Journal: Immunity

Article Title: Differential activation of the transcription factor IRF1 underlies the distinct immune responses elicited by type I and type III interferons

doi: 10.1016/j.immuni.2019.07.007

Figure Lengend Snippet: (A) Requirement of ISGF3 complex subunits in the induction of IRF1. PH5CH8 cells transfected with 20nM of IRF9, STAT1, and STAT2-targeting siRNA, or scramble siRNA and mock-treated (black) or IFNβ-treated for 6h. Relative mean ± SD expression changes of IRF1 mRNA is plotted relative to control siRNA transfected cells (100%). (B) Immunoblot IRF1, STAT1, STAT2, and β-Actin following of Wild-type (WT) and STAT1-deficient PH5CH8 cells with IFNβ or IFNλ3 for 4h. (C) Relative mean ± SD gene expression changes of IRF1 and CXCL10 mRNA expression by qPCR following 4h of IFNβ or IFNλ3 treatment. (D) Immunoblot analysis of IRF1 β-Actin following treatment with IFNβ or IFNλ3 or IFNγ in WT and STAT2-deficient PH5CH8 cells. (E) Immunoblot analysis of phosphorylated STAT1 (Y701), total STAT1, and β-Actin in WT and STAT2-deficientPH5CH8 cells treated with IFNβ (25 IU/ml or 250IU/ml), IFNλ3, or IFNγ for 0.5h. (D-E) Saturated pixels are highlighted in red. (F) Immunoblot of IRF1, STAT1, and STAT2 expression in 2fTGH, STAT1-deficient (U3A) and STAT2-deficient (U6A) cells treated with IFNβ (500 IU/ml) or IFNγ for 4h. (G) Electromobility shift assay with nuclear extracts from IFNγ, IFNβ (125 IU/ml), IFNλ3 (500 ng/ml) treated PH5CH8 incubated with radiolabeled IRF1 probe. (H) Supershift EMSA for the identification of transcriptional regulators of IRF1. Nuclear extracts from IFNγ, IFNβ (125 IU/ml), IFNλ3 (500 ng/ml) treated PH5CH8 were co-incubated with radiolabeled IRF1 probe with indicated antibodies. (I) Immunoblot IRF1 and GAPDH analysis expression after treatment of PH5CH8 cells with IFNβ and/or IFNλ3 for 4h. (J) Immunoblot of IRF1 and β-Actin after treatment with IFNβ or increasing concentrations of IFNλ3 for 6h. Changes in mRNA expression are represented relative to mock-treated cells and normalized to HPRT. Unless otherwise indicated, cells were stimulated with 25 IU/ml of IFNβ, 100 ng/ml IFNλ3, or 5ng/ml IFNγ. Unless otherwise indicated, data is representative of 3 independent experiments. See also Figure S3.

Article Snippet: STAT2 , Life Technologies , Hs01013123_m1.

Techniques: Transfection, Expressing, Control, Western Blot, Gene Expression, Electro Mobility Shift Assay, Incubation

Journal: Immunity

Article Title: Differential activation of the transcription factor IRF1 underlies the distinct immune responses elicited by type I and type III interferons

doi: 10.1016/j.immuni.2019.07.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: STAT2 , Life Technologies , Hs01013123_m1.

Techniques: Virus, Recombinant, Cell Recovery, RNA Sequencing, Negative Control, Software

Journal: Science advances

Article Title: N-MYC impairs innate immune signaling in high-grade serous ovarian carcinoma.

doi: 10.1126/sciadv.adj5428

Figure Lengend Snippet: Fig. 3. N-MYC represses IFN type I immune signaling. (A) qRT-PCR of IFN type I (IFNA2 and IFNB1) and III (IFNL1, IFNL2, and IFNL3) ligands in CaOV3 MYCN/GFP and GFP cells treated ± DOX (1 μg/ml) for 72 hours. P values were calculated using an unpaired two-tailed Student’s t test. (B) qRT-PCR of IFNAR1 and IFNLR1 (top) and ISGs (bottom) in CaOV3 wild-type cells transfected with scrambled negative control siRNA or siRNAs specific for IFNAR1 and IFNLR1 for 72 hours. (C) Immunoblot of P-STAT1, STAT1, P-STAT2, STAT2, IRF9, and α-tubulin levels in CaOV3 MYCN/GFP and CaOV3 GFP cells treated ± DOX for 24 and 72 hours. Data are representative of three independent experiments. Paired comparisons are shown in the same color for densitometry fold changes. (D) Immunoblot with the indicated antibodies in CaOV3 MYCN/GFP and GFP cells pretreated ± DOX for 72 hours and then treated with IFNB1 (10 ng/ml) for 2 hours. Nuclear and cytoplasmic fractions were prepared and subjected to Western blot. Data are representative of three independent experiments. (E) ChIP-qPCR analysis of IRF9 binding to the ISRE sequence of the ISG15 promoter in CaOV3 MYCN/GFP and GFP cells pretreated ± DOX for 72 hours and then treated with IFNB1 (50 ng/ml) for 30 min. P values were calculated using one-way analysis of variance (ANOVA) with Tukey posttest for pairwise comparison. (F) qRT-PCR of multiple ISGs in CaOV3 MYCN/GFP and GFP cells pretreated ± DOX (1 μg/ml) for 72 hours followed by IFNB1 pulse (10 ng/ml) for 2 hours and then 24-hour chase. P values were calculated using one-way ANOVA with Tukey posttest for pairwise comparison. Data in (A), (B), (E), and (F) are means ± SEM of n = 3 biological replicates. *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001. Densitometry analysis was performed using ImageJ software.

Article Snippet: The following antibodies were used in this study: anti- Hu Fc receptor binding inhibitor (#50- 112- 9053, eBioscience), P- STAT1 (Tyr701) (#9167, Cell Signaling Technology), STAT1 (#14994, Cell Signaling Technology), P- STAT2 (Tyr690) (#4441, Cell Signaling Technology), STAT2 (#72604, Cell Signaling Technology), IRF9 (#76684, Cell Signaling Technology), α- tubulin (#3873, Cell Signaling Technology), DYKDDDDK Tag rabbit monoclonal antibody (mAb) (#14793, Cell Signaling Technology), DYKDDDDK Tag mouse mAb (#8146, Cell Signaling Technology), STING (#13647, Cell Signaling Technology), TATA box–binding protein (#8515, Cell Signaling Technology), CD3- PECy7 (#341111, BD Bioscience Technology), RIG- I (#3743, Cell Signaling Technology), MDA- 5 (#5321, Cell Signaling Technology), P- TBK1 (Ser172) (#5483, Cell Signaling Technology), TBK1 (#3504, Cell Signaling Technology), P- IRF3 (Ser396) (#4947, Cell Signaling Technology), N- MYC (#51705, Cell Signaling Technology), VDAC (#4661, Cell Signaling Technology), MAVS (#24930, Cell Signaling Technology), c- MYC (#5605, Cell Signaling Technology), L- MYC (#76266, Cell Signaling Technology), P- STING (Ser366) (#50907, Cell Signaling Technology), hemagglutinin tag (#3724, Cell Signaling Technology), Myc tag (#2278, Cell Signaling Technology), cGAS (#15102, Cell Signaling Technology), anti- rabbit immunoglobulin G (IgG) (H+L) (DyLight 800 4× polyethylene glycol conjugate) (#5151, Cell Signaling Technology), and anti- mouse IgG (H+L) (DyLight 680 conjugate) (#5470, Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Two Tailed Test, Transfection, Negative Control, Western Blot, ChIP-qPCR, Binding Assay, Sequencing, Comparison, Software

Journal: eLife

Article Title: Multiple tumor suppressors regulate a HIF-dependent negative feedback loop via ISGF3 in human clear cell renal cancer

doi: 10.7554/eLife.37925

Figure Lengend Snippet:

Article Snippet: Antibody , STAT2 (rabbit polyclonal) , Bethyl A303-512A , , , , (1:1,000 for western blot, 1:25 for IHC).

Techniques: Western Blot, Recombinant

Journal: Molecular Metabolism

Article Title: Baricitinib counteracts metaflammation, thus protecting against diet-induced metabolic abnormalities in mice

doi: 10.1016/j.molmet.2020.101009

Figure Lengend Snippet: Baricitinib attenuates the HD-induced JAK-STAT pathway. Western blotting analysis for phosphorylation of Tyr 1007/1008 JAK1/2 in the skeletal muscle (A) and kidneys (B) and normalized to total JAK1/2 and for phosphorylation of Tyr 690 on STAT2 in the skeletal muscle (C) and kidneys (D) and normalized to total STAT2. All of the data are expressed as mean ± SEM for n = 6 per group. ∗p< 0.05 vs ND and •p< 0.05 vs HD.

Article Snippet: The antibodies used were rabbit anti-Tyr 1007/1008 JAK2 (#3776), rabbit anti-total JAK2 (#3230), rabbit anti-Tyr 690 STAT2 (Bioss Antibodies, bs-3428R), rabbit anti-total STAT2 (#72604), rabbit p21 (#2947), rabbit anti-Ser 307 IRS-1 (#2381), mouse anti-total IRS-1 (#3194), rabbit anti-Ser 473 AKT (#4060), rabbit anti-total AKT (#9272), rabbit anti-Ser 9 GSK-3β (#9332) and rabbit anti-total GSK-3β (9315).

Techniques: Western Blot