay. The survival of 0 - -GFP-infected mice and ICP4 - virus-infected mice is plotted over time (open red symbols).

    Article Snippet: Strain 129 mice, rag2 -/- mice, stat1 -/- mice, and rag2 -/- stat1 -/- mice were obtained from Taconic Farms (Germantown, NY).

    Techniques: Cell Culture, Mouse Assay, Plaque Assay, Infection

    Measurement of KOS and 0 - -GFP viral genome loads in the trigeminal ganglia of HSV-1 latently infected mice . A . Dotblot of HSV-1 VP16 PCR products. Each

    Journal: Virology Journal

    Article Title: ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency

    doi: 10.1186/1743-422X-3-44

    Figure Lengend Snippet: Measurement of KOS and 0 - -GFP viral genome loads in the trigeminal ganglia of HSV-1 latently infected mice . A . Dotblot of HSV-1 VP16 PCR products. Each "dot" contains VP16 PCR product amplified from the TG DNA of a single mouse, and the n-values indicate numbers of mice per group. TG harvested from uninfected (UI) mice served as negative controls for the PCR. TG harvested from mice dying of encephalitis (Day 9 p.i.) belonged to one of the following groups: rag2 -/- ifnar -/- mice, ifnar -/- ifngr -/- mice, or stat1 -/- mice inoculated with 2 × 10 4 pfu per eye of 0 - -GFP. TG harvested from mice that were latently infected with HSV-1 (Day 40 p.i.) belonged to one of the following groups: strain 129 mice inoculated with 2 × 10 5 pfu per eye of KOS; strain 129 mice, ifngr -/- mice, or ifnar -/- mice inoculated with 2 × 10 5 pfu per eye of 0 - -GFP; or stat1 -/- mice inoculated with 2 × 10 4 pfu per eye of 0 - -GFP. The standard curve on the right consists of PCR products amplified from a two-fold dilution series of VP16 plasmid DNA. B . The ratio of yields of VP16 to competitor PCR product yields (competitor dotblot not shown) was used to estimate viral genome copy number per PCR. The logarithm of viral genomes per TG , y, was plotted as a function of the mean logarithm of the ratio of VP16 PCR product yield: competitor PCR product yield , x, amplified from duplicate PCRs of each dilution of VP16 plasmid (error bars indicate the standard deviation between duplicate PCRs). The relationship between viral genome load and PCR product yields was described by the equation, y = 0.2556•x 3 + 0.1055•x 2 + 1.2079•x + 5.9309 (r 2 = 0.99). The number of HSV-1 genomes per TG in each sample was derived from fitting the data shown in panel A to the standard curve shown in panel B. C . Number of HSV-1 genomes per TG in mice that were uninfected or were latently infected with KOS or 0 - -GFP. The dashed line indicates the lower limit of detection of the PCR assay. Asterisks denote significant differences in viral genome load per TG relative to strain 129 mice latently infected with KOS (p

    Article Snippet: Strain 129 mice, rag2 -/- mice, stat1 -/- mice, and rag2 -/- stat1 -/- mice were obtained from Taconic Farms (Germantown, NY).

    Techniques: Infection, Mouse Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Standard Deviation, Derivative Assay

    Loss of IFN-α/β receptors or Stat 1 impairs an innate host response that represses KOS-GFP and 0 - -GFP at the site of inoculation . Mice were inoculated with 2 × 10 5 pfu per eye of A . HSV-1 strain KOS-GFP, or B . the ICP0 - virus, 0 - -GFP. GFP fluorescence was recorded in the right eyes of strain 129 mice, rag2 -/- mice (lymphocyte-deficient), ifngr -/- mice (IFN-γ receptor-null), ifnar -/- mice (IFN-α/β receptor-null), ifnar -/- ifngr -/- mice, stat1 -/- mice, rag2 -/- stat1 -/- mice, and rag2 -/- ifnar -/- mice. Representative photographs are shown of GFP fluorescence in the virus-infected eye of one mouse per group photographed over time at 36, 60, and 84 hours p.i. (4× magnification; 39 ms exposure for KOS-GFP; 63 ms exposure for 0 - -GFP).

    Journal: Virology Journal

    Article Title: ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency

    doi: 10.1186/1743-422X-3-44

    Figure Lengend Snippet: Loss of IFN-α/β receptors or Stat 1 impairs an innate host response that represses KOS-GFP and 0 - -GFP at the site of inoculation . Mice were inoculated with 2 × 10 5 pfu per eye of A . HSV-1 strain KOS-GFP, or B . the ICP0 - virus, 0 - -GFP. GFP fluorescence was recorded in the right eyes of strain 129 mice, rag2 -/- mice (lymphocyte-deficient), ifngr -/- mice (IFN-γ receptor-null), ifnar -/- mice (IFN-α/β receptor-null), ifnar -/- ifngr -/- mice, stat1 -/- mice, rag2 -/- stat1 -/- mice, and rag2 -/- ifnar -/- mice. Representative photographs are shown of GFP fluorescence in the virus-infected eye of one mouse per group photographed over time at 36, 60, and 84 hours p.i. (4× magnification; 39 ms exposure for KOS-GFP; 63 ms exposure for 0 - -GFP).

    Article Snippet: Strain 129 mice, rag2 -/- mice, stat1 -/- mice, and rag2 -/- stat1 -/- mice were obtained from Taconic Farms (Germantown, NY).

    Techniques: Mouse Assay, Fluorescence, Infection, Mass Spectrometry

    Loss of Stat 1 alleviates innate host repression of ICP0 - viruses in vivo . A . Strain 129 mice, rag2 -/- mice, PML -/- mice, or stat1 -/- mice were inoculated with 2 × 10 5 pfu per eye of the ICP0 - virus n212 (n = 4 mice per group). The mean ± sem of the logarithm of viral titers recovered from mouse eyes is plotted over time. B . Strain 129 mice, rag2 -/- mice, stat1 -/- mice, or rag2 -/- stat1 -/- mice were inoculated with 2 × 10 5 pfu per eye of the ICP0 - virus, 0 - -GFP (n = 4 mice per group). Dashed lines indicate the lower limit of detection of the plaque assay. C . Survival of strain 129 mice, rag2 -/- mice, PML -/- mice, stat1 -/- mice, or rag2 -/- stat1 -/- mice infected with the ICP0 - viruses, n212 or 0 - -GFP. Bars represent the mean ± sem of survival frequency of ICP0 - virus-infected mice at day 60 p.i. (n = 3 experiments; Σn = 14 mice per group).

    Journal: Virology Journal

    Article Title: ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency

    doi: 10.1186/1743-422X-3-44

    Figure Lengend Snippet: Loss of Stat 1 alleviates innate host repression of ICP0 - viruses in vivo . A . Strain 129 mice, rag2 -/- mice, PML -/- mice, or stat1 -/- mice were inoculated with 2 × 10 5 pfu per eye of the ICP0 - virus n212 (n = 4 mice per group). The mean ± sem of the logarithm of viral titers recovered from mouse eyes is plotted over time. B . Strain 129 mice, rag2 -/- mice, stat1 -/- mice, or rag2 -/- stat1 -/- mice were inoculated with 2 × 10 5 pfu per eye of the ICP0 - virus, 0 - -GFP (n = 4 mice per group). Dashed lines indicate the lower limit of detection of the plaque assay. C . Survival of strain 129 mice, rag2 -/- mice, PML -/- mice, stat1 -/- mice, or rag2 -/- stat1 -/- mice infected with the ICP0 - viruses, n212 or 0 - -GFP. Bars represent the mean ± sem of survival frequency of ICP0 - virus-infected mice at day 60 p.i. (n = 3 experiments; Σn = 14 mice per group).

    Article Snippet: Strain 129 mice, rag2 -/- mice, stat1 -/- mice, and rag2 -/- stat1 -/- mice were obtained from Taconic Farms (Germantown, NY).

    Techniques: In Vivo, Mouse Assay, Plaque Assay, Infection

    A Stat1-dependent host response restricts the spread of HSV-1 strain KOS-GFP into the central nervous system . Strain 129 mice, rag2 -/- mice, stat1 -/- mice, or rag2 -/- stat1 -/- mice were inoculated with 2 × 10 5 pfu per eye of HSV-1 strain KOS-GFP. A . The mean ± sem of the logarithm of viral titers recovered from homogenates of mouse eyes, TG, and hindbrain is plotted as a function of the time p.i. at which tissues were harvested (n = 5 per time point). Asterisks denote significant differences between stat1 +/+ versus stat1 -/- tissues (p

    Journal: Virology Journal

    Article Title: ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency

    doi: 10.1186/1743-422X-3-44

    Figure Lengend Snippet: A Stat1-dependent host response restricts the spread of HSV-1 strain KOS-GFP into the central nervous system . Strain 129 mice, rag2 -/- mice, stat1 -/- mice, or rag2 -/- stat1 -/- mice were inoculated with 2 × 10 5 pfu per eye of HSV-1 strain KOS-GFP. A . The mean ± sem of the logarithm of viral titers recovered from homogenates of mouse eyes, TG, and hindbrain is plotted as a function of the time p.i. at which tissues were harvested (n = 5 per time point). Asterisks denote significant differences between stat1 +/+ versus stat1 -/- tissues (p

    Article Snippet: Strain 129 mice, rag2 -/- mice, stat1 -/- mice, and rag2 -/- stat1 -/- mice were obtained from Taconic Farms (Germantown, NY).

    Techniques: Mouse Assay

    Effects of 4AAQB on the LPS-induced activation of MAP kinases, IkBα, NFκB p65 and STAT1 in RAW 264.7 macrophages and peritoneal macrophages. RAW264.7 cells were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Cells were harvested and total cell extracts were prepared. a Phosphorylated-ERK, phosphorylated-JNK, phosphorylated-p38, or b Phosphorylated-IκBα and NFκB p65 subunit and c Phosphorylated-STAT1 were detected by Western blot analysis. Total ERK, JNK, p38 and α-tubulin were used as internal standard. d Peritoneal macrophages were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Phosphorylated-ERK and total ERK were detected by Western blot analysis

    Journal: BMC Complementary and Alternative Medicine

    Article Title: 4-Acetylantroquinonol B inhibits lipopolysaccharide-induced cytokine release and alleviates sepsis through of MAPK and NFκB suppression

    doi: 10.1186/s12906-018-2172-2

    Figure Lengend Snippet: Effects of 4AAQB on the LPS-induced activation of MAP kinases, IkBα, NFκB p65 and STAT1 in RAW 264.7 macrophages and peritoneal macrophages. RAW264.7 cells were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Cells were harvested and total cell extracts were prepared. a Phosphorylated-ERK, phosphorylated-JNK, phosphorylated-p38, or b Phosphorylated-IκBα and NFκB p65 subunit and c Phosphorylated-STAT1 were detected by Western blot analysis. Total ERK, JNK, p38 and α-tubulin were used as internal standard. d Peritoneal macrophages were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Phosphorylated-ERK and total ERK were detected by Western blot analysis

    Article Snippet: Antibodies against phosphorylated IκBα, phosphorylated STAT1 were purchased from Cell Signaling Technologies (Boston, MA, USA).

    Techniques: Activation Assay, Concentration Assay, Western Blot

    Co-Immunoprecipitation and ubiquitination of P-STAT1 in CLP Cells were lysed and cleared by centrifugation. Aliquots were precleared by addition of normal IgG and 20 µl of appropriate agarose conjugate. After centrifugation, the supernatant was collected and incubated with 2 µg of P-Stat1 Ab. After washing, immunoblotting was performed using Ub AB. Blot is representative of four experiments.

    Journal: Surgery

    Article Title: Osteopontin Mediates Stat1 Degradation To Inhibit iNOS Transcription In A Cecal Ligation and Puncture Model of Sepsis

    doi: 10.1016/j.surg.2008.03.007

    Figure Lengend Snippet: Co-Immunoprecipitation and ubiquitination of P-STAT1 in CLP Cells were lysed and cleared by centrifugation. Aliquots were precleared by addition of normal IgG and 20 µl of appropriate agarose conjugate. After centrifugation, the supernatant was collected and incubated with 2 µg of P-Stat1 Ab. After washing, immunoblotting was performed using Ub AB. Blot is representative of four experiments.

    Article Snippet: Using BMM and liver tissue, western blot analysis was performed to determine iNOS, PSTAT1 and OPN expression in CLP in WT and OPN null animals at 0h, 12h and 24h. ( ) WT expression of iNOS and P-STAT1 in BMM and liver is unchanged or decreased from 12h to 24h in association with a 8–10 fold increase in cellular OPN. (p < 0.05; 24h vs. 12h for BMM and liver) In contrast, OPN null expression of iNOS and P-STAT1 is significantly greater in BMM and liver at 24h when compared to that found at 12h. (p < 0.01; 24h vs. 12h for both iNOS and P-STAT1 in BMM and liver) These results show that iNOS and P-STAT1 are significantly increased in the absence of OPN in the CLP model of sepsis.

    Techniques: Immunoprecipitation, Centrifugation, Incubation

    IP studies for Ub-Stat1 and iNOS in ex vivo treated BMM from OPN null and WT animals Ex vivo gain-of-function studies were performed using BMM from WT and OPN null mice. MG132 (10 µM), an inhibitor of 26S proteasome function, and/or exogenous OPN (50 µM) were added to assess Ub-P-STAT1 and iNOS expression. BMM cells were treated with LPS (50 ng/ml). The incubation period was 12 hours. Cells were lysed and cleared by centrifugation. Aliquots were precleared by addition of normal IgG and 20 µl of appropriate agarose conjugate. After centrifugation, the supernatant was collected and incubated with 2 µg of P-Stat1 Ab. After washing, immunoblotting was performed using Ub AB. Alternatively, IB was performed for iNOS. Blot is representative of four experiments.

    Journal: Surgery

    Article Title: Osteopontin Mediates Stat1 Degradation To Inhibit iNOS Transcription In A Cecal Ligation and Puncture Model of Sepsis

    doi: 10.1016/j.surg.2008.03.007

    Figure Lengend Snippet: IP studies for Ub-Stat1 and iNOS in ex vivo treated BMM from OPN null and WT animals Ex vivo gain-of-function studies were performed using BMM from WT and OPN null mice. MG132 (10 µM), an inhibitor of 26S proteasome function, and/or exogenous OPN (50 µM) were added to assess Ub-P-STAT1 and iNOS expression. BMM cells were treated with LPS (50 ng/ml). The incubation period was 12 hours. Cells were lysed and cleared by centrifugation. Aliquots were precleared by addition of normal IgG and 20 µl of appropriate agarose conjugate. After centrifugation, the supernatant was collected and incubated with 2 µg of P-Stat1 Ab. After washing, immunoblotting was performed using Ub AB. Alternatively, IB was performed for iNOS. Blot is representative of four experiments.

    Article Snippet: Using BMM and liver tissue, western blot analysis was performed to determine iNOS, PSTAT1 and OPN expression in CLP in WT and OPN null animals at 0h, 12h and 24h. ( ) WT expression of iNOS and P-STAT1 in BMM and liver is unchanged or decreased from 12h to 24h in association with a 8–10 fold increase in cellular OPN. (p < 0.05; 24h vs. 12h for BMM and liver) In contrast, OPN null expression of iNOS and P-STAT1 is significantly greater in BMM and liver at 24h when compared to that found at 12h. (p < 0.01; 24h vs. 12h for both iNOS and P-STAT1 in BMM and liver) These results show that iNOS and P-STAT1 are significantly increased in the absence of OPN in the CLP model of sepsis.

    Techniques: Ex Vivo, Mouse Assay, Expressing, Incubation, Centrifugation

    Western blot of iNOS, phosphorylated STAT1 (P-STAT1) and osteopontin (OPN) expression in CLP in WT and OPN null animals at 0h, 12h and 24h . Blot is presentative of four experiments.

    Journal: Surgery

    Article Title: Osteopontin Mediates Stat1 Degradation To Inhibit iNOS Transcription In A Cecal Ligation and Puncture Model of Sepsis

    doi: 10.1016/j.surg.2008.03.007

    Figure Lengend Snippet: Western blot of iNOS, phosphorylated STAT1 (P-STAT1) and osteopontin (OPN) expression in CLP in WT and OPN null animals at 0h, 12h and 24h . Blot is presentative of four experiments.

    Article Snippet: Using BMM and liver tissue, western blot analysis was performed to determine iNOS, PSTAT1 and OPN expression in CLP in WT and OPN null animals at 0h, 12h and 24h. ( ) WT expression of iNOS and P-STAT1 in BMM and liver is unchanged or decreased from 12h to 24h in association with a 8–10 fold increase in cellular OPN. (p < 0.05; 24h vs. 12h for BMM and liver) In contrast, OPN null expression of iNOS and P-STAT1 is significantly greater in BMM and liver at 24h when compared to that found at 12h. (p < 0.01; 24h vs. 12h for both iNOS and P-STAT1 in BMM and liver) These results show that iNOS and P-STAT1 are significantly increased in the absence of OPN in the CLP model of sepsis.

    Techniques: Western Blot, Expressing

    Time response of JAK/STAT pathway activation in RT4-D6P2T after BDNF administration. BDNF (100 pM) was administered to treat rat Schwann cells RT4-D6P2T at 0 min, 10 min, 30 min, 60 min, 120 min and 24 hr. STAT3/STAT1 were activated at 10 min after the treatment with BDNF (lane 2), and the phosphorylation level peaked at 30 min (lane 3) (*P

    Journal: Translational Andrology and Urology

    Article Title: Brain-derived neurotrophic factor promotes nerve regeneration by activating the JAK/STAT pathway in Schwann cells

    doi: 10.21037/tau.2016.02.03

    Figure Lengend Snippet: Time response of JAK/STAT pathway activation in RT4-D6P2T after BDNF administration. BDNF (100 pM) was administered to treat rat Schwann cells RT4-D6P2T at 0 min, 10 min, 30 min, 60 min, 120 min and 24 hr. STAT3/STAT1 were activated at 10 min after the treatment with BDNF (lane 2), and the phosphorylation level peaked at 30 min (lane 3) (*P

    Article Snippet: The primary antibodies used in these experiments were anti-JAK2 (1:500), anti-phospho-JAK2 (1:200) (Chemicon, Inc., Temecula, CA, USA), anti-STAT1 (1:500), anti-phospho-STAT1 (1:500), anti-STAT3 (1:500), and anti-phospho-STAT3 (1:500) (BD Biosciences, CA, USA).

    Techniques: Activation Assay

    BDNF activates JAK/STAT directly. The HSC and RT4-D6P2T cells were validated by immunofluorescence staining. (A) The cellular markers of Schwann cells, S100 and p75, were expressed in cells in the cytoplasm represented as green (FTIC) and nucleus as blue (DAPI) (×100); (B,C) different doses of BDNF were applied to treat BE( 2 )-C, SH-SY5Y and RT4-D6P2T (0, 1, 10, 100 pM) for 30 minutes. The phosphorylation of STAT3/STAT1 was extensively enhanced by BDNF at a dose of (100 pM) in RT4-D6P2T cells (*P

    Journal: Translational Andrology and Urology

    Article Title: Brain-derived neurotrophic factor promotes nerve regeneration by activating the JAK/STAT pathway in Schwann cells

    doi: 10.21037/tau.2016.02.03

    Figure Lengend Snippet: BDNF activates JAK/STAT directly. The HSC and RT4-D6P2T cells were validated by immunofluorescence staining. (A) The cellular markers of Schwann cells, S100 and p75, were expressed in cells in the cytoplasm represented as green (FTIC) and nucleus as blue (DAPI) (×100); (B,C) different doses of BDNF were applied to treat BE( 2 )-C, SH-SY5Y and RT4-D6P2T (0, 1, 10, 100 pM) for 30 minutes. The phosphorylation of STAT3/STAT1 was extensively enhanced by BDNF at a dose of (100 pM) in RT4-D6P2T cells (*P

    Article Snippet: The primary antibodies used in these experiments were anti-JAK2 (1:500), anti-phospho-JAK2 (1:200) (Chemicon, Inc., Temecula, CA, USA), anti-STAT1 (1:500), anti-phospho-STAT1 (1:500), anti-STAT3 (1:500), and anti-phospho-STAT3 (1:500) (BD Biosciences, CA, USA).

    Techniques: Immunofluorescence, Staining

    Time response of JAK/STAT pathway activation in HSCs after BDNF treatment. BDNF (100 pM) was administered to treat the human Schwann cells at 0 min, 10 min, 30 min, 60 min, 120 min, 24 hr and 72 hr. STAT3/STAT1 were activated at 10 min after the treatment with BDNF (lane 2), and the phosphorylation level peaked at 60 min (lane 4) (*P

    Journal: Translational Andrology and Urology

    Article Title: Brain-derived neurotrophic factor promotes nerve regeneration by activating the JAK/STAT pathway in Schwann cells

    doi: 10.21037/tau.2016.02.03

    Figure Lengend Snippet: Time response of JAK/STAT pathway activation in HSCs after BDNF treatment. BDNF (100 pM) was administered to treat the human Schwann cells at 0 min, 10 min, 30 min, 60 min, 120 min, 24 hr and 72 hr. STAT3/STAT1 were activated at 10 min after the treatment with BDNF (lane 2), and the phosphorylation level peaked at 60 min (lane 4) (*P

    Article Snippet: The primary antibodies used in these experiments were anti-JAK2 (1:500), anti-phospho-JAK2 (1:200) (Chemicon, Inc., Temecula, CA, USA), anti-STAT1 (1:500), anti-phospho-STAT1 (1:500), anti-STAT3 (1:500), and anti-phospho-STAT3 (1:500) (BD Biosciences, CA, USA).

    Techniques: Activation Assay

    Mechanism of JAK/STAT re-activation by BDNF in HSCs. BDNF (100 pM) was administered to treat the human Schwann cell HSCs for 0 min, 10 min, 30 min, 60 min, 120 min, 24 hr, 48 hr, and 72 hr. (A) JAK2 was activated by BDNF treatment and possessed two phosphorylation peaks, which was similar to the response of STAT3/STAT1; (B) the phosphorylation levels of JAK2, STAT1 and STAT3 were presented as a ratio of phosphorylated form to total expressed forms. P1: phosphorylation early peak. P2: phosphorylation late peak. The cell culture mediums from the above treatment were applied to assay the level of cytokines secreted from HSCs by ELISA; (C) 1 hr after the BDNF treatment, HSCs began to secrete OSM-M at levels reaching ~20±0.8 pg/mL at 24 hr ( # P > 0.05); (D) BDNF significantly increased the secretion of IL6 from HSCs after 1 hr of the treatment and reached ~360.9±74 pg/mL at 72 hr (*P

    Journal: Translational Andrology and Urology

    Article Title: Brain-derived neurotrophic factor promotes nerve regeneration by activating the JAK/STAT pathway in Schwann cells

    doi: 10.21037/tau.2016.02.03

    Figure Lengend Snippet: Mechanism of JAK/STAT re-activation by BDNF in HSCs. BDNF (100 pM) was administered to treat the human Schwann cell HSCs for 0 min, 10 min, 30 min, 60 min, 120 min, 24 hr, 48 hr, and 72 hr. (A) JAK2 was activated by BDNF treatment and possessed two phosphorylation peaks, which was similar to the response of STAT3/STAT1; (B) the phosphorylation levels of JAK2, STAT1 and STAT3 were presented as a ratio of phosphorylated form to total expressed forms. P1: phosphorylation early peak. P2: phosphorylation late peak. The cell culture mediums from the above treatment were applied to assay the level of cytokines secreted from HSCs by ELISA; (C) 1 hr after the BDNF treatment, HSCs began to secrete OSM-M at levels reaching ~20±0.8 pg/mL at 24 hr ( # P > 0.05); (D) BDNF significantly increased the secretion of IL6 from HSCs after 1 hr of the treatment and reached ~360.9±74 pg/mL at 72 hr (*P

    Article Snippet: The primary antibodies used in these experiments were anti-JAK2 (1:500), anti-phospho-JAK2 (1:200) (Chemicon, Inc., Temecula, CA, USA), anti-STAT1 (1:500), anti-phospho-STAT1 (1:500), anti-STAT3 (1:500), and anti-phospho-STAT3 (1:500) (BD Biosciences, CA, USA).

    Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    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

    Journal: Cancer Science

    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

    doi: 10.1111/cas.13964

    Figure Lengend 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

    Article Snippet: The following antibodies and dilutions were used: mouse anti‐beta‐catenin (1:10000, Transduction), rabbit anti‐STAT1 (1:300, Cell Signaling), rabbit anti‐Lysozyme (1:2000, Dako), goat anti‐EphB2 (1:2000, R & D), rabbit anti‐Cleaved Caspase3 (Asp175) (1:200, Cell Signaling) and rabbit anti‐SOX9 (1:1000, Millipore, Burlington, MA, USA).

    Techniques: 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

    Journal: Cancer Science

    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

    doi: 10.1111/cas.13964

    Figure Lengend 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

    Article Snippet: The following antibodies and dilutions were used: mouse anti‐beta‐catenin (1:10000, Transduction), rabbit anti‐STAT1 (1:300, Cell Signaling), rabbit anti‐Lysozyme (1:2000, Dako), goat anti‐EphB2 (1:2000, R & D), rabbit anti‐Cleaved Caspase3 (Asp175) (1:200, Cell Signaling) and rabbit anti‐SOX9 (1:1000, Millipore, Burlington, MA, USA).

    Techniques: Mouse Assay, Generated, Immunohistochemistry