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

FIG 4.

FIG 4

The ISRE motif plays a critical role in ISD-induced Ipr1 expression. (A) ISRE is the core motif in the Ipr1 promoter. RAW264.7 cells were transfected with wild-type and mutated Ipr1 promoter reporter plasmids for 24 h, and transcriptional activity was examined by dual-luciferase assay. The values of wild-type and mutated Ipr1 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). (B) Mutation of the ISRE motif impairs ISD-induced Ipr1 expression. RAW264.7 cells were transfected with wild-type and mutated Ipr1 promoter reporter plasmids for 24 h, followed by 8 h of transfection with ISD. Transcriptional activity was examined by dual-luciferase assay. The values of wild-type and mutated Ipr1 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 two-way ANOVA (**, P < 0.01). (C) Effect of STAT1 and/or ISD on the Ipr1 promoter. pCMV-HA or pCMV-HA-STAT1 was cotransfected with Ipr1-PRO-594 or Ipr1-P594-STATmut, respectively, into RAW264.7 cells for 36 h, and then the cells were treated with ISD for 8 h and the transcriptional activity was determined by luciferase assay. Values were normalized to the mean of the ISD− pCMV-HA+ group (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (D) Effect of STAT1 on Ipr1-P594-ISREmut. pCMV-HA or pCMV-HA-STAT1 was cotransfected with Ipr1-PRO-594 or Ipr1-P594-ISREmut, respectively, into RAW264.7 cells for 36 h, and the transcriptional activity was determined by luciferase assay. Values were normalized to the mean of the pGL4.10 pCMV-HA+ group (+SD). The statistical significance of differences in relative luciferase activity was assessed by two-way ANOVA (*, P < 0.05; **, P < 0.01). (E) Effect of IRF3 on the Ipr1 promoter. pCMV-myc or pCMV-IRF3 was cotransfected with Ipr1-PRO-594 or Ipr1-P594-ISREmut, respectively, into RAW264.7 cells for 36 h, and then transcriptional activity was determined by luciferase assay. The values of various treated groups were normalized to the mean of the Ipr1-PRO-594 group (+SD). The statistical significance of differences in relative luciferase activities was assessed by one-way ANOVA (*, P < 0.05; **, P < 0.01). (F) IRF3 increased Ipr1 transcription. RAW264.7 cells were transfected with pCMV-Myc and pCMV-IRF3 for 24 h, and then Ipr1 expression was measured by qRT-PCR (t test; **, P < 0.01). (G) ChIP analysis of IRF3 on the Ipr1 promoter region. RAW264.7 cells were transfected with pCMV-Flag-IRF3, and ChIP was performed using anti-Flag antibody, anti-H3 as a positive control antibody, and anti-IgG as a control antibody to detect enriched fragments. The values of ChIP groups were normalized to the mean of the IgG groups (+SD). The statistical significance of differences in relative enrichment was assessed by two-way ANOVA (**, P < 0.01). (H) the ISRE motif is critical for protein-promoter interaction. The sequence tgctcagtgtgctgagatcactttCATTTTTCTTTTCttgaagcctgact (lowercase letters are the flanking sequences of the ISRE motif) with biotinylation at the 5′ end was used as a nucleic acid probe (Ipr1-Biotin) and without modification was used as Ipr1-control, and the core sequence (CATTTTTCTTTTC) was mutated to CACCCTCACCTTTC and used as Ipr1-mut-Biotin. As shown, there was a shift band in the lane loaded with lysates and Ipr1-Biotin compared to Ipr1-mut-Biotin, as well as a significant decrease in the protein-probe complex when Ipr1-control was added.