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

Article Title: Mycobacterium avium inhibition of IFN-gamma signaling in mouse macrophages: Toll-like receptor 2 stimulation increases expression of dominant-negative STAT1 beta by mRNA stabilization.

doi: 10.4049/jimmunol.171.12.6766

Figure Lengend Snippet: FIGURE 2. Stimulation of TLR2 does not inhibit STAT1 DNA-binding activity. A, RAW264.7 macrophages were infected with M. avium (M.a) at 10:1 and 20:1 bacteria/macrophage or stimulated with TLR2 agonists li- poarabinomannan (LAM), 1 g/ml, and Pam3CSK4 (PCSK4), 1 g/ml, for 16 h. Cells were then stimulated for 45 min with IFN- (200 U/ml). Nu- clear extracts were prepared and STAT1 DNA binding was determined by EMSA. Results are representative of three experiments. B, Ab supershift experiment with anti-STAT1 Ab and nuclear extracts from RAW264.7 stimulated with lipoarabinomannan (LAM), Pam3CSK4 (PCSK4), and IFN-.

Article Snippet: A STAT1 expression vector was constructed by PCR amplification of the coding region of a STAT1 plasmid obtained from the American Type Culture Collection (I.M.A.G.E. clone 358731) and cloning of the PCR product into the pcDNA 3.1 expression vector (Invitrogen) using BamHI and XbaI sites.

Techniques: Binding Assay, Activity Assay, Infection, Bacteria

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

Article Title: Mycobacterium avium inhibition of IFN-gamma signaling in mouse macrophages: Toll-like receptor 2 stimulation increases expression of dominant-negative STAT1 beta by mRNA stabilization.

doi: 10.4049/jimmunol.171.12.6766

Figure Lengend Snippet: FIGURE 3. TLR2 stimulation increases phosphorylation of STAT1. A, RAW264.7 macrophages were infected with M. avium (M.a) at 10:1 and 20:1 bacteria/macrophage or stimulated with TLR2 agonists lipoarabinomannan (LAM), 1 g/ml; 19-kDa liproprotein (LP), 1 g/ml; and Pam3CSK4 (PCSK4), 1 g/ml, for 16 h. Cells were then stimulated for 45 min with IFN- (200 U/ml). Cell lysates were analyzed for phospho-STAT1 by Western blot. B, Densitometry analysis was plotted as percentage of increase in phospho-STAT1, and phospho-STAT1 levels relative to control cells were stimulated with IFN-. C, Densitometry analysis plotted as ratio of phospho-STAT1/phospho-STAT1. This experiment is representative of four separate experiments.

Article Snippet: A STAT1 expression vector was constructed by PCR amplification of the coding region of a STAT1 plasmid obtained from the American Type Culture Collection (I.M.A.G.E. clone 358731) and cloning of the PCR product into the pcDNA 3.1 expression vector (Invitrogen) using BamHI and XbaI sites.

Techniques: Phospho-proteomics, Infection, Bacteria, Western Blot, Control

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

Article Title: Mycobacterium avium inhibition of IFN-gamma signaling in mouse macrophages: Toll-like receptor 2 stimulation increases expression of dominant-negative STAT1 beta by mRNA stabilization.

doi: 10.4049/jimmunol.171.12.6766

Figure Lengend Snippet: FIGURE 4. Concentration-dependent effect of TLR2 stimulation on phosphorylation of STAT1 and STAT1. RAW264.7 cells were stimulated with the indicated concentrations of lipoarabinomannan for 16 h and stimulated with 200 U/ml IFN- for 45 min. A, Cell lysates were analyzed for phospho- STAT1 and STAT1 by Western blots. B, Densitometry analysis of the phospho-STAT1 blot plotted as percentage of increase relative to control cells stimulated with IFN-. C, Densitometry analysis of the STAT1 blot plotted as percentage of increase relative to control-unstimulated cells. Results are representative of three separate experiments.

Article Snippet: A STAT1 expression vector was constructed by PCR amplification of the coding region of a STAT1 plasmid obtained from the American Type Culture Collection (I.M.A.G.E. clone 358731) and cloning of the PCR product into the pcDNA 3.1 expression vector (Invitrogen) using BamHI and XbaI sites.

Techniques: Concentration Assay, Phospho-proteomics, Western Blot, Control

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

Article Title: Mycobacterium avium inhibition of IFN-gamma signaling in mouse macrophages: Toll-like receptor 2 stimulation increases expression of dominant-negative STAT1 beta by mRNA stabilization.

doi: 10.4049/jimmunol.171.12.6766

Figure Lengend Snippet: FIGURE 5. TLR2 stimulation increases mRNA level of STAT1. A, RAW264.7 macrophages were stimulated with the indicated concentrations of Pam3CSK4 for 16 h. RNA was isolated, and expression of STAT1 and STAT1 mRNA was determined by Northern blot hybridization. The blot was then stripped and probed with G3PDH. B, Densitometry analysis plot- ted as relative intensity after normalization with the G3PDH blot. These data are representative of three separate experiments.

Article Snippet: A STAT1 expression vector was constructed by PCR amplification of the coding region of a STAT1 plasmid obtained from the American Type Culture Collection (I.M.A.G.E. clone 358731) and cloning of the PCR product into the pcDNA 3.1 expression vector (Invitrogen) using BamHI and XbaI sites.

Techniques: Isolation, Expressing, Northern Blot, Hybridization

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

Article Title: Mycobacterium avium inhibition of IFN-gamma signaling in mouse macrophages: Toll-like receptor 2 stimulation increases expression of dominant-negative STAT1 beta by mRNA stabilization.

doi: 10.4049/jimmunol.171.12.6766

Figure Lengend Snippet: FIGURE 6. TLR2 stimulation increases mRNA stability of STAT1. A, RAW264.7 macrophages were stimulated for 16 h with 2 g/ml Pam3CSK4. Actinomycin D was added to Pam3CSK4-stimulated cells and control-unstimulated RAW264.7 cells. RNA was isolated at the indicated times, and mRNA expression was determined by Northern blot hybridiza- tion with STAT1 and G3PDH probes. B, Decay curves of STAT1 and STAT1 mRNA were determined from densitometry analysis of Northern blots hybridized with STAT1 and G3PDH. The data represent the means SEM of four separate experiments. C, t1/2 of STAT1 and STAT1 mRNA was determined from the decay curves. The data represent the means SEM of the four experiments. TLR2 stimulation significantly increased STAT1 mRNA t1/2 (p 0.05, Student’s t test).

Article Snippet: A STAT1 expression vector was constructed by PCR amplification of the coding region of a STAT1 plasmid obtained from the American Type Culture Collection (I.M.A.G.E. clone 358731) and cloning of the PCR product into the pcDNA 3.1 expression vector (Invitrogen) using BamHI and XbaI sites.

Techniques: Control, Isolation, Expressing, Northern Blot

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

Article Title: Mycobacterium avium inhibition of IFN-gamma signaling in mouse macrophages: Toll-like receptor 2 stimulation increases expression of dominant-negative STAT1 beta by mRNA stabilization.

doi: 10.4049/jimmunol.171.12.6766

Figure Lengend Snippet: FIGURE 7. Overexpression of STAT1 by transient transfection of RAW264.7 cells results in inhibition of IFN--induced gene expression. RAW264.7 cells were transiently transfected overnight with the indicated amounts of a STAT1-pcDNA 3.1 expression plasmid or 2 g of empty pcDNA 3.1 vector. The transfected cells were stimulated with IFN- (200 U/ml), and cell lysates were prepared after 45 min and analyzed for phospho-STAT1 by Western blotting (A). B, Densitometry analysis plotted as ratio of phospho-STAT1/phospho-STAT1. C, RNA was isolated from a duplicate set of wells after 8 h of stimulation, and gene induction by IFN- was determined by Northern blot hybridization with IRF1, TGTP, Eb, and G3PDH probes. D, Data plotted as percent inhibition relative to vector alone control. Results represent one of three similar experiments.

Article Snippet: A STAT1 expression vector was constructed by PCR amplification of the coding region of a STAT1 plasmid obtained from the American Type Culture Collection (I.M.A.G.E. clone 358731) and cloning of the PCR product into the pcDNA 3.1 expression vector (Invitrogen) using BamHI and XbaI sites.

Techniques: Over Expression, Transfection, Inhibition, Gene Expression, Expressing, Plasmid Preparation, Western Blot, Isolation, Northern Blot, Hybridization, Control

Journal: Journal of Virology

Article Title: Optimizing the Live Attenuated Influenza A Vaccine Backbone for High-Risk Patient Groups

doi: 10.1128/jvi.00871-22

Figure Lengend Snippet: OptiLAIV is attenuated and safe in Stat1 −/− mice. (A) Scheme of mouse immunization. (B) 8-week-old Stat1 −/− mice were vaccinated intranasally under anesthesia with 10 4 PFU LAIV or optiLAIV in 25 μL and body weight (left panel) and survival (right panel) were monitored for 14 days postvaccination ( n = 4 mock-vaccinated group; n = 8 per vaccinated group). (C) 8-week-old Stat1 −/− mice were vaccinated intranasally under anesthesia with 10 5 PFU LAIV or optiLAIV in 25 μL PBS. At 2, 4 and 6 days postvaccination snouts (left panel) and lungs (right panel) were collected and viral titers were determined by plaque assay ( n = 5 per group). The statistical significances between LAIV group and optiLAIV group were determined using two-way ANOVA with the Geisser-Greenhouse correction and post hoc Dunn’s multiple-comparison test in panel B and Mantel-Cox test for survival curve; multiple Mann-Whitney tests in panel C;. P-values are indicated in the figure. *, comparison between LAIV and optiLAIV. L.o.D. = limit of detection (5 PFU/mL). Graphs are representative of 2 independent experiments and indicate mean ± SEM for panel B and geometric mean for panel C. Symbols represent data from individual mice for panel B. In C, black dotted line represents 15% body weight loss cutoff.

Article Snippet: A549 and A549 STAT1 −/− (adenocarcinomic human alveolar basal epithelial cells, ATCC) were grown in DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, Gibco) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Gibco) and 100 U/mL Pen/Strep.

Techniques: Plaque Assay, Comparison, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Optimizing the Live Attenuated Influenza A Vaccine Backbone for High-Risk Patient Groups

doi: 10.1128/jvi.00871-22

Figure Lengend Snippet: OptiLAIV NS1 allows unfolded protein response activation in human cell models. (A–B) A549 cells were infected at an MOI of 5 with LAIV or optiLAIV or treated with tunicamycin at 5 μg/mL. At 16 h and 24 h postinfection RT-PCR was performed from isolated RNA for the spliced form of XBP1 mRNA (A). In parallel at 8 h, 16 h and 24 h postinfection, lysates were analyzed by Western blot for expression of sXBP1, actin, NP and NS1 proteins (B). (C) A549 wt cells (upper panel) or A549 Stat1 −/− cells (lower panel) were infected at an MOI of 5 with LAIV or optiLAIV and RT-qPCR for UPR-induced genes DNAJB9 and DDIT3 was performed in RNA lysates at 16 h and 24 h postinfection. (D) A549 wt cells (upper panel) or A549 Stat1 −/− cells (lower panel) were infected at an MOI of 0.01 with either LAIV or optiLAIV. Supernatants were collected at indicated time postinfection and viral titers determined by plaque assay. (E) Primary human nasal epithelial cells (Mucilair) were infected at an MOI of 5 with LAIV or optiLAIV. Cells were lysed at 24 h postinfection and RT-qPCR for each respective gene was performed. The statistical significances between LAIV group and optiLAIV group were determined using one-way ANOVA with post hoc Tukey’s multiple-comparison test for panels A, C and E; two-way ANOVA with the Geisser-Greenhouse correction and post hoc Dunn’s multiple-comparison test for panel D. *, comparison between LAIV and optiLAIV; # : comparison between mock and optiLAIV; ns = nonsignificant. Graphs are representative of 3 independent experiments and indicate mean ± SD.

Article Snippet: A549 and A549 STAT1 −/− (adenocarcinomic human alveolar basal epithelial cells, ATCC) were grown in DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, Gibco) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Gibco) and 100 U/mL Pen/Strep.

Techniques: Activation Assay, Infection, Reverse Transcription Polymerase Chain Reaction, Isolation, Western Blot, Expressing, Quantitative RT-PCR, Plaque Assay, Comparison

Journal:

Article Title: A Soluble Factor(s) Secreted from CD8 + T Lymphocytes Inhibits Human Immunodeficiency Virus Type 1 Replication through STAT1 Activation

doi: 10.1128/JVI.76.2.569-581.2002

Figure Lengend Snippet: STAT1 activation is induced by CAF from different HVS-transformed CD8+-T-cell lines. 1G5 cells were treated for 15 min with a 50% concentration of conditioned medium from the indicated lines. Whole-cell extracts were prepared for EMSA using a 32P-labeled m67 GAS probe. The presence of STAT1 in the complexes was confirmed by adding anti-STAT1 antibody in a supershift experiment (panel A, lane 6).

Article Snippet: Antibodies against STAT1 (N terminus, catalogue no. {"type":"entrez-nucleotide","attrs":{"text":"G16930","term_id":"1214356","term_text":"G16930"}} G16930 ) and IRF-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Activation Assay, Transformation Assay, Concentration Assay, Labeling

Journal:

Article Title: A Soluble Factor(s) Secreted from CD8 + T Lymphocytes Inhibits Human Immunodeficiency Virus Type 1 Replication through STAT1 Activation

doi: 10.1128/JVI.76.2.569-581.2002

Figure Lengend Snippet: Effects of CAF on HIV-1VSV replication and STAT1 activation in U3A and 2fTGH cells. (A) 2fTGH (STAT1-expressing) and U3A (STAT1-null) cells were incubated with (solid bar) or without (open bar) a 50% concentration of CAF (K#1 50K clone) for 16 h before infection with HIV-1VSV. The results (mean ± standard error) from triplicate determinations in a single experiment are presented as histograms. Similar results were obtained in two other independent experiments. The values represent luciferase activity compared to that derived from cells not treated with CAF, which was defined as 100%. This corresponded to absolute r.l.u. values of 11,391 ± 393 for 2fTGH and 18,600 ± 500 for U3A. The average background r.l.u. value was 1.5. (B) U3A and 2fTGH cells were treated with 50% CAF (K#1 50K clone) for 15 min before whole-cell extracts were prepared for EMSA using a 32P-labeled m67 GAS probe.

Article Snippet: Antibodies against STAT1 (N terminus, catalogue no. {"type":"entrez-nucleotide","attrs":{"text":"G16930","term_id":"1214356","term_text":"G16930"}} G16930 ) and IRF-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Activation Assay, Expressing, Incubation, Concentration Assay, Infection, Luciferase, Activity Assay, Derivative Assay, Labeling

Journal:

Article Title: A Soluble Factor(s) Secreted from CD8 + T Lymphocytes Inhibits Human Immunodeficiency Virus Type 1 Replication through STAT1 Activation

doi: 10.1128/JVI.76.2.569-581.2002

Figure Lengend Snippet: Effects of CAF on HIV-1 LTR activation in HeLa and U3A cells expressing a dominant negative STAT1 protein. (A) HeLa cells were transiently transfected with a plasmid expressing HIV-LTR-Luc and one expressing either no STAT1 (vector control), wild-type STAT1, or the STAT1 Y701F dominant negative mutant (STAT1 DN), as indicated. The transfected cells were then treated with CAF (50% concentration; K#1 50K line) for 16 h before PMA stimulation for 8 h and measurement of luciferase activity. (B) A similar experiment was performed using U3A (STAT1-null) cells. The results (mean ± standard error) from triplicate determinations in a single experiment are presented as histograms. Similar results were obtained in another independent experiment. The values represent luciferase activity compared to that derived from cells not treated with CAF, which was defined as 100%. This corresponded to absolute r.l.u. values of 1,850 ± 50 for HeLa cells and 4,650 ± 13 for U3A cells.

Article Snippet: Antibodies against STAT1 (N terminus, catalogue no. {"type":"entrez-nucleotide","attrs":{"text":"G16930","term_id":"1214356","term_text":"G16930"}} G16930 ) and IRF-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Activation Assay, Expressing, Dominant Negative Mutation, Transfection, Plasmid Preparation, Concentration Assay, Luciferase, Activity Assay, Derivative Assay

Journal:

Article Title: A Soluble Factor(s) Secreted from CD8 + T Lymphocytes Inhibits Human Immunodeficiency Virus Type 1 Replication through STAT1 Activation

doi: 10.1128/JVI.76.2.569-581.2002

Figure Lengend Snippet: IRF-1 was induced and bound to DBF sites within the HIV-1 LTR as a response to CAF. Different cell lines (A, 1G5; B, 2fTGH, U3A, and HeLa) were treated with CAF (50% concentration; K#1 50K line) for 16 h. Whole-cell extracts were prepared for EMSA using a 32P-labeled HIV-1 DBF probe. The composition of protein complexes was further assessed by adding anti-IRF-1, anti-IRF-2 or anti-STAT1 antibodies in a supershift experiment, as indicated. (C) HeLa cells were treated with conditioned media (50% concentration) from lines KP1#3 and K#1 50K, as indicated. Anti-IRF-1 or anti-IRF-2 antibodies were added in a supershift experiment, as indicated. X denotes a complex of unknown composition that is induced by all conditioned media, irrespective of the presence of CAF activity (see text). (D) Whole-cell extracts from 1G5 cells treated with CAF (50% concentration; K1 50K line) were analyzed by EMSA using a 32P-labeled ISG15 probe. Anti-IRF-1 or anti-IRF-2 antibodies were added in a supershift experiment, as indicated.

Article Snippet: Antibodies against STAT1 (N terminus, catalogue no. {"type":"entrez-nucleotide","attrs":{"text":"G16930","term_id":"1214356","term_text":"G16930"}} G16930 ) and IRF-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Concentration Assay, Labeling, Activity Assay