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

FIG 2.

FIG 2

ISD induces Ipr1 expression through its promoter. (A) ISD activates Ipr1 expression in murine macrophages (RAW264.7 and J774A.1 cells), BMDMs, and fibroblasts (NIH 3T3 cells). The cells were transfected with ISD (2 μg/ml). After 36 h, the expression of Ipr1 was determined by qRT-PCR analysis. The values of ISD+ groups were normalized to the mean of ISD− groups (+SD). The statistical significance of differences in relative mRNA expression was assessed by two-way ANOVA (*, P < 0.05; **, P < 0.01). (B) ISD induced Ipr1 expression in RAW264.7 cells and BMDMs by Western blotting in a time gradient. RAW264.7 cells and BMDMs were transfected with ISD for the indicated time intervals, and then protein levels were determined using anti-SP110 and anti-GAPDH by Western blotting. (C) ISD upregulates transcriptional activity of Ipr1 promoter reporter constructs in RAW264.7 and J774A.1 cells. Cells were transfected with promoter reporter constructs and the pGL4.73 vector for 24 h, and then ISD was transfected and the cells were incubated for 8 h. The transcriptional activity of reporter constructs was determined by luciferase assays. Values were normalized to the mean of the pGL4.10 (ISD−) group (+SD). The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (D) ISD activates luciferase activities of pathway reporter constructs ISRE-luc and NF-κB-luc. RAW264.7 cells were transfected with reporter plasmids pISRE-luc, pNF-κB-luc, pSRE-luc, pNFAT-luc, and pGRE-luc. After 24 h of transfection, the cells were transfected with 2 μg/ml ISD for 8 h, and then, the activity (+SD) of reporter constructs was determined by luciferase assays. The statistical significance of differences in relative luciferase activities was assessed by two-way ANOVA (**, P < 0.01). (E) Homology analysis of the Ipr1 promoter region in different species. The different species’ Ipr1 promoter regions were downloaded from the UCSC database, and the sequence alignment was done with DNAMAN. (F) Classified analysis of transcription factors in different species’ Ipr1 promoters. The transcription factors of various species’ Ipr1 promoters were predicted using the JASPAR database with a relative profile score threshold of 80%. The clustering analysis used the online tool Venny 2.1.0 (bioinfogp.cnb.csic.es/tools/venny). (G) ISD activates SP110 expression in THP-1 cells. THP-1 cells were transfected with ISD for 24 h. The expression of Ipr1 was determined by qRT-PCR analysis (t test; **, P < 0.01).