rabbit polyclonal anti total stat1  (Cell Signaling Technology Inc)

Journal: Cell reports

Article Title: XRN1 deletion induces PKR-dependent cell lethality in interferon-activated cancer cells

doi: 10.1016/j.celrep.2023.113600

Figure Lengend Snippet: (A) Correlation of expression levels of individual genes with XRN1 genetic dependency based on CRISPR-Cas9-mediated gene essentiality screens. Each dot represents one gene, and the top ten gene expression correlates are labeled in red. Pearson correlations and corresponding p adj values were computed for each feature in the Cancer Dependency Map Public 22Q4 dataset using all cancer cell lines. (B) Representative immunoblots from two independent biological replicates showing phospho-STAT1, total STAT1, total PKR, MDA5, and β-actin protein levels in a panel of XRN1 KO-sensitive cancer cell lines treated with either DMSO control or ruxolitinib (1 μM) for 24 h. Molecular weight (MW) markers are shown in kDa. (C, E, and G) Representative immunoblots showing XRN1, phospho-STAT1, total STAT1, phospho-PKR, total PKR, and β-actin protein levels in control and XRN1 -deleted NCI-H1650 (C), HCC1438 (E), and SW900 (G) cells treated with either DMSO control or ruxolitinib (1 μM). MW markers are shown in kDa. Three independent biological replicates were performed for each cell line. (D, F, and H) Cell viability was assessed by either ATP bioluminescence (left panels) or crystal violet staining (right panels) after CRISPR-Cas9 targeting of control loci or XRN1 in NCI-H1650 (D), HCC1438 (F), and SW900 (H) cells treated with DMSO control or ruxolitinib (1 μM). ATP bioluminescence values were normalized to the control sg1 sample within each cell line. Each dot represents the average of three technical replicates from one of three independent biological replicates in (D), (F), and (H). Error bars represent standard deviation from the mean. *p < 0.05 and ***p < 0.001, as calculated by repeated measures two-way ANOVA. Crystal violet images are representative of three independent biological replicates. See also .

Article Snippet: Rabbit polyclonal anti-total STAT1 , Cell Signaling Technology , Cat# 9172; RRID: AB_2198300.

Techniques: Expressing, CRISPR, Gene Expression, Labeling, Western Blot, Control, Molecular Weight, Staining, Standard Deviation

Journal: Cell reports

Article Title: XRN1 deletion induces PKR-dependent cell lethality in interferon-activated cancer cells

doi: 10.1016/j.celrep.2023.113600

Figure Lengend Snippet: (A) Representative immunoblots showing XRN1, phospho-STAT1, total STAT1, MDA5, phospho-PKR, total PKR, and β-actin protein levels in control or XRN1 KO A549 (left) or NCI-H1299 (right) cells after 24 h of treatment with vehicle control (sterile water) or interferon-β (10 ng/mL). Molecular weight (MW) markers are shown in kDa. (B) Cell viability was assessed by ATP bioluminescence in control or XRN1 KO A549 (left) or NCI-H1299 (right) cells 5 days after treatment with vehicle control (sterile water) or the indicated concentrations of interferon-β. Each dot represents the average of three technical replicates from one independent experiment. (C) Representative immunoblots showing XRN1, phospho-PKR, total PKR, and β-actin protein levels in control, XRN1 single KO, PKR single KO, or XRN1/PKR double KO (DKO) A549 (left) or NCI-H1299 (right) cells after 24 h of treatment with vehicle control (sterile water) or interferon-β (10 ng/mL). MW markers are shown in kDa. (D) Cell viability was assessed by ATP bioluminescence in control, XRN1 single KO, PKR single KO, or XRN1/PKR double KO (DKO) A549 (left) or NCI-H1299 (right) cells 5 days after treatment with vehicle control (sterile water) or the indicated concentrations of interferon-β. Each dot represents the average of three technical replicates from one independent experiment. Three independent biological replicates were performed for each cell line in (A)–(D). ATP bioluminescence values were normalized to the vehicle control sample for each isogenic cell line in (B) and (D). Error bars represent standard deviation from the mean. See also .

Article Snippet: Rabbit polyclonal anti-total STAT1 , Cell Signaling Technology , Cat# 9172; RRID: AB_2198300.

Techniques: Western Blot, Control, Sterility, Molecular Weight, Standard Deviation

Journal: Cell reports

Article Title: XRN1 deletion induces PKR-dependent cell lethality in interferon-activated cancer cells

doi: 10.1016/j.celrep.2023.113600

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-total STAT1 , Cell Signaling Technology , Cat# 9172; RRID: AB_2198300.

Techniques: Recombinant, Protease Inhibitor, Western Blot, Stripping, Bicinchoninic Acid Protein Assay, Transfection, Cell Viability Assay, Gene Expression, Control, CRISPR, Gene Knockout, Clone Assay, Plasmid Preparation, Expressing, Software

Journal: Respiratory Research

Article Title: Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

doi: 10.1186/1465-9921-11-64

Figure Lengend Snippet: Cigarette smoke extract decreases type II interferon-induced protein expression . A : ICAM-1 cell surface protein levels were determined using an enzyme-linked immunoassay with hTBE cell monolayers that were first treated with media without or with CSE at the indicated concentration for 4 hours. Cells were then incubated for 20 hours in media containing the same CSE concentration without or with IFN-γ. Values are expressed as mean ± S.D. ( n = 3 replicates), and a significant difference ( p < 0.05) in IFN-γ-induced levels between cells treated versus not treated with CSE is indicated by an asterisk . B : IRF-9, ICAM-1, HSP90, and β-actin protein levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentration for 4 hours, followed by incubation for 8 hours in media containing the same CSE concentration without or with IFN-γ. C : Total Stat1 and β-actin protein levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentration for 4 hours, followed by incubation for 8 hours in media containing the same CSE concentration without or with IFN-γ. In B and C , protein levels were quantified using band densitometry of immunoblot analyses from separate experiments with the value for cells not exposed to CSE but treated with IFN-γ set at 1 in each experiment. Values are expressed as mean ± S.D. ( n = 4 experiments) in the bar graph adjacent to a representative immunoblot analysis, and a significant difference ( p < 0.05) in IFN-γ-induced levels between cells treated versus not treated with CSE is indicated by an asterisk .

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone 6 against human interferon regulatory factor-9 (IRF-9) from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal IgG 4915 against human ICAM-1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9171 against tyrosine-701 phosphorylated human Stat1 from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI); mouse IgG2a mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG ab4742 against serine-727 phosphorylated human Stat1 from Abcam (Cambridge, MA); goat polyclonal IgG against human RSV proteins from Biodesign International (Saco, ME).

Techniques: Expressing, Concentration Assay, Incubation, Western Blot

Journal: Respiratory Research

Article Title: Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

doi: 10.1186/1465-9921-11-64

Figure Lengend Snippet: Cigarette smoke extract inhibits type II interferon-induced Stat1 activation . A : Tyrosine-701 and serine-727 phosphorylated and total Stat1, and β-actin levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 30 minutes in media containing the same CSE concentration without or with IFN-γ. B : Tyrosine-701 and serine-727 phosphorylated and total Stat1, ICAM-1, and β-actin levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 20 hours in media containing the same CSE concentration without or with IFN-γ. In A and B , protein levels were quantified using band densitometry of immunoblot analyses from separate experiments with the value for cells not exposed to CSE but treated with IFN-γ set at 1 in each experiment. Values are expressed as mean ± S.D. ( n = 3 experiments) in the bar graph adjacent to a representative immunoblot analysis, and a significant difference ( p < 0.05) in IFN-γ-induced levels between cells treated versus not treated with CSE is indicated by an asterisk .

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone 6 against human interferon regulatory factor-9 (IRF-9) from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal IgG 4915 against human ICAM-1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9171 against tyrosine-701 phosphorylated human Stat1 from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI); mouse IgG2a mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG ab4742 against serine-727 phosphorylated human Stat1 from Abcam (Cambridge, MA); goat polyclonal IgG against human RSV proteins from Biodesign International (Saco, ME).

Techniques: Activation Assay, Western Blot, Incubation, Concentration Assay

Journal: Respiratory Research

Article Title: Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

doi: 10.1186/1465-9921-11-64

Figure Lengend Snippet: Cigarette smoke extract has a delayed effect on epithelial cell Stat1 activation . A : Tyrosine-701 phosphorylated and total Stat1, and β-actin levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 8 hours in media containing the same CSE concentration without or with IFN-γ. Protein levels were quantified using band densitometry of immunoblot analyses from separate experiments with the value for cells not exposed to CSE but treated with IFN-γ set at 1 in each experiment. Values are expressed as mean ± S.D. ( n = 3 experiments) in the bar graph adjacent to a representative immunoblot analysis, and a significant difference ( p < 0.05) in IFN-γ-induced levels between cells treated versus not treated with CSE is indicated by an asterisk . B : Tyrosine-701 phosphorylated and total Stat1, and β-actin levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentrations for 12 hours, followed by incubation for 30 minutes in media containing the same CSE concentration without or with IFN-γ. C : Tyrosine-701 phosphorylated and total Stat1, and β-actin levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentrations for 12 hours, followed by incubation for 30 minutes in media without or with IFN-γ that had been preincubated without or with the same CSE concentration alone for 8 hours.

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone 6 against human interferon regulatory factor-9 (IRF-9) from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal IgG 4915 against human ICAM-1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9171 against tyrosine-701 phosphorylated human Stat1 from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI); mouse IgG2a mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG ab4742 against serine-727 phosphorylated human Stat1 from Abcam (Cambridge, MA); goat polyclonal IgG against human RSV proteins from Biodesign International (Saco, ME).

Techniques: Activation Assay, Western Blot, Incubation, Concentration Assay

Journal: Respiratory Research

Article Title: Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

doi: 10.1186/1465-9921-11-64

Figure Lengend Snippet: N-acetylcysteine inhibits cigarette smoke effects on type II interferon-induced responses . A : ICAM-1 cell surface protein levels were determined using an enzyme-linked immunoassay with hTBE cells that were first treated in media without or with NAC for 1 hour. Cells were then incubated in media containing the antioxidant without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 20 hours with the same compounds without or with IFN-γ. Values are expressed as mean ± S.D. ( n = 3 replicates) and a significant difference ( p < 0.05) in comparable CSE-treated levels between cells not incubated versus incubated with NAC is indicated by an asterisk . B : Tyrosine-701 phosphorylated and total Stat1, ICAM-1, and β-actin protein levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated in media without or with NAC for 1 hour. Cells were then incubated in media containing the antioxidant without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 8 hours with the same compounds without or with IFN-γ.

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone 6 against human interferon regulatory factor-9 (IRF-9) from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal IgG 4915 against human ICAM-1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9171 against tyrosine-701 phosphorylated human Stat1 from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI); mouse IgG2a mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG ab4742 against serine-727 phosphorylated human Stat1 from Abcam (Cambridge, MA); goat polyclonal IgG against human RSV proteins from Biodesign International (Saco, ME).

Techniques: Incubation, Western Blot

Journal: Respiratory Research

Article Title: Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

doi: 10.1186/1465-9921-11-64

Figure Lengend Snippet: GSH monoethyl ester inhibits cigarette smoke effects on type II interferon-induced responses . A : ICAM-1 cell surface protein levels were determined using an enzyme-linked immunoassay with hTBE cells that were first treated in media without or with GSH-MEE for 2 hours. Cells were then incubated in media containing the antioxidant without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 20 hours with the same compounds without or with IFN-γ. Values are expressed as mean ± S.D. ( n = 3 replicates) and a significant difference ( p < 0.01) in comparable CSE-treated levels between cells not incubated versus incubated with GSH-MEE is indicated by an asterisk . B : Tyrosine-701 phosphorylated and total Stat1, ICAM-1, and β-actin protein levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated in media without or with GSH-MEE for 2 hours. Cells were then incubated in media containing the antioxidant without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 8 hours with the same compounds without or with IFN-γ.

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone 6 against human interferon regulatory factor-9 (IRF-9) from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal IgG 4915 against human ICAM-1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9171 against tyrosine-701 phosphorylated human Stat1 from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI); mouse IgG2a mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG ab4742 against serine-727 phosphorylated human Stat1 from Abcam (Cambridge, MA); goat polyclonal IgG against human RSV proteins from Biodesign International (Saco, ME).

Techniques: Incubation, Western Blot

Journal: Respiratory Research

Article Title: Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

doi: 10.1186/1465-9921-11-64

Figure Lengend Snippet: Model for cigarette smoke effects on type II interferon signal transduction . Cigarette smoke inhibits type II interferon-dependent gene expression by decreasing Stat1 phosphorylation. A portion of this effect is mediated by reactive oxygen species (ROS). Decreased antiviral gene expression decreases epithelial cell responses to IFN-γ that inhibit viral infection.

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone 6 against human interferon regulatory factor-9 (IRF-9) from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal IgG 4915 against human ICAM-1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9171 against tyrosine-701 phosphorylated human Stat1 from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI); mouse IgG2a mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG ab4742 against serine-727 phosphorylated human Stat1 from Abcam (Cambridge, MA); goat polyclonal IgG against human RSV proteins from Biodesign International (Saco, ME).

Techniques: Transduction, Gene Expression, Phospho-proteomics, Infection

Journal: Respiratory Research

Article Title: Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia

doi: 10.1186/1465-9921-10-96

Figure Lengend Snippet: Cytokine-induced Stat1 activation does not persist during subsequent TLR2 stimulation . A) Phosphorylated and total Stat1 cellular protein levels were assessed using immunoblot analysis of extracts from human airway epithelia that were treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 30 min. B). Phosphorylated and total Stat1 cellular protein levels were assessed using immunoblot analysis of extracts from human airway epithelia that were first treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 24 hours, followed by incubation without or with Pam3CSK4 (25 mg/ml) for 30 min.

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone L35A5 against human IκBα, rabbit polyclonal IgG 9171 against human Stat1 phosphorylated on tyrosine-701, rabbit polyclonal IgG 9172 against human total Stat1, rabbit IgG mAb clone 3D7 against human p38 MAP kinase phosphorylated on threonine-180 and tyrosine-182, rabbit IgG mAb clone 7D6 against human total p38 MAP kinase from Cell Signaling Technology (Beverly, MA); mouse IgG2α mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI).

Techniques: Activation Assay, Western Blot, Incubation