Journal: Nature communications

Article Title: Heterozygous missense variant of the proteasome subunit β-type 9 causes neonatal-onset autoinflammation and immunodeficiency.

doi: 10.1038/s41467-021-27085-y

Figure Lengend Snippet: Fig. 1 Clinical figures and IFN signature of patients. a, b Pernio-like violaceous rash in patient 1 at 7-month-old (a) and patient 2 at 1-month-old (b). c–e myositis by leg magnetic resonance imaging (c), pulmonary hypertension by echocardiogram(d), and basal ganglia calcification by head computed tomography (CT) (e) in patient 1. f IFN scores of peripheral bloods of 11 healthy participants as controls, three Aicardi–Goutières syndrome (AGS) patients with IFIH1 variant and patient 2 at 84-day old. The score was regarded as positive if it exceeded +2 SD (5.04, dotted line) of the average (solid line) IFN score from healthy controls. Statistical analysis was performed with two-sided Mann–Whitney test. g The skin biopsy samples of a healthy participant as a control and patient 2 at 52-day-old stained with anti-p-STAT1 Ab. A scale bar, 100 μm. h The STAT1 phosphorylation assay with SV40-transformed dermal fibroblasts from healthy volunteers as controls and Patient 2. The expression levels of IFN-γ-induced p-STAT1 were enhanced in patient 2 fibroblasts compared to control fibroblasts.

Article Snippet: Expression of p-STAT1, STAT1, and β-actin was evaluated with anti-p-STAT1 (pY701) Ab (58D6, Cell Signaling 9167, 1:1000), rabbit polyclonal anti-STAT1 Abs (Stat1α (C-24), SANTA CRUZ sc-345, 1:1000) and anti-β-actin Ab (AC-74, Sigma-Aldrich A2228, AC-74, 1:2000), respectively.

Techniques: Magnetic Resonance Imaging, Computed Tomography, Variant Assay, MANN-WHITNEY, Control, Staining, Phospho-proteomics, Transformation Assay, Expressing

Journal: PLoS ONE

Article Title: Toxoplasma gondii Clonal Strains All Inhibit STAT1 Transcriptional Activity but Polymorphic Effectors Differentially Modulate IFNγ Induced Gene Expression and STAT1 Phosphorylation

doi: 10.1371/journal.pone.0051448

Figure Lengend Snippet: HFFs were infected with RH(I), RH GRA15 II , Pru(II), PruΔ gra15 , or CEP(III) parasites and/or stimulated with 100 U/ml IFNγ, subsequently fixed and permeabilized and stained with an antibody against IRF1 (red) and with Hoechst dye (blue, nucleus). A, B. HFFs were infected for three or 24 hours with GFP-expressing parasites (green), or left uninfected, and stimulated with IFNγ for the last two or six hours, or left unstimulated. Nuclear localization of IRF1 was quantified in at least 12 randomly selected cells per condition and normalized to uninfected, unstimulated cells (A) and a representative cell for each condition is shown (B). Scale bar represents 10 µm. This experiment was performed three times with similar results. Data and standard deviation from one representative experiment are shown. Asterisk (*) indicates p<0.05 compared to uninfected cells, dollar sign ($) indicates p<0.05 compared to type II infected cells. C. HFFs were infected with an RH(I) or RH GRA15 II strain, left uninfected, or left uninfected and stimulated with IFNγ for 24 hours. Nuclear localization of IRF1 was quantified in at least 30 randomly selected cells and normalized to uninfected, unstimulated cells. Data and standard deviation from one experiment are shown. Asterisk (*) indicates p<0.05 compared to uninfected cells or as represented by brackets. D. U3A STAT1-deficient cells were infected with Pru(II) or PruΔ gra15 parasites for 20 hours, left uninfected, or stimulated with IFNγ for 1 hour. Nuclear localization of IRF1 was quantified in at least 8 randomly selected cells per condition, and normalized to uninfected, unstimulated cells. This experiment was performed twice with similar results, data from one representative experiment are shown. Asterisk (*) indicates p<0.05 compared to uninfected cells, dollar sign ($) indicates p<0.05 compared to type II infected cells.

Article Snippet: Antibodies against IRF1 (BD Biosciences #612046), phospho-STAT1 Tyr701 58D6 (Cell Signaling #9167), phospho-STAT1 Ser727 (Cell Signaling #9177), total STAT1α p91 (C-24) (Santa Cruz #345), GAPDH (6C5) (Santa Cruz #32233), and Toxoplasma surface antigen (SAG)-1 (kindly provided by John Boothroyd, Stanford University) were used in immunofluorescence and Western blot assays.

Techniques: Infection, Staining, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: Toxoplasma gondii Clonal Strains All Inhibit STAT1 Transcriptional Activity but Polymorphic Effectors Differentially Modulate IFNγ Induced Gene Expression and STAT1 Phosphorylation

doi: 10.1371/journal.pone.0051448

Figure Lengend Snippet: A, B. HFFs were infected with a GFP (green) expressing RH(I), RHΔ rop16 , Pru(II), Pru ROP16 I , or CEP(III) strain, or left uninfected, for 3 hours, and 100 U/ml IFNγ was added for the last two hours of infection, or cells were left unstimulated. Cells were fixed, permeabilized, and stained with anti-phospho-STAT1 Tyr (red) and Hoechst dye (nucleus, blue). Nuclear localization of phospho-STAT1 Tyr was quantified in at least 30 randomly selected cells infected with at least three parasites (A) and a representative cell for each condition is shown (B). Scale bar represents 10 µm. This experiment was performed for each condition at least two times with similar results. Data and standard deviation from one representative experiment are shown. Asterisk (*) indicates p<0.05 compared to uninfected cells or as indicated by brackets.

Article Snippet: Antibodies against IRF1 (BD Biosciences #612046), phospho-STAT1 Tyr701 58D6 (Cell Signaling #9167), phospho-STAT1 Ser727 (Cell Signaling #9177), total STAT1α p91 (C-24) (Santa Cruz #345), GAPDH (6C5) (Santa Cruz #32233), and Toxoplasma surface antigen (SAG)-1 (kindly provided by John Boothroyd, Stanford University) were used in immunofluorescence and Western blot assays.

Techniques: Infection, Expressing, Staining, Standard Deviation

Journal: PLoS ONE

Article Title: Toxoplasma gondii Clonal Strains All Inhibit STAT1 Transcriptional Activity but Polymorphic Effectors Differentially Modulate IFNγ Induced Gene Expression and STAT1 Phosphorylation

doi: 10.1371/journal.pone.0051448

Figure Lengend Snippet: HFFs were infected with RH(I) or RHΔ rop16 parasites at an MOI ∼7, or left uninfected, for four hours. Cells were stimulated, or not, with 100 U/ml human IFNγ for the last hour of infection and cell lysates were collected, run on an SDS-PAGE gel, and Western blotted for phospho-STAT1 Ser727 , phospho-STAT1 Tyr701 , total STAT1α, IRF1, GAPDH (host cell loading control) and SAG1 (parasite loading control). This experiment has been performed three times with similar results. B. HFFs were infected with RH(I) or RHΔ rop16 parasites for four hours, stimulated with 100 U/ml IFNγ for the last two hours of infection, fixed, and stained with anti-IRF1 (red), anti-phospho-STAT1 Tyr (green), and Hoechst dye (nucleus, blue). Parasites also express GFP (green). Scale bar represents 10 µm. This experiment was performed three times with similar results. C. HFFs were infected with RH(I), RHΔ rop16 , Pru(II), or CEP(III) parasites at an MOI ∼1, or left uninfected, for four hours. Cells were stimulated, or not, with 100 U/ml human IFNγ for the last hour of infection and cell lysates were collected, run on an SDS-PAGE gel, and Western blotted for phospho-STAT1 Ser727 , phospho-STAT1 Tyr701 , total STAT1α, IRF1, GAPDH (host cell loading control) and SAG1 (parasite loading control). This experiment has been performed two times with similar results.

Article Snippet: Antibodies against IRF1 (BD Biosciences #612046), phospho-STAT1 Tyr701 58D6 (Cell Signaling #9167), phospho-STAT1 Ser727 (Cell Signaling #9177), total STAT1α p91 (C-24) (Santa Cruz #345), GAPDH (6C5) (Santa Cruz #32233), and Toxoplasma surface antigen (SAG)-1 (kindly provided by John Boothroyd, Stanford University) were used in immunofluorescence and Western blot assays.

Techniques: Infection, SDS Page, Western Blot, Control, Staining

Journal: PLoS ONE

Article Title: Toxoplasma gondii Clonal Strains All Inhibit STAT1 Transcriptional Activity but Polymorphic Effectors Differentially Modulate IFNγ Induced Gene Expression and STAT1 Phosphorylation

doi: 10.1371/journal.pone.0051448

Figure Lengend Snippet: HFFs were infected with RH(I), RH GRA15 II , Pru(II), PruΔ gra15 , or CEP(III) parasites and/or stimulated with 100 U/ml IFNγ, subsequently fixed and permeabilized and stained with an antibody against IRF1 (red) and with Hoechst dye (blue, nucleus). A, B. HFFs were infected for three or 24 hours with GFP-expressing parasites (green), or left uninfected, and stimulated with IFNγ for the last two or six hours, or left unstimulated. Nuclear localization of IRF1 was quantified in at least 12 randomly selected cells per condition and normalized to uninfected, unstimulated cells (A) and a representative cell for each condition is shown (B). Scale bar represents 10 µm. This experiment was performed three times with similar results. Data and standard deviation from one representative experiment are shown. Asterisk (*) indicates p<0.05 compared to uninfected cells, dollar sign ($) indicates p<0.05 compared to type II infected cells. C. HFFs were infected with an RH(I) or RH GRA15 II strain, left uninfected, or left uninfected and stimulated with IFNγ for 24 hours. Nuclear localization of IRF1 was quantified in at least 30 randomly selected cells and normalized to uninfected, unstimulated cells. Data and standard deviation from one experiment are shown. Asterisk (*) indicates p<0.05 compared to uninfected cells or as represented by brackets. D. U3A STAT1-deficient cells were infected with Pru(II) or PruΔ gra15 parasites for 20 hours, left uninfected, or stimulated with IFNγ for 1 hour. Nuclear localization of IRF1 was quantified in at least 8 randomly selected cells per condition, and normalized to uninfected, unstimulated cells. This experiment was performed twice with similar results, data from one representative experiment are shown. Asterisk (*) indicates p<0.05 compared to uninfected cells, dollar sign ($) indicates p<0.05 compared to type II infected cells.

Article Snippet: Antibodies against IRF1 (BD Biosciences #612046), phospho-STAT1 Tyr701 58D6 (Cell Signaling #9167), phospho-STAT1 Ser727 (Cell Signaling #9177), total STAT1α p91 (C-24) (Santa Cruz #345), GAPDH (6C5) (Santa Cruz #32233), and Toxoplasma surface antigen (SAG)-1 (kindly provided by John Boothroyd, Stanford University) were used in immunofluorescence and Western blot assays.

Techniques: Infection, Staining, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: Toxoplasma gondii Clonal Strains All Inhibit STAT1 Transcriptional Activity but Polymorphic Effectors Differentially Modulate IFNγ Induced Gene Expression and STAT1 Phosphorylation

doi: 10.1371/journal.pone.0051448

Figure Lengend Snippet: A, B. HFFs were infected with a GFP (green) expressing RH(I), RHΔ rop16 , Pru(II), Pru ROP16 I , or CEP(III) strain, or left uninfected, for 3 hours, and 100 U/ml IFNγ was added for the last two hours of infection, or cells were left unstimulated. Cells were fixed, permeabilized, and stained with anti-phospho-STAT1 Tyr (red) and Hoechst dye (nucleus, blue). Nuclear localization of phospho-STAT1 Tyr was quantified in at least 30 randomly selected cells infected with at least three parasites (A) and a representative cell for each condition is shown (B). Scale bar represents 10 µm. This experiment was performed for each condition at least two times with similar results. Data and standard deviation from one representative experiment are shown. Asterisk (*) indicates p<0.05 compared to uninfected cells or as indicated by brackets.

Article Snippet: Antibodies against IRF1 (BD Biosciences #612046), phospho-STAT1 Tyr701 58D6 (Cell Signaling #9167), phospho-STAT1 Ser727 (Cell Signaling #9177), total STAT1α p91 (C-24) (Santa Cruz #345), GAPDH (6C5) (Santa Cruz #32233), and Toxoplasma surface antigen (SAG)-1 (kindly provided by John Boothroyd, Stanford University) were used in immunofluorescence and Western blot assays.

Techniques: Infection, Expressing, Staining, Standard Deviation

Journal: PLoS ONE

Article Title: Toxoplasma gondii Clonal Strains All Inhibit STAT1 Transcriptional Activity but Polymorphic Effectors Differentially Modulate IFNγ Induced Gene Expression and STAT1 Phosphorylation

doi: 10.1371/journal.pone.0051448

Figure Lengend Snippet: HFFs were infected with RH(I) or RHΔ rop16 parasites at an MOI ∼7, or left uninfected, for four hours. Cells were stimulated, or not, with 100 U/ml human IFNγ for the last hour of infection and cell lysates were collected, run on an SDS-PAGE gel, and Western blotted for phospho-STAT1 Ser727 , phospho-STAT1 Tyr701 , total STAT1α, IRF1, GAPDH (host cell loading control) and SAG1 (parasite loading control). This experiment has been performed three times with similar results. B. HFFs were infected with RH(I) or RHΔ rop16 parasites for four hours, stimulated with 100 U/ml IFNγ for the last two hours of infection, fixed, and stained with anti-IRF1 (red), anti-phospho-STAT1 Tyr (green), and Hoechst dye (nucleus, blue). Parasites also express GFP (green). Scale bar represents 10 µm. This experiment was performed three times with similar results. C. HFFs were infected with RH(I), RHΔ rop16 , Pru(II), or CEP(III) parasites at an MOI ∼1, or left uninfected, for four hours. Cells were stimulated, or not, with 100 U/ml human IFNγ for the last hour of infection and cell lysates were collected, run on an SDS-PAGE gel, and Western blotted for phospho-STAT1 Ser727 , phospho-STAT1 Tyr701 , total STAT1α, IRF1, GAPDH (host cell loading control) and SAG1 (parasite loading control). This experiment has been performed two times with similar results.

Article Snippet: Antibodies against IRF1 (BD Biosciences #612046), phospho-STAT1 Tyr701 58D6 (Cell Signaling #9167), phospho-STAT1 Ser727 (Cell Signaling #9177), total STAT1α p91 (C-24) (Santa Cruz #345), GAPDH (6C5) (Santa Cruz #32233), and Toxoplasma surface antigen (SAG)-1 (kindly provided by John Boothroyd, Stanford University) were used in immunofluorescence and Western blot assays.

Techniques: Infection, SDS Page, Western Blot, Control, Staining