irf1 d5e4  (Cell Signaling Technology Inc)

Journal: Nucleic Acids Research

Article Title: ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA

doi: 10.1093/nar/gkaf545

Figure Lengend Snippet: Identification of ERH and other novel positive regulators of IFNγ signaling by genome-wide genetic screening. ( A ) Schematic of the IFNγ-induced JAK/STAT signaling pathway, which stimulates expression of many genes, including IRF1. ( B ) Overview of FACS-based CRISPR-Cas9 knockout screen. Human RKO cells with dox-inducible iCas9 were transduced with a lentiviral genome-wide sgRNA library. Cas9 expression was induced for 2.5 or 5 days, after which cells were treated with IFNγ, and IRF1 induction detected by intracellular staining. Cells with the lowest or highest IRF1 levels were collected by FACS, and disrupted genes were identified by analyzing sgRNA-targeted coding sequences. ( C ) sgRNA enrichment in the IRF1 low cell population was plotted. Dashed lines indicate significance ( P ≤ 0.05) and enrichment (log2 fold change ≥ 1). Significantly enriched genes involved in the JAK/STAT pathway, the exon junction complex, or RNA splicing and export are highlighted. ( D ) Heatmap of selected IRF1 regulators as in ( C ) or MYC regulators involved in JAK/STAT signaling, the EJC, splicing and export, nonsense mediated decay, or type I interferon signaling. For each gene, the time point with the strongest enrichment is plotted. ( E ) RKO-iCas9 cells were transduced with vectors expressing the indicated sgRNAs. After 5 days of dox-induced Cas9 expression, cells were stimulated with IFNγ, after which endogenous IRF1 or MYC were detected by intra-cellular staining and flow cytometry. Representative samples from four (sg MAGOH ) or five (sg AAVS1 , sg ERH , and sg JAK2 ), n = 4 or 5 biological replicates, are shown. ( F ) Quantification of median fluorescence intensity (MFI) from panel E. Data represent means and sd; n = 4 or 5 biological replicates. One-way ANOVA with Bonferroni’s multiple comparison correction (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). ( G ) In parallel, IRF1 mRNA levels were measured by RT-qPCR. Data represent the mean and sd; n = 3 biological replicates. Two-tailed t-test with Benjamini–Hochberg correction (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001).

Article Snippet: IRF1 , FACS , 1:100 , PE , Cell Signaling Technology , 12 732 , D5E4 XP , Primary.

Techniques: Genome Wide, Expressing, CRISPR, Knock-Out, Transduction, Staining, Flow Cytometry, Fluorescence, Comparison, Quantitative RT-PCR, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Macrophages are activated toward phagocytic lymphoma cell clearance by pentose phosphate pathway inhibition

doi: 10.1016/j.xcrm.2024.101830

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-IRF1 (D5E4) , Cell Signaling Technology , Cat#8478; RRID: AB_10949108.

Techniques: Staining, Virus, Recombinant, Bicinchoninic Acid Protein Assay, Cell Viability Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Blocking Assay, Phospho-proteomics, Purification, Mass Spectrometry, Software, Modification

Journal: Nature

Article Title: Epithelial IFNγ signalling and compartmentalized antigen presentation orchestrate gut immunity

doi: 10.1038/s41586-023-06721-1

Figure Lengend Snippet: C57BL/6 J mice were infected with Citrobacter rodentium. (A) Citrobacter colonization in the colon (n = 5 per group) and (B) quantification of Ifng transcript in colon tissue by RT-PCR (n = 4 for d0 and d21, n = 6 for d6 and n = 5 for d12). STAT1 pTyr701 analyses in IECs by (C) immunoblotting, representative of two independent experiments and (D) flow cytometry (n = 3 for d0, n = 6 for d6 and n = 5 for d12 and n = 4 for d21). (E) Quantification of Irf1 transcript in purified IECs (EpCAM+) by RT-PCR (n = 4 for d0, d6 and d21, n = 5 for d12). Flow cytometric analyses for (F and G) MHCI (n = 4 for d0, n = 5 for d6 and d12) and (H and I) MHCII (n = 4 for d0, n = 5 for d6 and d12) in the IECs from colons of mice at indicated day post-infection. (J) Colon histology score in Ifngr1fl/fl and Ifngr1fl/fl VilCre mice. The horizontal bar represents the median, and each symbol and each lane in the immunoblot represents an individual mouse. Data were analyzed by (A, B, D, E, G and I) Brown-Forsthye and Welsch ANOVA tests followed by the Dunnet’s post-hoc test.

Article Snippet: Blocking was performed in 3% bovine serum albumin in trisbuffered saline with Tween-20 for 1 h, and membranes were incubated with primary antibodies Phospho-Stat1 (Tyr701) (58D6) no. 9167, IRF1 (D5E4) XP no. 8478 or GAPDH (D16H11) XP no. 5174 from Cell Signalling technology at 1:1,000 dilution overnight at 4 °C.

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Purification

Journal: Nature

Article Title: Epithelial IFNγ signalling and compartmentalized antigen presentation orchestrate gut immunity

doi: 10.1038/s41586-023-06721-1

Figure Lengend Snippet: Ifngr1fl/fl and Ifngr1fl/flVilCre mice were infected with C. rodentium. a–c, Citrobacter colonization in colon (a), colon length (b) and enumeration (c) of the CD11b+Ly6G+ cells in the epithelium at the indicated day p.i. and representative flow cytometry plot gated on CD45+Lin− (EpCAM, Ter119, CD19, TCR−) cells at day 12 (n = 5 per group). d, Representative pictures of H&E-stained colon sections at ×100 magnification and histology scores. e, STAT1 pTyr701 analyses in EpCAM+ cells by flow cytometry and representative flow cytometry plot gated on EpCAM+CD45− cells at day 12 (n = 5 per group). f, Quantification of Irf1 transcript in purified EpCAM+ IECs (n = 4 Ifngr1fl/fl and n = 6 Ifngr1fl/flVilCre). g, Ifngr1fl/fl, Ifngr1fl/flVilCreERT2 and Ifngr1fl/flVilCreERT2R26LSLIrf1 mice were infected with C. rodentium on day 0, injected with tamoxifen at days 3–6 p.i., and analysed on day 12 (n = 5 per group). h, Immunoblotting for IRF1 in the purified IECs (representative of two independent experiments). i, Flow cytometric analyses of IFNγR1 in IECs. j, Colon length. k, Enumeration of the proportion of CD11b+Ly6G+ cells in CD45+Lin− (EpCAM, Ter119, CD19, TCR−) cells in the epithelial compartment. l, Histology score and representative H&E-stained colon section at ×100 magnification (n = 5 per group). The horizontal bar represents the median, and each symbol and lane in the immunoblot represents an individual mouse. Data were analysed by analysis of variance (ANOVA) tests (a–f,k) followed by the Dunnet’s or Sidak post hoc test or Kruskal–Walis test followed by Dunn’s post-test (j,l). Scale bar, 100 μm. CFU, colony-forming unit; MFI, median fluorescence intensity.

Article Snippet: Blocking was performed in 3% bovine serum albumin in trisbuffered saline with Tween-20 for 1 h, and membranes were incubated with primary antibodies Phospho-Stat1 (Tyr701) (58D6) no. 9167, IRF1 (D5E4) XP no. 8478 or GAPDH (D16H11) XP no. 5174 from Cell Signalling technology at 1:1,000 dilution overnight at 4 °C.

Techniques: Infection, Flow Cytometry, Staining, Purification, Injection, Western Blot, Fluorescence

Journal: Nature

Article Title: Epithelial IFNγ signalling and compartmentalized antigen presentation orchestrate gut immunity

doi: 10.1038/s41586-023-06721-1

Figure Lengend Snippet: Ifngr1fl/fl and Ifngr1fl/flVilCre mice were infected with C. rodentium. a, Quantification of MHCI expression on the surface of IECs (n = 5 per group). b, Ifngr1fl/fl and Ifngr1fl/flVilCre mice were infected with CitroOva, and IECs were analysed for the presentation of H2Kb:Ova complex on the surface by flow cytometry (n = 5 per group). c–g, B2m fl/fl and B2m fl/flVilCreERT2 mice were infected with ova-expressing Citrobacter on day 0, treated with tamoxifen on days 3–6, and sacrificed on day 12 p.i. Expression of CD39 by CD8αβ+ (c) and CD4+ (d) IE-T cells (n = 5 per group). Entpd5 expression in purified CD8αβ+ and CD4+ IE-T cells (e) (n = 6 per group), GM-CSF production from ova-specific CD4+ IE-T cells (f) and colon length (g) (n = 5 per group). Each symbol represents an individual mouse, and the bar represents the median. h, Structural prediction by motif analyses. i–k, Flow cytometric staining for IFNγR2 in WT and Ifngr2P32L cell lines (four biological replicates) (i). These cells were stimulated with IFNγ and evaluated for STAT1 pTyr701 and IRF1 induction (representative of two independent experiments) (j) and MHCI expression on the cell surface by flow cytometry (k). Each symbol represents an individual human patient, and the bar represents the median. Data were analysed by ANOVA tests (a,b) followed by the Dunnet’s post hoc test, Mann–Whitney U test (two-sided) (c–g) or Kruskal–Wallis test followed by Dunn’s post-test (h,l). Scale bar, 100 μm. MFI, median fluorescence intensity.

Article Snippet: Blocking was performed in 3% bovine serum albumin in trisbuffered saline with Tween-20 for 1 h, and membranes were incubated with primary antibodies Phospho-Stat1 (Tyr701) (58D6) no. 9167, IRF1 (D5E4) XP no. 8478 or GAPDH (D16H11) XP no. 5174 from Cell Signalling technology at 1:1,000 dilution overnight at 4 °C.

Techniques: Infection, Expressing, Flow Cytometry, Purification, Staining, MANN-WHITNEY, Fluorescence

Journal: Nature

Article Title: Epithelial IFNγ signalling and compartmentalized antigen presentation orchestrate gut immunity

doi: 10.1038/s41586-023-06721-1

Figure Lengend Snippet: Ovalbumin coding sequence was cloned into the NocI site of pOBX18 plasmid containing the artificial and constitutive Enterobacteriaceae-specific promoter and terminator sequence into the temperature-sensitive transposase 7 attachment site (attTn7) targeting vector (vector described in59) (A) plasmid map and (B) gel picture of the transformed vector. Then, this vector was electroporated into C. rodentium. Growth at permissive temperature along with constitutive expression of the recombination machinery led to the integration of the promoter-ova-terminator sequence into the attTn7 site. Next, the plasmid was cured by subsequent growth at non-permissive temperatures. Site-specific integration was confirmed by (C) PCR and sequencing from the neighboring glmS region of the attTntn7 locus and the expression of ova was confirmed by (D) RT-PCR, representative of two independent clones. (E) Ifngr1fl/fl and Ifngr1 fl/fl VilCre mice were infected with ova+ Citrobacter and (F) Ifngr fl/fl VilcreERT2 OvaTg and Ifngr fll+ VilcreERT2 OvaTg mice were infected with wild-type Citrobacter and administered tamoxifen on days 3–6 p.i and evaluated for IFN-γ production from the ova-specific CD4+ IE-T cells (n = 4 per group). (G) Flow cytometric phenotyping of ova-specific CD4+ IE-T cells at day 12 post-infection with ova+ Citrobacter. (H-K) Ifngr1fl/fl, Ifngr1fl/fl VilCreERT2, and Ifngr1fl/fl VilCreERT2 R26LSL Irf1 mice were infected with ova+ Citrobacter rodentium on day 0, injected with tamoxifen at days 3–6 post-infection, and analyzed on day 12 for CD4+ IE-T cells and (I) Citrobacter colonization in the colon (n = 5 per group). (L) Ifngr1fl/fl and Ifngr1 fl/fl VilCre mice were infected with Citrobacter and treated with isotype or anti-CD4 antibody every 2 days starting at day 7 analyzed for CD4+ T cells on day 12 p.i. (n = 8 per group). The horizontal bar represents the median, and each symbol represents an individual mouse. Data were analyzed by (E and F) Mann-Whitney U test (two-sided) or (H-L) Kruskal-Wallis test followed by Dunn’s post-test.

Article Snippet: Blocking was performed in 3% bovine serum albumin in trisbuffered saline with Tween-20 for 1 h, and membranes were incubated with primary antibodies Phospho-Stat1 (Tyr701) (58D6) no. 9167, IRF1 (D5E4) XP no. 8478 or GAPDH (D16H11) XP no. 5174 from Cell Signalling technology at 1:1,000 dilution overnight at 4 °C.

Techniques: Expressing, Sequencing, Clone Assay, Plasmid Preparation, Transformation Assay, Reverse Transcription Polymerase Chain Reaction, Infection, Injection, MANN-WHITNEY