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

Article Title: Mycobacterium tuberculosis inhibits autocrine type I interferon signaling to increase intracellular survival.

doi: 10.4049/jimmunol.1801303

Figure Lengend Snippet: (A) Schematic representation of experiment. (B) IRF-3 deficient RAW264.7 macrophages transfected with an IFN-β responsive luciferase gene (Invivogen) were treated with 20μg/ml of the indicated neutralizing antibodies. 1ng/ml IFN-β was added to the cells every 24 hours, and the amount of secreted luciferase was quantified at the indicated timepoints. (C-D) Ifnβ−/− BMDMs were infected Mtb H37Rv at MOI of 3 for 4h and treated with IFN-β as in Figure 1. (C) CFUs were enumerated at the indicated timepoints. (D) Nitrite levels from culture supernatants were determined using the Griess reagent. (E-F) C57BL6/6 or Ifnar1−/− BMDMs were infected with Mtb H37Rv at MOI of 3 for 4h and treated with IFN-β as in Figure 1. (E) CFUs were enumerated at the indicated timepoints. (F) Nitrite levels from culture supernatants were determined using the Griess reagent. All data shown are presented as mean ± S.E.M. of at least three independent experiments.

Article Snippet: Experiments with neutralizing antibodies were performed using InVivo Plus anti-mouse IFNAR1 and InVivo mAB mouse IgG1 isotype control (Bio Xcell).

Techniques: Transfection, Luciferase, Infection

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

Article Title: Mycobacterium tuberculosis inhibits autocrine type I interferon signaling to increase intracellular survival.

doi: 10.4049/jimmunol.1801303

Figure Lengend Snippet: (A) Ifn-β−/− BMDMs were infected with Mtb H37Rv at MOI of 3 for 4h and flow cytometry was conducted at 4hpi to measure surface receptor expression levels of IFNAR1 and IFNAR2. (B and C) Ifn-β−/− BMDMs were infected as described in the presence of 300pg/ml IFN-β. Cell lysates were collected at 20 min post infection and immunoblotted for pJAK1 (Y1022/1023), total JAK1, pTYK2 (Y1054/1055), and total TYK2. (D and E) Ifn-β−/− BMDMs were infected as described in the presence of 300pg/ml IFN-γ. Cell lysates were collected at 20 min post infection and immunoblotted for pJAK1 (Y1022/1023), total JAK1, pTYK2 (Y1054/1055), and total TYK2. (F) Ifn-β BMDMs were treated with DMSO or increasing amounts of a JAK inhibitor for 1 hour, then stimulated with 200pg/ml IFN-β for 4 hours. Cell lysates were then collected and immunoblotted for pSTAT1 (Y701), total STAT1, and β-actin. Densitometry was performed using ImageJ software and phosphorylated protein bands were normalized to loading control for each condition. Densitometric ratios are relative to the UI +IFNβ or the UI +IFNγ conditions. Data and densities shown represent one representative experiment out of three.

Article Snippet: Experiments with neutralizing antibodies were performed using InVivo Plus anti-mouse IFNAR1 and InVivo mAB mouse IgG1 isotype control (Bio Xcell).

Techniques: Infection, Flow Cytometry, Expressing, Software

Journal: bioRxiv

Article Title: Amyloid-β precursor protein promotes tumor growth by establishing an immune-exclusive tumor microenvironment

doi: 10.1101/2025.02.10.637339

Figure Lengend Snippet: A, Volcano plot showing increased (red) and decreased (blue) chemokine gene expression (FDR-adjusted P < 0.05) in APP sgRNA-1 versus Ctrl sgRNA LG1233 tumors. The significantly differential chemokines were labelled. B, Ctrl or APP sgRNA-1 LG1233 tumor cells were injected into male C57BL/6J mice and treated with Cxcr3 neutralizing antibodies. The percentage of CD4+ T, CD8+ T and NK cells were analyzed by flow cytometry. n=6. C, Ctrl or APP sgRNA-1 LG1233 tumor cells were injected into C57BL/6J mice and treated with Cxcr3 neutralizing antibodies. Tumor growth curves were plotted. n=6. D, Tumor growth curves of DBA/2J mice subcutaneously injected with Ctrl or APP sgRNA-1 KLN205 tumor cells and treated with Cxcr3 neutralizing antibodies. n=6-8. E-F, Gene set enrichment analysis of RNA-seq data from bulk KLN205 (E) and LG1233 (F) tumor tissues. Type I interferon signaling was compared between APP sgRNA-1 versus Ctrl sgRNA tumors. G-H, Ctrl or APP sgRNA-1 KLN205 tumor cells were injected into male DBA/2J mice and treated with Ifnar1 neutralizing antibodies. Tumor growth curves (G) and representative photos of tumors (H). n =7. I, Tumor growth curves of C57BL/6J mice subcutaneously injected with LG1233 cells with control or APP sgRNA-1 and treated with Ifnar1 neutralizing antibodies. n = 7-9. J, Ctrl or APP sgRNA-1 KLN205 tumor cells were injected into male DBA/2J mice and treated with Ifnar1 neutralizing antibodies. The percentage of CD8+ T were analyzed by flow cytometry. K, the expression level of phosphorylated TBK1 (S172) in Ctrl sgRNA and APP sgRNA-1 LG1233 tumor cells was evaluated by flow cytometry. L, The expression level of phosphorylated IRF3 (S396) in Ctrl sgRNA and APP sgRNA-1 LG1233 tumor cells was evaluated by flow cytometry. M, The expression level of phosphorylated Stat1 (Y701) in Ctrl sgRNA and APP sgRNA-1 LG1233 tumor cells was evaluated by flow cytometry. N-O, Ctrl or APP sgRNA-1 LG1233 tumor cells were treated with LPS for 2 hr. The expression of type I interferon (N) and chemokines (O) was assessed by RT-PCR. P-Q, WT LG1233 tumor cells were treated with recombinant APP E1-Fc peptide for 12 hr, followed by treatment with LPS for 2 hr. The expression of type I interferon (P) and chemokines (Q) was assessed by RT-PCR. Data are mean ± s.d. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Neutralization of Ifnar1 was accomplished by intraperitoneal injection of anti-Ifnar1 antibody (200 μg, twice a week, Bio X Cell) one day ahead of tumor injection.

Techniques: Expressing, Injection, Flow Cytometry, RNA Sequencing Assay, Control, Reverse Transcription Polymerase Chain Reaction, Recombinant