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. 2020 Mar 30;40(8):e00471-19. doi: 10.1128/MCB.00471-19

FIG 7.

FIG 7

P53 plays a critical role in Ipr1-regulated Irgm1 expression. (A) Knockdown of Ipr1 impairs the luciferase activity of signal pathway reporter construct p53-luc. RAW264.7 cells were cotransfected with siIpr1-1, siIpr1-2, and siNC and the reporter plasmid p53-luc, and then, the cells were transfected with pcDNA3.1 or pcDNA3.1-Ipr1. After 36 h, the luciferase activity was determined by DLR. The values were normalized to the mean of the pcDNA3.1 siNC group (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (B) Knockdown of p53 led to downregulation of Irgm1 and p21 transcription in RAW264.7 cells. RAW264.7 cells were transfected with sip53-1 or siNC for 48 h. Then, Act D (5 nM) was added for 8 h. Transcription of Irgm1 and p21 was determined by qRT-PCR. The values were normalized to the mean of those in the DMSO siNC groups (+SD). The statistical significance of differences in relative mRNA expression was assessed by two-way ANOVA. (C) PFT-α inhibited the expression of p21 and Irgm1. The cells were treated with ADR (200 nM) for 24 h. Then, PFT-α (30 μM) was added for 8 h. Transcription of Irgm1 and p21 was determined by qRT-PCR. The values were normalized to the mean of those in the PBS DMSO groups (+SD). The statistical significance of differences in relative mRNA expression was assessed by two-way ANOVA (**, P < 0.01). (D) P53 increased Irgm1 promoter activity. RAW264.7 cells were cotransfected with pCMV-p53, pCMV-myc, and Irgm1-PRO-500, Irgm1-PRO-900, and Irgm1-PRO-1400 for 36 h. Promoter transcriptional activities were examined by luciferase assay. Values were normalized to the mean of the Irgm1-PRO-500 pCMV-myc group (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (E) P53 induced Irgm1 and p21 expression. RAW264.7 cells were transfected with pCMV-myc and pCMV-p53 for 36 h. Gene expression was examined by qRT-PCR. The values of pCMV-p53 groups were normalized to the mean of those in the pCMV-myc groups (+SD). The statistical significance of differences in relative mRNA expression was assessed by two-way ANOVA (**, P < 0.01). (F) Mutation of the p53 element in the Irgm1 promoter region. The p53 motif was predicted in the Irgm1 promoter using the JASPAR database. Nucleotide mutation relied on the conservation of specific sites in the p53 motif.