FIG 8.

The p53 motif plays a critical role in Irgm1 transcriptional activation. (A) Mutation of the p53 motif impairs Irgm1 promoter transcription activity. RAW264.7 cells were transfected with the luciferase reporter plasmid Irgm1-PRO-900 and the mutated plasmid Irgm1-P900-p53mut. After 36 h of transfection, Act D and ISD were added to the culture medium for 8 h. Then, the transcriptional activity was determined by luciferase assay. Values were normalized to the mean of those in the Irgm1-PRO-900-DMSO group (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (*, P < 0.05; **, P < 0.01). (B) The p53 motif plays an important role in the Irgm1 promoter. The gene expression plasmid pCMV-p53 was cotransfected with the reporter plasmids Irgm1-PRO-900 and Irgm1-P900-p53mut for 36 h. Promoter transcriptional activity was determined by luciferase assay. Values were normalized to the mean of those in the Irgm1-PRO-900-pCMV-myc group (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (C) pCMV-p53 or pCMV-myc was cotransfected with siNC or siIpr1-1 into RAW264.7 cells. After 48 h, cells were harvested and analyzed by Western blotting with anti-SP110 for IPR1 and anti-myc for myc-P53. Ipr1 siRNA, but not siNC, reduced the expression of Ipr1 successfully. (D) p53 rescued Irgm1 expression that was decreased by siIpr1-1. siNC, siIpr1-1, pCMV-myc, and pCMV-p53 were co-transfected into RAW264.7 cells for 36 h. The expression of Irgm1 was determined by qRT-PCR. Values were normalized to the mean of the siNC⁺ pCMV-myc group (+SD). The statistical significance of differences in relative mRNA expression was assessed by one-way ANOVA (**, P < 0.01).