Figure 6.
Increased IFN-γ signaling in A20-deficient HSCs. (A) Real-time PCR data indicating expression levels of Ifn-γ transcripts in the BM cells and splenocytes of 14-d-old A20Hem-KO and control mice. Expression levels of Ifn-γ were normalized to Hprt levels. (B) FACS plots indicating YFP expression in the BM cells and splenocytes of IFN-γ–YFP reporter (GREAT) mice, crossed with either control or A20Hem-KO mice. (A and B) Data are representative of three independent experiments. (C) Diagrammatic representation of the Ifn-γ gene indicating the presence of five NF-κB–binding sites in its promoter. (D) NF-κB–binding site in the promoter of Ifn-γ gene of the indicated species. (E) ChIP analysis of NF-κB (p65) binding to the Ifn-γ promoter in the BM cells of 14-d-old A20Hem-KO and control mice. Shown are the real-time PCR data of p65 immunoprecipitates, which were normalized to IgG control immunoprecipitates. Data are representative of two independent experiments. (F) Real-time PCR data indicating expression of IFN-γ target genes in CD150+LSK cells from 14-d-old A20Hem-KO and control mice. Expression levels of target genes were normalized to Hprt levels. Data are pooled from two independent experiments with six mice per group. (G and H) Representative FACS plots (G) and cumulative frequencies (H) indicating expression levels of Sca1 in Lin−c-Kit+CD150+ cells of the BM from 14-d-old A20Hem-KO and control mice. Data are representative of 10 independent experiments (G) and are pooled from six mice per group (H). (I) Real-time PCR data indicating expression levels of IFN-γ–controlled cell cycle regulators in HSCs of 14-d-old A20Hem-KO and control mice. Expression levels of target genes were normalized to Hprt levels. Data are representative of two independent experiments with six mice per group. All data represent mean ± SEM. Two-tailed Student’s t tests were used to assess statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).