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  • 95
    Thermo Fisher stat1
    Expression of <t>STAT1</t> in THP-1 cells after infection with different adenoviruses. (A) The expression of total (pan-STAT1) and phosphorylated STAT1 in THP-1 cells infected with Ad5, dl 309, of dl 312 or mock infected. Total protein from THP-1 cells was extracted
    Stat1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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 1490 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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    93
    Becton Dickinson stat1
    <t>STAT1</t> P696S is associated with low levels of STAT1 in the patients’ cells. EBV-B cells from H, C +/+ , P1, P2, an individual with a single WT STAT1 allele encoding a protein (C +/– ), and a patient homozygous for a mutated STAT1 allele, resulting in an absence of the protein (C –/– ), were stimulated with 10 5 IU/ml IFN-α or IFN-γ or were left unstimulated (NSt) for 30 minutes and subjected to FACS analysis ( A and C ) or Western blotting ( B ), with specific antibodies against P-Tyr701-STAT1 (P-T-STAT1) ( B and C ), the STAT1 N terminus (STAT1 N-ter) ( A and B ) and C terminus (STAT1 C-ter) ( B ), P-Tyr690-STAT2 (P-T-STAT2), and STAT2 ( B ). The results shown are representative of 2 or 3 independent experiments.
    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> P696S is associated with low levels of STAT1 in the patients’ cells. EBV-B cells from H, C +/+ , P1, P2, an individual with a single WT STAT1 allele encoding a protein (C +/– ), and a patient homozygous for a mutated STAT1 allele, resulting in an absence of the protein (C –/– ), were stimulated with 10 5 IU/ml IFN-α or IFN-γ or were left unstimulated (NSt) for 30 minutes and subjected to FACS analysis ( A and C ) or Western blotting ( B ), with specific antibodies against P-Tyr701-STAT1 (P-T-STAT1) ( B and C ), the STAT1 N terminus (STAT1 N-ter) ( A and B ) and C terminus (STAT1 C-ter) ( B ), P-Tyr690-STAT2 (P-T-STAT2), and STAT2 ( B ). The results shown are representative of 2 or 3 independent experiments.
    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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    99
    Cell Signaling Technology Inc phospho stat1 tyr701
    <t>STAT1</t> P696S is associated with low levels of STAT1 in the patients’ cells. EBV-B cells from H, C +/+ , P1, P2, an individual with a single WT STAT1 allele encoding a protein (C +/– ), and a patient homozygous for a mutated STAT1 allele, resulting in an absence of the protein (C –/– ), were stimulated with 10 5 IU/ml IFN-α or IFN-γ or were left unstimulated (NSt) for 30 minutes and subjected to FACS analysis ( A and C ) or Western blotting ( B ), with specific antibodies against P-Tyr701-STAT1 (P-T-STAT1) ( B and C ), the STAT1 N terminus (STAT1 N-ter) ( A and B ) and C terminus (STAT1 C-ter) ( B ), P-Tyr690-STAT2 (P-T-STAT2), and STAT2 ( B ). The results shown are representative of 2 or 3 independent experiments.
    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
    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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    92
    Becton Dickinson anti stat1
    Normal Activation of <t>STAT1</t> Mutants in Stable Transfectants (A) Western blot of total protein extracts (100 μg) from a parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele, with antibodies specific for phosphorylated-Tyr-701-STAT1, STAT1, and STAT3. The cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNA or IFNG. (B) Immunofluorescence staining, with a STAT1-specific antibody, of STAT1-deficient U3C fibrosarcoma cell clones, stably cotransfected with a zeocin-resistance vector and a vector containing a WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. For (A) and (B), one experiment representative of three independent experiments is shown.
    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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    99
    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


    Expression of STAT1 in THP-1 cells after infection with different adenoviruses. (A) The expression of total (pan-STAT1) and phosphorylated STAT1 in THP-1 cells infected with Ad5, dl 309, of dl 312 or mock infected. Total protein from THP-1 cells was extracted

    Journal: Journal of Virology

    Article Title: STAT1 Interaction with E3-14.7K in Monocytes Affects the Efficacy of Oncolytic Adenovirus

    doi: 10.1128/JVI.02829-13

    Figure Lengend Snippet: Expression of STAT1 in THP-1 cells after infection with different adenoviruses. (A) The expression of total (pan-STAT1) and phosphorylated STAT1 in THP-1 cells infected with Ad5, dl 309, of dl 312 or mock infected. Total protein from THP-1 cells was extracted

    Article Snippet: The cells were then incubated with the secondary antibodies, Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen) (for STAT1) and Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen) (for the E3B antibodies), diluted 1:1,000 in PBS, for 1 h at room temperature.

    Techniques: Expressing, Infection

    Expression of STAT 1 protein in H460 cells primed with 100 ng/ml IFN-γ after adenovirus infection. (A) The expression of total and phosphorylated STAT1 in human lung carcinoma H460 cells primed with 100 ng/ml IFN-γ for 24 h prior to infection

    Journal: Journal of Virology

    Article Title: STAT1 Interaction with E3-14.7K in Monocytes Affects the Efficacy of Oncolytic Adenovirus

    doi: 10.1128/JVI.02829-13

    Figure Lengend Snippet: Expression of STAT 1 protein in H460 cells primed with 100 ng/ml IFN-γ after adenovirus infection. (A) The expression of total and phosphorylated STAT1 in human lung carcinoma H460 cells primed with 100 ng/ml IFN-γ for 24 h prior to infection

    Article Snippet: The cells were then incubated with the secondary antibodies, Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen) (for STAT1) and Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen) (for the E3B antibodies), diluted 1:1,000 in PBS, for 1 h at room temperature.

    Techniques: Expressing, Infection

    Effects of STAT1 inhibitor (fludarabine) on chemokine expression, macrophage infiltration, and antitumor efficacy by E3B-deleted adenovirus. (A) Expression of CCL-5 and CXCL-10 was assayed by ELISA on RAW 264.7 cells untreated or pretreated with 10 μM

    Journal: Journal of Virology

    Article Title: STAT1 Interaction with E3-14.7K in Monocytes Affects the Efficacy of Oncolytic Adenovirus

    doi: 10.1128/JVI.02829-13

    Figure Lengend Snippet: Effects of STAT1 inhibitor (fludarabine) on chemokine expression, macrophage infiltration, and antitumor efficacy by E3B-deleted adenovirus. (A) Expression of CCL-5 and CXCL-10 was assayed by ELISA on RAW 264.7 cells untreated or pretreated with 10 μM

    Article Snippet: The cells were then incubated with the secondary antibodies, Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen) (for STAT1) and Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen) (for the E3B antibodies), diluted 1:1,000 in PBS, for 1 h at room temperature.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay

    Identification of interaction of E3B-14.7K with STAT1. (A) STAT1 protein expression in whole-cell extracts from E3B-14.7K, RID, or empty vector stably transfected cell lines primed with IFN-γ for 24 h and subsequently infected with Ad5, dl 309,

    Journal: Journal of Virology

    Article Title: STAT1 Interaction with E3-14.7K in Monocytes Affects the Efficacy of Oncolytic Adenovirus

    doi: 10.1128/JVI.02829-13

    Figure Lengend Snippet: Identification of interaction of E3B-14.7K with STAT1. (A) STAT1 protein expression in whole-cell extracts from E3B-14.7K, RID, or empty vector stably transfected cell lines primed with IFN-γ for 24 h and subsequently infected with Ad5, dl 309,

    Article Snippet: The cells were then incubated with the secondary antibodies, Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen) (for STAT1) and Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen) (for the E3B antibodies), diluted 1:1,000 in PBS, for 1 h at room temperature.

    Techniques: Expressing, Plasmid Preparation, Stable Transfection, Transfection, Infection

    Expression of STAT1, adenovirus E3B-14.7K protein, and chemokines in primary human monocytes and the effects of fludarabine on chemokine expression. (A) Confocal images showing STAT1 and E3B-14.7K protein distribution in human primary monocytes 48 h after

    Journal: Journal of Virology

    Article Title: STAT1 Interaction with E3-14.7K in Monocytes Affects the Efficacy of Oncolytic Adenovirus

    doi: 10.1128/JVI.02829-13

    Figure Lengend Snippet: Expression of STAT1, adenovirus E3B-14.7K protein, and chemokines in primary human monocytes and the effects of fludarabine on chemokine expression. (A) Confocal images showing STAT1 and E3B-14.7K protein distribution in human primary monocytes 48 h after

    Article Snippet: The cells were then incubated with the secondary antibodies, Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen) (for STAT1) and Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen) (for the E3B antibodies), diluted 1:1,000 in PBS, for 1 h at room temperature.

    Techniques: Expressing

    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

    BRISC-SHMT2 interaction is important for interferon signaling a b) Immunoprecipitation (IP) was performed using anti-Flag antibody in 293T cells transiently transfected with Flag-HA epitope-tagged Abraxas2 wild-type (WT) or mutants. Immunoblotting was performed for the indicated proteins. c) Abraxas2 –/– MEFs and Abraxas2 –/– MEFs stably reconstituted with WT or mutants were infected with Herpes Simplex Virus (HSV) lacking the lytic phase gene ICP0. IFN signalling was assessed by immunoblotting for STAT1 phosphorylated at Y701 (pSTAT1). d) WT MEFs overexpressing Abraxas2 WT or E144R were challenged with LPS and IFNβ. IFN signaling was assessed as in c . Data in a - d are representative of three independent experiments. For gel source data, see Supplementary Figure 1 . e) Volcano plots illustrating the fold change in gene expression of IFN type I-stimulated genes relative to Abraxas2 –/– KO MEFs without LPS treatment. p-values were calculated using a Student’s t-test (two-tail distribution and equal variances between the two samples) on the triplicate 2^(-ΔCT) values for each gene in each treatment group compared to the control group. Genes below the p=0.05 threshold were upregulated in WT+LPS, but represented as below statistical significance due to the relative expression in KO samples being almost zero. Regulated genes are highlighted in Extended Data Fig. 9a .

    Journal: Nature

    Article Title: Metabolic control of BRISC-SHMT2 assembly regulates immune signaling

    doi: 10.1038/s41586-019-1232-1

    Figure Lengend Snippet: BRISC-SHMT2 interaction is important for interferon signaling a b) Immunoprecipitation (IP) was performed using anti-Flag antibody in 293T cells transiently transfected with Flag-HA epitope-tagged Abraxas2 wild-type (WT) or mutants. Immunoblotting was performed for the indicated proteins. c) Abraxas2 –/– MEFs and Abraxas2 –/– MEFs stably reconstituted with WT or mutants were infected with Herpes Simplex Virus (HSV) lacking the lytic phase gene ICP0. IFN signalling was assessed by immunoblotting for STAT1 phosphorylated at Y701 (pSTAT1). d) WT MEFs overexpressing Abraxas2 WT or E144R were challenged with LPS and IFNβ. IFN signaling was assessed as in c . Data in a - d are representative of three independent experiments. For gel source data, see Supplementary Figure 1 . e) Volcano plots illustrating the fold change in gene expression of IFN type I-stimulated genes relative to Abraxas2 –/– KO MEFs without LPS treatment. p-values were calculated using a Student’s t-test (two-tail distribution and equal variances between the two samples) on the triplicate 2^(-ΔCT) values for each gene in each treatment group compared to the control group. Genes below the p=0.05 threshold were upregulated in WT+LPS, but represented as below statistical significance due to the relative expression in KO samples being almost zero. Regulated genes are highlighted in Extended Data Fig. 9a .

    Article Snippet: Phospho-STAT1 and STAT1 antibodies were purchased from Cell Signaling Corporation and used according to the manufacturer’s specifications.

    Techniques: Immunoprecipitation, Transfection, Stable Transfection, Infection, Expressing

    Purification and analysis of SHMT2 mutants a) Elution profile of the indicated SHMT2ΔN forms from an S75 10/300 size exclusion chromatography column (single experiment). b) Coomassie-stained SDS-PAGE analysis of the indicated SHMT2ΔN protein preparations (data are representative of two independent experiments). c) BRISC DUB activity against a fluorogenic K63-linked diUb substrate in the presence of the indicated SHMT2ΔN mutants. Data are average +/-SEM of three independent experiments carried out in duplicate. d) BRISC DUB activity against K63-linked hexa-Ub chains in the presence of the SHMT2ΔN or SHMT1 forms. e) Ubiquitylation levels of IFNAR1 after IFNα stimulation in 293T cells overexpressing the indicated Abraxas2 and SHMT2ΔN forms (LL- > RR = L211R+L215R). IFNAR1 immunoprecipitation (IP) was performed under denaturing conditions and ubiquitin levels were detected using the vu-1 antibody. Mock IP was performed using a generic rabbit IgG antibody. f) Immunoprecipitation (IP) performed using anti-Flag antibody in MEFs transiently transfected with Flag-HA epitope-tagged SHMT2ΔN or mutants. Immunoblot was performed for Abraxas2 and SHMT2 as indicated. UTF = untransfected cells used as control. g) MEFs overexpressing the indicated SHMT2ΔN or mutants were challenged with LPS and interferon receptor-dependent signal transduction response was assessed by immunoblot for STAT1 phosphorylated at Y701 (pSTAT1). Data shown in d - e are representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: Metabolic control of BRISC-SHMT2 assembly regulates immune signaling

    doi: 10.1038/s41586-019-1232-1

    Figure Lengend Snippet: Purification and analysis of SHMT2 mutants a) Elution profile of the indicated SHMT2ΔN forms from an S75 10/300 size exclusion chromatography column (single experiment). b) Coomassie-stained SDS-PAGE analysis of the indicated SHMT2ΔN protein preparations (data are representative of two independent experiments). c) BRISC DUB activity against a fluorogenic K63-linked diUb substrate in the presence of the indicated SHMT2ΔN mutants. Data are average +/-SEM of three independent experiments carried out in duplicate. d) BRISC DUB activity against K63-linked hexa-Ub chains in the presence of the SHMT2ΔN or SHMT1 forms. e) Ubiquitylation levels of IFNAR1 after IFNα stimulation in 293T cells overexpressing the indicated Abraxas2 and SHMT2ΔN forms (LL- > RR = L211R+L215R). IFNAR1 immunoprecipitation (IP) was performed under denaturing conditions and ubiquitin levels were detected using the vu-1 antibody. Mock IP was performed using a generic rabbit IgG antibody. f) Immunoprecipitation (IP) performed using anti-Flag antibody in MEFs transiently transfected with Flag-HA epitope-tagged SHMT2ΔN or mutants. Immunoblot was performed for Abraxas2 and SHMT2 as indicated. UTF = untransfected cells used as control. g) MEFs overexpressing the indicated SHMT2ΔN or mutants were challenged with LPS and interferon receptor-dependent signal transduction response was assessed by immunoblot for STAT1 phosphorylated at Y701 (pSTAT1). Data shown in d - e are representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Article Snippet: Phospho-STAT1 and STAT1 antibodies were purchased from Cell Signaling Corporation and used according to the manufacturer’s specifications.

    Techniques: Purification, Size-exclusion Chromatography, Staining, SDS Page, Activity Assay, Immunoprecipitation, Transfection, Transduction

    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 P696S is associated with low levels of STAT1 in the patients’ cells. EBV-B cells from H, C +/+ , P1, P2, an individual with a single WT STAT1 allele encoding a protein (C +/– ), and a patient homozygous for a mutated STAT1 allele, resulting in an absence of the protein (C –/– ), were stimulated with 10 5 IU/ml IFN-α or IFN-γ or were left unstimulated (NSt) for 30 minutes and subjected to FACS analysis ( A and C ) or Western blotting ( B ), with specific antibodies against P-Tyr701-STAT1 (P-T-STAT1) ( B and C ), the STAT1 N terminus (STAT1 N-ter) ( A and B ) and C terminus (STAT1 C-ter) ( B ), P-Tyr690-STAT2 (P-T-STAT2), and STAT2 ( B ). The results shown are representative of 2 or 3 independent experiments.

    Journal: The Journal of Clinical Investigation

    Article Title: A partial form of recessive STAT1 deficiency in humans

    doi: 10.1172/JCI37083

    Figure Lengend Snippet: STAT1 P696S is associated with low levels of STAT1 in the patients’ cells. EBV-B cells from H, C +/+ , P1, P2, an individual with a single WT STAT1 allele encoding a protein (C +/– ), and a patient homozygous for a mutated STAT1 allele, resulting in an absence of the protein (C –/– ), were stimulated with 10 5 IU/ml IFN-α or IFN-γ or were left unstimulated (NSt) for 30 minutes and subjected to FACS analysis ( A and C ) or Western blotting ( B ), with specific antibodies against P-Tyr701-STAT1 (P-T-STAT1) ( B and C ), the STAT1 N terminus (STAT1 N-ter) ( A and B ) and C terminus (STAT1 C-ter) ( B ), P-Tyr690-STAT2 (P-T-STAT2), and STAT2 ( B ). The results shown are representative of 2 or 3 independent experiments.

    Article Snippet: They were then washed and incubated for 1 hour at 4°C with antibodies against P-Tyr701-STAT1 (catalog no. 612132; BD Biosciences — Transduction Laboratories) or STAT1 (catalog no. 610116; BD Biosciences — Transduction Laboratories) or with the corresponding isotypic antibodies.

    Techniques: FACS, Western Blot

    STAT1 P696S is associated with a partial recessive defect in late responses to IFN-α and IFN-γ. ( A ) Abundance of mRNA corresponding to genes induced by IFN-α and/or IFN-γ ( MX1 , ISG15 , and IRF1 ) in EBV-B cells from C +/+ , P1, P2, and C –/– , either not stimulated or stimulated with 10 5 IU/ml IFN-α or IFN-γ for 2 hours. The results are normalized with respect to GUS mRNA and are expressed as multiples (fold induction) of the unstimulated value ± SD. * P

    Journal: The Journal of Clinical Investigation

    Article Title: A partial form of recessive STAT1 deficiency in humans

    doi: 10.1172/JCI37083

    Figure Lengend Snippet: STAT1 P696S is associated with a partial recessive defect in late responses to IFN-α and IFN-γ. ( A ) Abundance of mRNA corresponding to genes induced by IFN-α and/or IFN-γ ( MX1 , ISG15 , and IRF1 ) in EBV-B cells from C +/+ , P1, P2, and C –/– , either not stimulated or stimulated with 10 5 IU/ml IFN-α or IFN-γ for 2 hours. The results are normalized with respect to GUS mRNA and are expressed as multiples (fold induction) of the unstimulated value ± SD. * P

    Article Snippet: They were then washed and incubated for 1 hour at 4°C with antibodies against P-Tyr701-STAT1 (catalog no. 612132; BD Biosciences — Transduction Laboratories) or STAT1 (catalog no. 610116; BD Biosciences — Transduction Laboratories) or with the corresponding isotypic antibodies.

    Techniques:

    STAT1 P696S amino acid substitution does not affect STAT1 activation. ( A and B ) Immunoprecipitation with a V5-specific (S) or isotypic (I) antibody from total protein extracts (1 mg) of parental fibrosarcoma WT cell line (2C4) or STAT1-deficient U3C fibrosarcoma cells transfected with a V5-tagged vector containing a mock allele, the WTα, or P696SαA (P696S) STAT1 alleles. ( A ) Total protein extracts and ( B ) immunoprecipitates were studied by Western blotting with STAT1- and STAT2-specific antibodies. Cells were not stimulated or were stimulated for 30 minutes with 10 5 IU/ml IFN-α. ( C and D ) Immunoprecipitation with a V5-specific antibody from total protein extracts (1 mg) of STAT1-deficient U3C fibrosarcoma cells cotransfected with vectors containing a mock allele, WTα, and P696SαA STAT1 alleles tagged with V5 or with c-myc. ( C ) Total protein extracts and ( D ) immunoprecipitates were analyzed by Western blotting with specific antibodies against phosphorylated-Tyr701 STAT1, V5-specific, and c-myc–specific antibodies. The cells were not stimulated (data not shown) or stimulated for 30 minutes with 10 5 IU/ml IFN-α. For C and D , each blot was obtained from a different gel. Each result shown is representative of 2 independent experiments ( A – D ).

    Journal: The Journal of Clinical Investigation

    Article Title: A partial form of recessive STAT1 deficiency in humans

    doi: 10.1172/JCI37083

    Figure Lengend Snippet: STAT1 P696S amino acid substitution does not affect STAT1 activation. ( A and B ) Immunoprecipitation with a V5-specific (S) or isotypic (I) antibody from total protein extracts (1 mg) of parental fibrosarcoma WT cell line (2C4) or STAT1-deficient U3C fibrosarcoma cells transfected with a V5-tagged vector containing a mock allele, the WTα, or P696SαA (P696S) STAT1 alleles. ( A ) Total protein extracts and ( B ) immunoprecipitates were studied by Western blotting with STAT1- and STAT2-specific antibodies. Cells were not stimulated or were stimulated for 30 minutes with 10 5 IU/ml IFN-α. ( C and D ) Immunoprecipitation with a V5-specific antibody from total protein extracts (1 mg) of STAT1-deficient U3C fibrosarcoma cells cotransfected with vectors containing a mock allele, WTα, and P696SαA STAT1 alleles tagged with V5 or with c-myc. ( C ) Total protein extracts and ( D ) immunoprecipitates were analyzed by Western blotting with specific antibodies against phosphorylated-Tyr701 STAT1, V5-specific, and c-myc–specific antibodies. The cells were not stimulated (data not shown) or stimulated for 30 minutes with 10 5 IU/ml IFN-α. For C and D , each blot was obtained from a different gel. Each result shown is representative of 2 independent experiments ( A – D ).

    Article Snippet: They were then washed and incubated for 1 hour at 4°C with antibodies against P-Tyr701-STAT1 (catalog no. 612132; BD Biosciences — Transduction Laboratories) or STAT1 (catalog no. 610116; BD Biosciences — Transduction Laboratories) or with the corresponding isotypic antibodies.

    Techniques: Activation Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot

    Whole-blood assay on patients’ cells shows a defect of response to IFN-γ. Cytokine concentrations in the whole-blood supernatant from our cohort of healthy controls (C +/+ ), patients with complete IL-12B deficiency (cIL-12B), patients with complete IL-12Rβ1 deficiency (cIL-12Rβ1), patients with complete IFN-γR1 deficiency (cIFN-γR1), patients with partial IFN-γR1 deficiency (pIFN-γR1), patients with complete IFN-γR2 deficiency (cIFN-γR2), patients with complete STAT1 deficiency (cSTAT1), patients with partial dominant STAT1 deficiency (pdSTAT1), P1 and P2, and H when not stimulated or stimulated for 48 hours with live BCG alone or supplemented with IL-12 or IFN-γ. The concentrations of IFN-γ and IL-12p70 (pg/ml) in the supernatant were determined by ELISA. Each individual is represented by an open circle, and the median is represented by a thick horizontal bar.

    Journal: The Journal of Clinical Investigation

    Article Title: A partial form of recessive STAT1 deficiency in humans

    doi: 10.1172/JCI37083

    Figure Lengend Snippet: Whole-blood assay on patients’ cells shows a defect of response to IFN-γ. Cytokine concentrations in the whole-blood supernatant from our cohort of healthy controls (C +/+ ), patients with complete IL-12B deficiency (cIL-12B), patients with complete IL-12Rβ1 deficiency (cIL-12Rβ1), patients with complete IFN-γR1 deficiency (cIFN-γR1), patients with partial IFN-γR1 deficiency (pIFN-γR1), patients with complete IFN-γR2 deficiency (cIFN-γR2), patients with complete STAT1 deficiency (cSTAT1), patients with partial dominant STAT1 deficiency (pdSTAT1), P1 and P2, and H when not stimulated or stimulated for 48 hours with live BCG alone or supplemented with IL-12 or IFN-γ. The concentrations of IFN-γ and IL-12p70 (pg/ml) in the supernatant were determined by ELISA. Each individual is represented by an open circle, and the median is represented by a thick horizontal bar.

    Article Snippet: They were then washed and incubated for 1 hour at 4°C with antibodies against P-Tyr701-STAT1 (catalog no. 612132; BD Biosciences — Transduction Laboratories) or STAT1 (catalog no. 610116; BD Biosciences — Transduction Laboratories) or with the corresponding isotypic antibodies.

    Techniques: Whole Blood Assay, Enzyme-linked Immunosorbent Assay

    STAT1 P696S is associated with a predominant splicing defect. ( A ) PCR of full-length STAT1A, STAT1B, and ACTIN amplified from cDNA from C +/+ , P1 (homozygous for the P696S STAT1 mutation), and H. The sizes of the amplicons are indicated. This result is representative of 5 independent experiments. ( B ) Schematic representation of the splicing events identified in STAT1A and STAT1B WT or P696S mutant. The ends of the WT and P696S STAT1 mRNAs are shown, with their corresponding spliced forms. The exons are numbered with Roman numerals and represented in gray boxes, with the introns between them shown in white boxes, with the exception of exon 23, shown in red and purple boxes in the α and β isoforms, respectively. The hatched bars at the beginning of each sequence represent the 5′ region of each mRNA. At the end of the STAT1A form, the hatched red bars represent the STAT1A polyadenylation site at the end of exon 25. At the end of STAT1B forms, the hatched purple bars represent the STAT1B polyadenylation site at the end of exon 23, which is longer than STAT1A exon 23. The open brackets represent the splicing events. Events shown in red predominate and are associated with the P696S STAT1 mutation.

    Journal: The Journal of Clinical Investigation

    Article Title: A partial form of recessive STAT1 deficiency in humans

    doi: 10.1172/JCI37083

    Figure Lengend Snippet: STAT1 P696S is associated with a predominant splicing defect. ( A ) PCR of full-length STAT1A, STAT1B, and ACTIN amplified from cDNA from C +/+ , P1 (homozygous for the P696S STAT1 mutation), and H. The sizes of the amplicons are indicated. This result is representative of 5 independent experiments. ( B ) Schematic representation of the splicing events identified in STAT1A and STAT1B WT or P696S mutant. The ends of the WT and P696S STAT1 mRNAs are shown, with their corresponding spliced forms. The exons are numbered with Roman numerals and represented in gray boxes, with the introns between them shown in white boxes, with the exception of exon 23, shown in red and purple boxes in the α and β isoforms, respectively. The hatched bars at the beginning of each sequence represent the 5′ region of each mRNA. At the end of the STAT1A form, the hatched red bars represent the STAT1A polyadenylation site at the end of exon 25. At the end of STAT1B forms, the hatched purple bars represent the STAT1B polyadenylation site at the end of exon 23, which is longer than STAT1A exon 23. The open brackets represent the splicing events. Events shown in red predominate and are associated with the P696S STAT1 mutation.

    Article Snippet: They were then washed and incubated for 1 hour at 4°C with antibodies against P-Tyr701-STAT1 (catalog no. 612132; BD Biosciences — Transduction Laboratories) or STAT1 (catalog no. 610116; BD Biosciences — Transduction Laboratories) or with the corresponding isotypic antibodies.

    Techniques: Polymerase Chain Reaction, Amplification, Mutagenesis, Sequencing

    STAT1 P696S would be associated with an ESE defect. ( A ). Nucleotides from exon 23 are shown, with the C2086 nucleotide in the WT sequence and the C2086T substitution in the P696S sequence shown in red. The horizontal blue and green bars show the significance threshold homology score for the binding of SC35 and SRp40 proteins, respectively. The predicted binding sites of these proteins are shown as rectangles along the length of the nucleotide sequence at the height of the homology score. ( B ) The genomic STAT1 region from nucleotide 36989 to 38523 (NC_000002) was inserted into an exon-trapping vector using Xho I and BamH 1, with or without the C2086T (P696S) nucleotide substitution. The exons are numbered in Roman numerals and shown in gray boxes, with the introns between them in white boxes, with the exception of exon 23, which is shown in a red box. The vector is shown as black boxes. HEK293 and COS-7 cells were transfected with the various constructs, the exon-trapping pSPL3 mock vector (pmock-p), or no vector (–). RNA was isolated, and the various spliced products were amplified and are shown on an agarose gel with GADPH amplification. The various products were isolated and sequenced, and the resulting sequences are also shown with corresponding exons and MW. These results are representative of 2 independent experiments.

    Journal: The Journal of Clinical Investigation

    Article Title: A partial form of recessive STAT1 deficiency in humans

    doi: 10.1172/JCI37083

    Figure Lengend Snippet: STAT1 P696S would be associated with an ESE defect. ( A ). Nucleotides from exon 23 are shown, with the C2086 nucleotide in the WT sequence and the C2086T substitution in the P696S sequence shown in red. The horizontal blue and green bars show the significance threshold homology score for the binding of SC35 and SRp40 proteins, respectively. The predicted binding sites of these proteins are shown as rectangles along the length of the nucleotide sequence at the height of the homology score. ( B ) The genomic STAT1 region from nucleotide 36989 to 38523 (NC_000002) was inserted into an exon-trapping vector using Xho I and BamH 1, with or without the C2086T (P696S) nucleotide substitution. The exons are numbered in Roman numerals and shown in gray boxes, with the introns between them in white boxes, with the exception of exon 23, which is shown in a red box. The vector is shown as black boxes. HEK293 and COS-7 cells were transfected with the various constructs, the exon-trapping pSPL3 mock vector (pmock-p), or no vector (–). RNA was isolated, and the various spliced products were amplified and are shown on an agarose gel with GADPH amplification. The various products were isolated and sequenced, and the resulting sequences are also shown with corresponding exons and MW. These results are representative of 2 independent experiments.

    Article Snippet: They were then washed and incubated for 1 hour at 4°C with antibodies against P-Tyr701-STAT1 (catalog no. 612132; BD Biosciences — Transduction Laboratories) or STAT1 (catalog no. 610116; BD Biosciences — Transduction Laboratories) or with the corresponding isotypic antibodies.

    Techniques: Sequencing, Binding Assay, Plasmid Preparation, Transfection, Construct, Isolation, Amplification, Agarose Gel Electrophoresis

    Only the normal splicing STAT1 P696S form is translated. Human cells completely lacking STAT1 (U3C cells) were transfected with the WTα, P696SαA, and P696SαB STAT1 alleles or with the V5-tagged vector containing a mock allele (pmock-V5) or were not transfected (–). ( A ) These cells were subjected to Western blotting analysis with specific STAT1 and STAT2 antibodies; ( B ) subjected to full-length PCR amplification of the STAT1A isoform and ACTIN cDNAs; and ( C ) subjected to relative real-time STAT1 PCR. The results were normalized with respect to GUS mRNA and are expressed as a percentage of the amount of WT STAT1 mRNA. SD from triplication of a single experiment is indicated. These results are representative of 2 independent experiments.

    Journal: The Journal of Clinical Investigation

    Article Title: A partial form of recessive STAT1 deficiency in humans

    doi: 10.1172/JCI37083

    Figure Lengend Snippet: Only the normal splicing STAT1 P696S form is translated. Human cells completely lacking STAT1 (U3C cells) were transfected with the WTα, P696SαA, and P696SαB STAT1 alleles or with the V5-tagged vector containing a mock allele (pmock-V5) or were not transfected (–). ( A ) These cells were subjected to Western blotting analysis with specific STAT1 and STAT2 antibodies; ( B ) subjected to full-length PCR amplification of the STAT1A isoform and ACTIN cDNAs; and ( C ) subjected to relative real-time STAT1 PCR. The results were normalized with respect to GUS mRNA and are expressed as a percentage of the amount of WT STAT1 mRNA. SD from triplication of a single experiment is indicated. These results are representative of 2 independent experiments.

    Article Snippet: They were then washed and incubated for 1 hour at 4°C with antibodies against P-Tyr701-STAT1 (catalog no. 612132; BD Biosciences — Transduction Laboratories) or STAT1 (catalog no. 610116; BD Biosciences — Transduction Laboratories) or with the corresponding isotypic antibodies.

    Techniques: Transfection, Plasmid Preparation, Western Blot, Polymerase Chain Reaction, Amplification

    STAT1 P696S is associated with a partial recessive defect in the activation of both ISGF3 and GAF, following stimulation with IFN-α and IFN-γ. EMSA with nuclear extracts (5 μg) from EBV-B cells from H, C +/+ , P1, P2, C +/– , and C –/– not stimulated or stimulated for 30 minutes with 10 3 and 10 5 IU/ml IFN-α ( A ) or IFN-γ ( C ). Radiolabeled ISRE ( A ) or GAS ( C ) probes were used. These results are representative of 6 or 7 independent experiments ( A and C ). Quantification of these 6 or 7 independent experiments by PhosphoImager SI (Molecular Dynamics), using the ISRE ( B ) or GAS ( D ) probe, in response to IFN-α ( B ) or IFN-γ ( D ). Each independent experiment (Exp) is shown in a different color. The responses are expressed as percentages of patients’ response versus a healthy control’s response (defined as 100%). Vertical lines have been drawn between maximum and minimum values. Various lanes from the same gel have been inverted in A ; white dividing lines show where the image was cut.

    Journal: The Journal of Clinical Investigation

    Article Title: A partial form of recessive STAT1 deficiency in humans

    doi: 10.1172/JCI37083

    Figure Lengend Snippet: STAT1 P696S is associated with a partial recessive defect in the activation of both ISGF3 and GAF, following stimulation with IFN-α and IFN-γ. EMSA with nuclear extracts (5 μg) from EBV-B cells from H, C +/+ , P1, P2, C +/– , and C –/– not stimulated or stimulated for 30 minutes with 10 3 and 10 5 IU/ml IFN-α ( A ) or IFN-γ ( C ). Radiolabeled ISRE ( A ) or GAS ( C ) probes were used. These results are representative of 6 or 7 independent experiments ( A and C ). Quantification of these 6 or 7 independent experiments by PhosphoImager SI (Molecular Dynamics), using the ISRE ( B ) or GAS ( D ) probe, in response to IFN-α ( B ) or IFN-γ ( D ). Each independent experiment (Exp) is shown in a different color. The responses are expressed as percentages of patients’ response versus a healthy control’s response (defined as 100%). Vertical lines have been drawn between maximum and minimum values. Various lanes from the same gel have been inverted in A ; white dividing lines show where the image was cut.

    Article Snippet: They were then washed and incubated for 1 hour at 4°C with antibodies against P-Tyr701-STAT1 (catalog no. 612132; BD Biosciences — Transduction Laboratories) or STAT1 (catalog no. 610116; BD Biosciences — Transduction Laboratories) or with the corresponding isotypic antibodies.

    Techniques: Activation Assay

    STAT1 P696S is associated with salmonellosis and viral disease. ( A ) Human STAT1a and STAT1b isoforms are shown, with their known pathogenic mutations. Coding exons are numbered with Roman numerals and delimited by a vertical bar. Regions corresponding to the coiled-coil domain (CC), DNA-binding domain (DNA-B), linker domain (L), SH2 domain (SH2), tail segment domain (TS), and transactivator domain (TA) are indicated in gray, together with their amino acid boundaries, and are delimited by gray dotted lines. Tyr701 (Y) and Ser727 (S) are indicated. Mutations in red are recessive and associated with complete STAT1 deficiency in homozygous individuals. Mutations in green are associated with partial STAT1 deficiency in heterozygous individuals. The mutation in blue is a partial recessive mutation associated with partial STAT1 deficiency in homozygous individuals. The mutation reported here for what we believe to be the first time is indicated in italics. ( B ) STAT1 genotype and clinical phenotype of the kindred. Members II.1 and II.3 presented salmonellosis, and II.3 also presented viral diseases. These 2 individuals are subsequently referred to as P1 and P2, respectively. Individuals with clinical disease are indicated in black, and healthy individuals are shown in white. The mother, who is heterozygous for the STAT1 P696S mutation, is subsequently referred to as H. STAT1 genotypes are indicated under each individual. The index case is indicated with an arrow. ( C ) Genomic sequences in the sense orientation of exon 23 of STAT1 in a healthy control and in P1. C +/+ indicates a healthy control.

    Journal: The Journal of Clinical Investigation

    Article Title: A partial form of recessive STAT1 deficiency in humans

    doi: 10.1172/JCI37083

    Figure Lengend Snippet: STAT1 P696S is associated with salmonellosis and viral disease. ( A ) Human STAT1a and STAT1b isoforms are shown, with their known pathogenic mutations. Coding exons are numbered with Roman numerals and delimited by a vertical bar. Regions corresponding to the coiled-coil domain (CC), DNA-binding domain (DNA-B), linker domain (L), SH2 domain (SH2), tail segment domain (TS), and transactivator domain (TA) are indicated in gray, together with their amino acid boundaries, and are delimited by gray dotted lines. Tyr701 (Y) and Ser727 (S) are indicated. Mutations in red are recessive and associated with complete STAT1 deficiency in homozygous individuals. Mutations in green are associated with partial STAT1 deficiency in heterozygous individuals. The mutation in blue is a partial recessive mutation associated with partial STAT1 deficiency in homozygous individuals. The mutation reported here for what we believe to be the first time is indicated in italics. ( B ) STAT1 genotype and clinical phenotype of the kindred. Members II.1 and II.3 presented salmonellosis, and II.3 also presented viral diseases. These 2 individuals are subsequently referred to as P1 and P2, respectively. Individuals with clinical disease are indicated in black, and healthy individuals are shown in white. The mother, who is heterozygous for the STAT1 P696S mutation, is subsequently referred to as H. STAT1 genotypes are indicated under each individual. The index case is indicated with an arrow. ( C ) Genomic sequences in the sense orientation of exon 23 of STAT1 in a healthy control and in P1. C +/+ indicates a healthy control.

    Article Snippet: They were then washed and incubated for 1 hour at 4°C with antibodies against P-Tyr701-STAT1 (catalog no. 612132; BD Biosciences — Transduction Laboratories) or STAT1 (catalog no. 610116; BD Biosciences — Transduction Laboratories) or with the corresponding isotypic antibodies.

    Techniques: Binding Assay, Mutagenesis, Genomic Sequencing

    The IL-27 and IFN-λ1 pathways are STAT1 dependent. ( A ) IFIT1 mRNA in EBV-B cells from C +/+ , P1, and C –/– after stimulation for 1.5, 2, and 2.5 hours with 20 ng/ml IFN-λ1 or no stimulation. Results are normalized with respect to GUS mRNA and are expressed as multiples (fold induction) of the unstimulated value ± SD. * P

    Journal: The Journal of Clinical Investigation

    Article Title: A partial form of recessive STAT1 deficiency in humans

    doi: 10.1172/JCI37083

    Figure Lengend Snippet: The IL-27 and IFN-λ1 pathways are STAT1 dependent. ( A ) IFIT1 mRNA in EBV-B cells from C +/+ , P1, and C –/– after stimulation for 1.5, 2, and 2.5 hours with 20 ng/ml IFN-λ1 or no stimulation. Results are normalized with respect to GUS mRNA and are expressed as multiples (fold induction) of the unstimulated value ± SD. * P

    Article Snippet: They were then washed and incubated for 1 hour at 4°C with antibodies against P-Tyr701-STAT1 (catalog no. 612132; BD Biosciences — Transduction Laboratories) or STAT1 (catalog no. 610116; BD Biosciences — Transduction Laboratories) or with the corresponding isotypic antibodies.

    Techniques:

    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

    Normal Activation of STAT1 Mutants in Stable Transfectants (A) Western blot of total protein extracts (100 μg) from a parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele, with antibodies specific for phosphorylated-Tyr-701-STAT1, STAT1, and STAT3. The cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNA or IFNG. (B) Immunofluorescence staining, with a STAT1-specific antibody, of STAT1-deficient U3C fibrosarcoma cell clones, stably cotransfected with a zeocin-resistance vector and a vector containing a WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. For (A) and (B), one experiment representative of three independent experiments is shown.

    Journal: PLoS Genetics

    Article Title: Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease

    doi: 10.1371/journal.pgen.0020131

    Figure Lengend Snippet: Normal Activation of STAT1 Mutants in Stable Transfectants (A) Western blot of total protein extracts (100 μg) from a parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele, with antibodies specific for phosphorylated-Tyr-701-STAT1, STAT1, and STAT3. The cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNA or IFNG. (B) Immunofluorescence staining, with a STAT1-specific antibody, of STAT1-deficient U3C fibrosarcoma cell clones, stably cotransfected with a zeocin-resistance vector and a vector containing a WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. For (A) and (B), one experiment representative of three independent experiments is shown.

    Article Snippet: The following antibodies were used: anti-phospho-STAT1 (9171; Cell Signaling Technology), anti-STAT1 (610116; BD Transduction Laboratories, San Diego, California, United States), anti-STAT3 (sc-7179; Santa-Cruz Biotechnology, Santa Cruz, California, United States), anti-phospho-STAT2 (4441; Cell Signaling), and anti-STAT2 (sc-476; Santa-Cruz Biotechnology, Santa Cruz, California, United States).

    Techniques: Activation Assay, Western Blot, Clone Assay, Stable Transfection, Plasmid Preparation, Immunofluorescence, Staining

    Novel STAT1 Mutations in Two Kindreds (A) STAT1 genotype and clinical phenotype of three kindreds. In kindred A, members I.1 and II.1 had tuberculosis, and III.2 and IV.1 (P1) had severe BCG disease. In kindred B, members I.1 and III. 2 (P2) were infected with M. tuberculosis and M. avium, respectively. Kindred C has been described elsewhere; II.2 (P3) developed disseminated BCG disease. Individuals with clinical disease caused by weakly virulent (BCG or M. avium ) and more virulent (M. tuberculosis) mycobacteria are indicated in black and gray, respectively, and healthy individuals are shown in white. The index cases are indicated with an arrow. Genetically affected individuals (heterozygous for any of the three STAT1 mutations) with no clinical phenotype at the time of this study are indicated by a vertical line. Known STAT1 genotypes (WT, E320Q, Q463H, L706S) are indicated under each individual, with a question mark indicating unknown genotype. (B) The human STAT1 coding region is shown, with its known pathogenic mutations. The coiled-coil domain (CC), DNA-binding domain (DNA-B), linker domain (L), SH2 domain (SH2), tail segment domain (TS), and trans -activator domain (TA) are indicated, together with their amino-acid boundaries. Tyrosine 701 (Y) is also indicated. Mutations in red are recessive mutations associated with complete STAT1 deficiency (due to a lack of STAT1 production), impaired IFNG-induced GAF activation and IFNA-induced ISGF3 activation, and a syndrome of predisposition to mycobacterial and severe viral disease in homozygous individuals. Mutations in green are associated, in heterozygous individuals, with partial STAT1 deficiency (normal STAT1 expression), impaired IFNG-induced GAS-binding activity but normal IFNA-induced ISRE-binding activity, and MSMD (predisposition to mycobacterial but not viral disease). Mutations reported for the first time in this study are indicated in italics.

    Journal: PLoS Genetics

    Article Title: Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease

    doi: 10.1371/journal.pgen.0020131

    Figure Lengend Snippet: Novel STAT1 Mutations in Two Kindreds (A) STAT1 genotype and clinical phenotype of three kindreds. In kindred A, members I.1 and II.1 had tuberculosis, and III.2 and IV.1 (P1) had severe BCG disease. In kindred B, members I.1 and III. 2 (P2) were infected with M. tuberculosis and M. avium, respectively. Kindred C has been described elsewhere; II.2 (P3) developed disseminated BCG disease. Individuals with clinical disease caused by weakly virulent (BCG or M. avium ) and more virulent (M. tuberculosis) mycobacteria are indicated in black and gray, respectively, and healthy individuals are shown in white. The index cases are indicated with an arrow. Genetically affected individuals (heterozygous for any of the three STAT1 mutations) with no clinical phenotype at the time of this study are indicated by a vertical line. Known STAT1 genotypes (WT, E320Q, Q463H, L706S) are indicated under each individual, with a question mark indicating unknown genotype. (B) The human STAT1 coding region is shown, with its known pathogenic mutations. The coiled-coil domain (CC), DNA-binding domain (DNA-B), linker domain (L), SH2 domain (SH2), tail segment domain (TS), and trans -activator domain (TA) are indicated, together with their amino-acid boundaries. Tyrosine 701 (Y) is also indicated. Mutations in red are recessive mutations associated with complete STAT1 deficiency (due to a lack of STAT1 production), impaired IFNG-induced GAF activation and IFNA-induced ISGF3 activation, and a syndrome of predisposition to mycobacterial and severe viral disease in homozygous individuals. Mutations in green are associated, in heterozygous individuals, with partial STAT1 deficiency (normal STAT1 expression), impaired IFNG-induced GAS-binding activity but normal IFNA-induced ISRE-binding activity, and MSMD (predisposition to mycobacterial but not viral disease). Mutations reported for the first time in this study are indicated in italics.

    Article Snippet: The following antibodies were used: anti-phospho-STAT1 (9171; Cell Signaling Technology), anti-STAT1 (610116; BD Transduction Laboratories, San Diego, California, United States), anti-STAT3 (sc-7179; Santa-Cruz Biotechnology, Santa Cruz, California, United States), anti-phospho-STAT2 (4441; Cell Signaling), and anti-STAT2 (sc-476; Santa-Cruz Biotechnology, Santa Cruz, California, United States).

    Techniques: Infection, Binding Assay, Activation Assay, Expressing, Activity Assay

    Normal Activation but Impaired DNA-Binding Activity of STAT1 in Heterozygous Cells from Patients (A) Western blot of total protein extracts (100 μg) from EBV-transformed B cells derived from a healthy control (C), three patients under study (P1, P2, P3), and a patient with recessive complete STAT1 deficiency (P4) homozygous for the 1758_1759delAG mutation, probed with specific antibodies against phosphorylated-Tyr-701-STAT1, STAT1, and STAT3. EBV-transformed B cells were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. (B) Immunofluorescence staining with a STAT1-specific antibody of skin-derived SV40-transformed fibroblasts from a healthy control (C) and three patients under study (P1, P2, P3). Fibroblasts were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. (C and E) EMSA of nuclear extracts (5 μg) from EBV-transformed B cells derived from a healthy control (C), three patients under study (P1, P2, P3), and the patient with complete STAT1 deficiency (P4). EBV-transformed B cells were not stimulated (NS) or were stimulated for 30 min with 10 3 and 10 5 IU/ml of IFNG (C) and IFNA (E), respectively. Radiolabeled GAS (C) or ISRE (E) probes were used. (D) Quantification of four to six independent experiments by PhosphoImager SI (Molecular Dynamics, Piscataway, New Jersey, United States) using the GAS probe in response to 10 5 IU/ml of IFNG is also presented. The mean, minimum, and maximum values are expressed with respect to the positive control response (100%). For (A–C) and (E), one experiment representative of three to five independent experiments is shown.

    Journal: PLoS Genetics

    Article Title: Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease

    doi: 10.1371/journal.pgen.0020131

    Figure Lengend Snippet: Normal Activation but Impaired DNA-Binding Activity of STAT1 in Heterozygous Cells from Patients (A) Western blot of total protein extracts (100 μg) from EBV-transformed B cells derived from a healthy control (C), three patients under study (P1, P2, P3), and a patient with recessive complete STAT1 deficiency (P4) homozygous for the 1758_1759delAG mutation, probed with specific antibodies against phosphorylated-Tyr-701-STAT1, STAT1, and STAT3. EBV-transformed B cells were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. (B) Immunofluorescence staining with a STAT1-specific antibody of skin-derived SV40-transformed fibroblasts from a healthy control (C) and three patients under study (P1, P2, P3). Fibroblasts were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. (C and E) EMSA of nuclear extracts (5 μg) from EBV-transformed B cells derived from a healthy control (C), three patients under study (P1, P2, P3), and the patient with complete STAT1 deficiency (P4). EBV-transformed B cells were not stimulated (NS) or were stimulated for 30 min with 10 3 and 10 5 IU/ml of IFNG (C) and IFNA (E), respectively. Radiolabeled GAS (C) or ISRE (E) probes were used. (D) Quantification of four to six independent experiments by PhosphoImager SI (Molecular Dynamics, Piscataway, New Jersey, United States) using the GAS probe in response to 10 5 IU/ml of IFNG is also presented. The mean, minimum, and maximum values are expressed with respect to the positive control response (100%). For (A–C) and (E), one experiment representative of three to five independent experiments is shown.

    Article Snippet: The following antibodies were used: anti-phospho-STAT1 (9171; Cell Signaling Technology), anti-STAT1 (610116; BD Transduction Laboratories, San Diego, California, United States), anti-STAT3 (sc-7179; Santa-Cruz Biotechnology, Santa Cruz, California, United States), anti-phospho-STAT2 (4441; Cell Signaling), and anti-STAT2 (sc-476; Santa-Cruz Biotechnology, Santa Cruz, California, United States).

    Techniques: Activation Assay, Binding Assay, Activity Assay, Western Blot, Transformation Assay, Derivative Assay, Mutagenesis, Immunofluorescence, Staining, Positive Control

    Pathways of IFNG-Induced GAF-Mediated Immunity and IFNA-Induced ISGF3-Mediated Immunity Patients homozygous for null STAT1 mutations [ 36 , 37 ] suffer from both mycobacterial and viral diseases. Patients heterozygous for STAT1 mutations L706S, Q463H, and E320Q suffer from mycobacterial but not viral diseases ([ 17 ] and this report).

    Journal: PLoS Genetics

    Article Title: Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease

    doi: 10.1371/journal.pgen.0020131

    Figure Lengend Snippet: Pathways of IFNG-Induced GAF-Mediated Immunity and IFNA-Induced ISGF3-Mediated Immunity Patients homozygous for null STAT1 mutations [ 36 , 37 ] suffer from both mycobacterial and viral diseases. Patients heterozygous for STAT1 mutations L706S, Q463H, and E320Q suffer from mycobacterial but not viral diseases ([ 17 ] and this report).

    Article Snippet: The following antibodies were used: anti-phospho-STAT1 (9171; Cell Signaling Technology), anti-STAT1 (610116; BD Transduction Laboratories, San Diego, California, United States), anti-STAT3 (sc-7179; Santa-Cruz Biotechnology, Santa Cruz, California, United States), anti-phospho-STAT2 (4441; Cell Signaling), and anti-STAT2 (sc-476; Santa-Cruz Biotechnology, Santa Cruz, California, United States).

    Techniques:

    Impaired DNA-Binding Activity of STAT1 Mutants in Stable Transfectants (A–D) EMSA of nuclear extracts (5 μg [A and B]; 30 μg [C and D]) from a parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated for 30 min with the indicated doses of IFNG (A and B) or IFNA (C and D). We used the radiolabeled GAS probe FCGR1 (A and B) or an ISRE probe (C and D). For (A–D), one experiment representative of three to five independent experiments is shown. (E) Quantification of three independent experiments by PhosphoImager SI (Molecular Dynamics), using the ISRE probe, in response to 10 4 and 10 5 IU/ml IFNA is also presented. The mean, minimum, and maximum values are expressed with respect to the WT stable transfectant clone response (100%).

    Journal: PLoS Genetics

    Article Title: Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease

    doi: 10.1371/journal.pgen.0020131

    Figure Lengend Snippet: Impaired DNA-Binding Activity of STAT1 Mutants in Stable Transfectants (A–D) EMSA of nuclear extracts (5 μg [A and B]; 30 μg [C and D]) from a parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated for 30 min with the indicated doses of IFNG (A and B) or IFNA (C and D). We used the radiolabeled GAS probe FCGR1 (A and B) or an ISRE probe (C and D). For (A–D), one experiment representative of three to five independent experiments is shown. (E) Quantification of three independent experiments by PhosphoImager SI (Molecular Dynamics), using the ISRE probe, in response to 10 4 and 10 5 IU/ml IFNA is also presented. The mean, minimum, and maximum values are expressed with respect to the WT stable transfectant clone response (100%).

    Article Snippet: The following antibodies were used: anti-phospho-STAT1 (9171; Cell Signaling Technology), anti-STAT1 (610116; BD Transduction Laboratories, San Diego, California, United States), anti-STAT3 (sc-7179; Santa-Cruz Biotechnology, Santa Cruz, California, United States), anti-phospho-STAT2 (4441; Cell Signaling), and anti-STAT2 (sc-476; Santa-Cruz Biotechnology, Santa Cruz, California, United States).

    Techniques: Binding Assay, Activity Assay, Clone Assay, Stable Transfection, Plasmid Preparation, Transfection

    Molecular Representation of STAT1 Mutants (A) Ribbon representation of the WT STAT1 homodimer complexed with DNA. Secondary structure elements representing the β strands are shown in cyan, and the helices are shown in blue-magenta. Atoms of residues in mutated positions L706, E320, and Q463 (indicated by arrows) are shown in space-filling models. Atoms are shown in red (for oxygen), blue (for nitrogen), and gray (for carbon). (B) Magnified focus on the region containing the three mutated residues.

    Journal: PLoS Genetics

    Article Title: Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease

    doi: 10.1371/journal.pgen.0020131

    Figure Lengend Snippet: Molecular Representation of STAT1 Mutants (A) Ribbon representation of the WT STAT1 homodimer complexed with DNA. Secondary structure elements representing the β strands are shown in cyan, and the helices are shown in blue-magenta. Atoms of residues in mutated positions L706, E320, and Q463 (indicated by arrows) are shown in space-filling models. Atoms are shown in red (for oxygen), blue (for nitrogen), and gray (for carbon). (B) Magnified focus on the region containing the three mutated residues.

    Article Snippet: The following antibodies were used: anti-phospho-STAT1 (9171; Cell Signaling Technology), anti-STAT1 (610116; BD Transduction Laboratories, San Diego, California, United States), anti-STAT3 (sc-7179; Santa-Cruz Biotechnology, Santa Cruz, California, United States), anti-phospho-STAT2 (4441; Cell Signaling), and anti-STAT2 (sc-476; Santa-Cruz Biotechnology, Santa Cruz, California, United States).

    Techniques:

    Impact of STAT1 Mutations on Transcription (A) Levels of mRNA corresponding to STAT1, IFNG- and/or IFNA-inducible genes (IRF1 and ISG15) and GADP in EBV-transformed B cells from a control (C), the three affected individuals (P1, P2, and P3) and a STAT1-deficient individual (P4), or in the 2C4 parental fibrosarcoma cell line, STAT1-deficient U3C cell line, and U3C cells stably transfected with a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele, either not stimulated (NS), or stimulated for 2 h with 10 3 of IFNG or 10 4 of IFNA for EBV-transformed B cells and 10 5 IU/ml of IFNG or IFNA for fibroblasts, as detected by Northern blotting. (B) Relative real-time PCR of IRF1, ISG15, and MX1, and cDNAs from EBV-transformed B cells derived from a healthy control (C), three patients under study (P1, P2, P3), and a patient with recessive complete STAT1 deficiency (P5) homozygous for the 1928insA STAT1 mutation or from parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock, WT, E320Q, Q463H, or L706S STAT1 allele stimulated or not stimulated with 10 5 IU/ml of IFNG or IFNA for 1 h and 2 h for EBV-transformed B cells and fibroblasts, respectively, for IRF1, and for 6 h for both cellular types for ISG15 and MX1 . Means values of duplicates of one experiment are shown with their respective standard variations.

    Journal: PLoS Genetics

    Article Title: Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease

    doi: 10.1371/journal.pgen.0020131

    Figure Lengend Snippet: Impact of STAT1 Mutations on Transcription (A) Levels of mRNA corresponding to STAT1, IFNG- and/or IFNA-inducible genes (IRF1 and ISG15) and GADP in EBV-transformed B cells from a control (C), the three affected individuals (P1, P2, and P3) and a STAT1-deficient individual (P4), or in the 2C4 parental fibrosarcoma cell line, STAT1-deficient U3C cell line, and U3C cells stably transfected with a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele, either not stimulated (NS), or stimulated for 2 h with 10 3 of IFNG or 10 4 of IFNA for EBV-transformed B cells and 10 5 IU/ml of IFNG or IFNA for fibroblasts, as detected by Northern blotting. (B) Relative real-time PCR of IRF1, ISG15, and MX1, and cDNAs from EBV-transformed B cells derived from a healthy control (C), three patients under study (P1, P2, P3), and a patient with recessive complete STAT1 deficiency (P5) homozygous for the 1928insA STAT1 mutation or from parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock, WT, E320Q, Q463H, or L706S STAT1 allele stimulated or not stimulated with 10 5 IU/ml of IFNG or IFNA for 1 h and 2 h for EBV-transformed B cells and fibroblasts, respectively, for IRF1, and for 6 h for both cellular types for ISG15 and MX1 . Means values of duplicates of one experiment are shown with their respective standard variations.

    Article Snippet: The following antibodies were used: anti-phospho-STAT1 (9171; Cell Signaling Technology), anti-STAT1 (610116; BD Transduction Laboratories, San Diego, California, United States), anti-STAT3 (sc-7179; Santa-Cruz Biotechnology, Santa Cruz, California, United States), anti-phospho-STAT2 (4441; Cell Signaling), and anti-STAT2 (sc-476; Santa-Cruz Biotechnology, Santa Cruz, California, United States).

    Techniques: Transformation Assay, Stable Transfection, Transfection, Northern Blot, Real-time Polymerase Chain Reaction, Derivative Assay, Mutagenesis, Clone Assay, Plasmid Preparation

    Mechanism of Dominance of the STAT1 Alleles for GAS-Binding Activity and of Recessivity of the STAT1 Alleles for ISRE-Binding Activity (A) Western blot of total protein extracts (100 μg) from unstimulated EBV-transformed B-cell lines from a healthy control (+/+), P4′s father (+/−) (heterozygous for the loss-of-expression, loss-of-function STAT1 1758_1759delAG allele), and P5 (−/−) (homozygous for the loss-of-expression, loss-of-function STAT1 1928insA allele), using antibodies specific for STAT1 and STAT3. (B) EMSA of nuclear extracts (5 μg) from EBV-transformed B cells derived from a healthy control (+/+), P5 (−/−) (homozygous for the loss-of-expression, loss-of-function STAT1 1928insA allele), and P4′s father (+/−) (heterozygous for the loss-of-expression, loss-of-function STAT1 1758_1759delAG allele). EBV-transformed B cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNG. A radiolabeled GAS (FCGR1) probe was used. (C) (a) Whole-cell extracts of 10 7 STAT1-deficient U3C fibrosarcoma cell clones stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, or L706S STAT1 allele were subjected to immunoprecipitation with the following biotinylated peptides: TSFGYDKPHVLV (1), corresponding to the intracellular part of IFNGR1 around the unphosphorylated Tyr-440 residue (Y); TSFG(pTyr)DKPHVLV (2), corresponding to the intracellular part of IFNGR1 around the phosphorylated Tyr-440 residue (pTyr); and SLIG(pTyr)RPTEDSK (3), corresponding to an irrelevant peptide similar to peptide 2. (b) 20 μL of each extract was taken before immunoprecipitation, and Western blotting was performed with STAT1- and STAT3-specific antibodies. (D) EMSA of nuclear extracts (5 μg) from EBV-transformed B cells derived from a healthy control (C), P4′s father (+/−) (heterozygous for the loss-of-expression, loss-of-function STAT1 1758_1759delAG allele) and P5 (−/−) (homozygous for the loss-of-expression, loss-of-function STAT1 1928insA allele). EBV-transformed B cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml of IFNA. A radiolabeled ISRE probe was used. (E) Immunoprecipitation with a STAT1-specific antibody, followed by Western blotting with Tyr701-phospho-STAT1–specific, Tyr690-phospho-STAT2–specific, STAT1-specific, and STAT2-specific antibodies, of total protein extracts (1 mg) from a parental fibrosarcoma cell line (2C4), a STAT2-deficient U6A fibrosarcoma cell line, and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock, WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNA. For (B–E), one experiment representative of two independent experiments is shown.

    Journal: PLoS Genetics

    Article Title: Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease

    doi: 10.1371/journal.pgen.0020131

    Figure Lengend Snippet: Mechanism of Dominance of the STAT1 Alleles for GAS-Binding Activity and of Recessivity of the STAT1 Alleles for ISRE-Binding Activity (A) Western blot of total protein extracts (100 μg) from unstimulated EBV-transformed B-cell lines from a healthy control (+/+), P4′s father (+/−) (heterozygous for the loss-of-expression, loss-of-function STAT1 1758_1759delAG allele), and P5 (−/−) (homozygous for the loss-of-expression, loss-of-function STAT1 1928insA allele), using antibodies specific for STAT1 and STAT3. (B) EMSA of nuclear extracts (5 μg) from EBV-transformed B cells derived from a healthy control (+/+), P5 (−/−) (homozygous for the loss-of-expression, loss-of-function STAT1 1928insA allele), and P4′s father (+/−) (heterozygous for the loss-of-expression, loss-of-function STAT1 1758_1759delAG allele). EBV-transformed B cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNG. A radiolabeled GAS (FCGR1) probe was used. (C) (a) Whole-cell extracts of 10 7 STAT1-deficient U3C fibrosarcoma cell clones stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, or L706S STAT1 allele were subjected to immunoprecipitation with the following biotinylated peptides: TSFGYDKPHVLV (1), corresponding to the intracellular part of IFNGR1 around the unphosphorylated Tyr-440 residue (Y); TSFG(pTyr)DKPHVLV (2), corresponding to the intracellular part of IFNGR1 around the phosphorylated Tyr-440 residue (pTyr); and SLIG(pTyr)RPTEDSK (3), corresponding to an irrelevant peptide similar to peptide 2. (b) 20 μL of each extract was taken before immunoprecipitation, and Western blotting was performed with STAT1- and STAT3-specific antibodies. (D) EMSA of nuclear extracts (5 μg) from EBV-transformed B cells derived from a healthy control (C), P4′s father (+/−) (heterozygous for the loss-of-expression, loss-of-function STAT1 1758_1759delAG allele) and P5 (−/−) (homozygous for the loss-of-expression, loss-of-function STAT1 1928insA allele). EBV-transformed B cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml of IFNA. A radiolabeled ISRE probe was used. (E) Immunoprecipitation with a STAT1-specific antibody, followed by Western blotting with Tyr701-phospho-STAT1–specific, Tyr690-phospho-STAT2–specific, STAT1-specific, and STAT2-specific antibodies, of total protein extracts (1 mg) from a parental fibrosarcoma cell line (2C4), a STAT2-deficient U6A fibrosarcoma cell line, and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock, WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNA. For (B–E), one experiment representative of two independent experiments is shown.

    Article Snippet: The following antibodies were used: anti-phospho-STAT1 (9171; Cell Signaling Technology), anti-STAT1 (610116; BD Transduction Laboratories, San Diego, California, United States), anti-STAT3 (sc-7179; Santa-Cruz Biotechnology, Santa Cruz, California, United States), anti-phospho-STAT2 (4441; Cell Signaling), and anti-STAT2 (sc-476; Santa-Cruz Biotechnology, Santa Cruz, California, United States).

    Techniques: Binding Assay, Activity Assay, Western Blot, Transformation Assay, Expressing, Derivative Assay, Clone Assay, Stable Transfection, Plasmid Preparation, Immunoprecipitation

    Impact of Mutant STAT1 Alleles on IFNG- and IFNA-Mediated Immunity (A) Cytokine production in the supernatant of whole blood from healthy controls (C) and patients (P1′s mother, P2, and P3) and their respective “travel” control, not stimulated (NS) or stimulated for 72 h with live BCG alone or BCG plus IL12 or IFNG. The levels of IFNG and IL12 in the supernatant were determined by enzyme-linked immunosorbent assay. One experiment representative of two independent experiments is shown. (B) Skin-derived SV40-transformed fibroblasts from a healthy control (C), the three patients under study (P1, P2, P3), a parental fibrosarcoma cell line (2C4), STAT1-deficient U3C fibrosarcoma cell line (U3C), and U3C clones stably transfected with a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 alleles, were infected with HSV-1 or VSV, with or without priorstimulation with IFNA (10 5 IU/ml) for 24 h. Viral titers were determined after 48 h of infection. Five independent experiments are shown for the patient's cells and three independent experiments are shown for sarcoma fibroblasts. Each assay is symbolized by a different character.

    Journal: PLoS Genetics

    Article Title: Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease

    doi: 10.1371/journal.pgen.0020131

    Figure Lengend Snippet: Impact of Mutant STAT1 Alleles on IFNG- and IFNA-Mediated Immunity (A) Cytokine production in the supernatant of whole blood from healthy controls (C) and patients (P1′s mother, P2, and P3) and their respective “travel” control, not stimulated (NS) or stimulated for 72 h with live BCG alone or BCG plus IL12 or IFNG. The levels of IFNG and IL12 in the supernatant were determined by enzyme-linked immunosorbent assay. One experiment representative of two independent experiments is shown. (B) Skin-derived SV40-transformed fibroblasts from a healthy control (C), the three patients under study (P1, P2, P3), a parental fibrosarcoma cell line (2C4), STAT1-deficient U3C fibrosarcoma cell line (U3C), and U3C clones stably transfected with a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 alleles, were infected with HSV-1 or VSV, with or without priorstimulation with IFNA (10 5 IU/ml) for 24 h. Viral titers were determined after 48 h of infection. Five independent experiments are shown for the patient's cells and three independent experiments are shown for sarcoma fibroblasts. Each assay is symbolized by a different character.

    Article Snippet: The following antibodies were used: anti-phospho-STAT1 (9171; Cell Signaling Technology), anti-STAT1 (610116; BD Transduction Laboratories, San Diego, California, United States), anti-STAT3 (sc-7179; Santa-Cruz Biotechnology, Santa Cruz, California, United States), anti-phospho-STAT2 (4441; Cell Signaling), and anti-STAT2 (sc-476; Santa-Cruz Biotechnology, Santa Cruz, California, United States).

    Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Derivative Assay, Transformation Assay, Clone Assay, Stable Transfection, Transfection, Infection

    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