Journal: mBio

Article Title: STAT1 Signaling in Astrocytes Is Essential for Control of Infection in the Central Nervous System

doi: 10.1128/mBio.01881-16

Figure Lengend Snippet: Astrocyte expression of STAT1 is required for IFN-γ-mediated control of parasite replication. (A) Primary astrocytes were stimulated with IFN-γ (100 U/ml), type I IFN (100 U/ml), IL-6 (10 ng/ml), or IL-27 (10 ng/ml) for 1 h and then assayed for phospho-STAT1. (B) Cells were infected with the Pru-Venusluc strain of T. gondii (parasite/cell ratio, 1:1) for 24 h and then assayed for phospho-STAT1. (C) Cells were infected with Pru-Venusluc for 24 h, stimulated with IFN-γ for 20 h, and then assayed for phospho-STAT1. (D) Cells were stimulated with 100 U/ml IFN-γ before the Pru-Venusluc strain of T. gondii (parasite/cell ratio, 1:1) for 24 h and then assayed for STAT1 phosphorylation by FACS. (E) To assay the ability of astrocytes to limit parasite growth, cells were stimulated with IFN-γ (100 U/ml) for 24 h and then infected with T. gondii (parasite/cell ratio, 1:1), and the percentage of infected cells was assessed microscopically after 20 h. (F) Cells from WT or STAT1 −/− mice were stimulated with 100 U/ml IFN-γ before the Pru-GFP strain of T. gondii for 1.5 h and stained for T. gondii Pru-GFP (green) and anti-IGTP (red) antibodies, with DAPI (blue) as a nuclear counterstain. Bars, 7 µm (WT) and 13 µm (STAT1 −/− ).

Article Snippet: Primary astrocytes were plated at a density of 1 × 10 5 cells/cm 2 in 12-well plates, fixed with 90% methanol, and then blocked in 2% normal goat serum (NGS) prior to incubation with antibodies for GFAP (2A5; Dako, Carpinteria, CA; 1:400), STAT1 (E-23; Santa Cruz Biotechnology, Dallas, TX; 1:200), iNOS (ab15323; Abcam, Cambridge, MA; 1:200), CD11b (M1/70; BD Bioscience; 1:200), and IGTP (BD Bioscience; 1:200), followed with appropriate secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594.

Techniques: Expressing, Infection, Staining