FIG 5.

ISD induces Irgm1 expression through its promoter. (A and B) ISD induced Irgm1 expression in a time course in RAW264.7 cells. RAW264.7 cells were transfected with ISD (2 μg/ml). The mRNA was harvested after 0 h, 8 h, 16 h, and 24 h of treatment, and the total protein was harvested at 0 h, 24 h, 48 h, and 72 h. The expression of Irgm1 was determined using qRT-PCR and Western blotting. The values of the ISD-treated groups were normalized to the mean of the ISD 0-h group (+SD). The statistical significance of differences in relative mRNA expression was assessed by one-way ANOVA (**, P < 0.01). The proteins were determined with anti-LRG47 and anti-GAPDH. (C) Analysis of Irgm1 promoter activities. Irgm1 promoters with different lengths were transfected into RAW264.7 cells for 36 h. The relative luciferase activities of Irgm1 promoter groups were normalized to the mean of the pGL4.10 group (+SD). The statistical significance of differences in relative luciferase activities was assessed by one-way ANOVA (**, P < 0.01). (D and E) The Irgm1 promoter region has higher H3K4me3 and H3K27ac peaks in bone marrow macrophages, spleen, and thymus. The peaks of H3K4me3 and H3K27ac were obtained from the WashU EpiGenome Browser, and the peak scales were fixed from a minimum of 0 to a maximum of 30. (F) Effect of ISD on the Irgm1 promoter. Irgm1 promoter reporter plasmids were transfected into RAW264.7 cells for 24 h. Then, ISD (2 μg/ml) was transfected into RAW264.7 cells for 8 h. The transcriptional activity was examined by luciferase assay. Values were normalized to the mean of the Irgm1-PRO-500 (ISD⁻) group (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (*, P < 0.05; **, P < 0.01).