Fig 1. IFN-I upregulates the lysine methyltransferase Setdb2 in Mϕ.
(A) Comparison of chromatin-modifying enzymes in unstimulated (control) and IFN-I-stimulated BM-Mϕ by RT-PCR. Values on scatter plot represent log10 (ΔCt) at 24 hours post-stimulation. The gray lines represent a 4-fold change in gene expression; upregulated genes (red dots), downregulated genes (green dots). (B) RT-PCR of SET-domain superfamily genes in BM-Mϕ treated with a vehicle control (dotted line) or IFN-I for 24 hours. (C-E) RT-PCR of Setdb2 by BM-Mϕ stimulated with 1, 10, and 100 units/mL of IFN-I for 5 hours (C), stimulated with IFN-I for 5, 24, and 48 hours (D), and stimulated with IFN-I, IFN-II, and IFN-III for 5 hours (E). (B-E) Data are mean ± SEM relative to unstimulated BM-Mϕ, n = 3–4 independent experiments. (F, G) lacZ expression (F) and MFI (G) in BM-Mϕ isolated from Setdb2 LacZ reporter mice was determined by flow cytometry 24 hours post-IFN-I stimulation. Data are mean ± SEM gating on CD11b+ cells, n = 3 independent experiments. (H, I) C57BL/6 (H) and Setdb2 LacZ reporter (I) mice were inoculated with PBS or IAV (1 x 104 PFU). (H) RT-PCR of Ifnb1 and Setdb2 in IAV-infected lungs on day 4 post-infection. Data represent fold-change relative to uninfected lungs; data are mean ± SEM, n = 8–12 mice. (I) lacZ expression by CD11b+ cells in the lungs and spleen was determined by flow cytometry; PBS (black), IAV (red). Histograms are a representative example from 3 independent experiments (n = 8–12 mice). (J, K) RT-PCR of Setdb2 in human PBMCs (J) and M-CSF-skewed Mϕ (K) stimulated with IFN-I over the indicated time course. (L) RT-PCR of SET-domain superfamily genes in human Mɸtreated with a vehicle control (dotted line) or IFN-I for 72 hours. (M, N) RT-PCR of Setdb2 in murine AECs (M) and NHBEs (N) stimulated with IFN-I for the indicated time course. (J-N) Data are mean ± SEM relative to unstimulated cells (dotted line), n = 3–5 independent experiments. N.D.; not detected. *p<0.05; **p<0.01; ***p<0.001.