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

FIG 6.

FIG 6

Knockdown of Ipr1 decreases Irgm1 transcription. (A) Knockdown of Ipr1 downregulates Irgm1. siIpr1-1, siIpr1-2, and siNC were transfected into RAW264.7 cells with or without ISD treatment. The mRNAs were harvested after 36 h, and expression of Ipr1 and Irgm1 was examined by qRT-PCR. Values were normalized to the mean of the siNC ISD− group (+SD). The statistical significance of differences in relative mRNA expression was assessed by two-way ANOVA (*, P < 0.05; **, P < 0.01). (B) Knockdown of Ipr1 downregulates Irgm1 at the protein level. siIpr1-1, siIpr1-2, and siNC were transfected into RAW264.7 cells with or without ISD, and the total protein was harvested after 48 h and detected with anti-SP110, anti-LRG47, and anti-GAPDH by Western blotting. (C) Knockdown of Ipr1 decreases transcriptional activity of the Irgm1 promoter. Irgm1 promoter reporter plasmids were cotransfected with siIpr1-1 or siIpr1-2 for 48 h, and the transcriptional activities were examined by luciferase assays. Values were normalized to the mean of the siNC-treated groups (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (D) Ipr1 enhances the transcriptional activity of the Irgm1 promoter. Irgm1 promoter reporter plasmids were cotransfected with pEGFP-C1 or pEGFP-Ipr1 into RAW264.7 and J774A.1 cells for 36 h, and then, the transcriptional activities were examined by luciferase assays. The Tnf-α promoter reporter plasmid was used as a positive control. The values of the pEGFP-Ipr1 groups were normalized to the mean of the pEGFP-C1 groups (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (E) Downregulation of Irgm1 and p21 after Ipr1 knockdown. RAW264.7 cells were transfected with siIpr1-1 and siNC for 36 h, and then the cells were treated with ISD for 8 h. The expression was determined by qRT-PCR. Values were normalized to the mean of those of the siNC (ISD−) groups (+SD). The statistical significance of differences in relative mRNA expression was assessed by two-way ANOVA (**, P < 0.01). (F) Proportion (percent) of G0/G1 cells (t test; **, P < 0.01).