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

FIG 10.

FIG 10

IPR1 promotes P53 release by increasing interaction between RPL11 and MDM2, and the model of Ipr1 functions in signal transduction from ISD to Irgm1. (A) IPR1 enhanced interaction between RPL11 and MDM2. Flag-Rpl11 and HA-Mdm2 were transiently coexpressed in HEK293T cells with pEGFP-Ipr1 (lanes ii) or pEGFP-C1 (lanes i) for 48 h, and interactions were detected using anti-Flag for co-IP and anti-HA and anti-GFP for Western blotting. (B) IPR1 slightly decreased interaction between P53 and MDM2. Flag-p53 and HA-Mdm2 were transiently coexpressed in HEK293T cells with pEGFP-Ipr1 (lanes ii) or pEGFP-C1 (lanes i) for 48 h, and interactions were detected using anti-HA for co-IP and anti-Flag and anti-GFP for Western blotting. (C) IPR1 increased interaction between RPL11 and MDM2 and decreased interaction between P53 and MDM2 significantly. Flag-p53, Flag-Rpl11, and HA-Mdm2 were transiently coexpressed in HEK293T cells with pEGFP-Ipr1 (lanes ii) or pEGFP-C1 (lanes i) for 48 h, and interactions were detected using anti-HA for co-IP and anti-Flag and anti-GFP for Western blotting. (D) Knockdown of Rpl11 downregulates p53. siRpl11-1, siRpl11-2, and siNC were transfected into RAW264.7 cells with or without ISD treatment. Cells were harvested after 72 h and detected by anti-P53, anti-Rpl11, and anti-GAPDH by Western blotting. (E) ISD enhanced interaction between RPL11/IPR1 and MDM2. RAW264.7 cells were treated with ISD (lanes ii) or not (lanes i) for 60 h. Anti-MDM2 antibody was used for immunoprecipitation, and anti-Rpl11 and anti-SP110 were used for Western blotting. (F) Ubiquitination status of Flag-P53. Flag-p53, Flag-Rpl11, and HA-Mdm2 were cotransfected into HEK293T cells with pEGFP-Ipr1 or pEGFP-C1 for 60 h; 4 h prior to harvesting, cells were treated or not with MG132 (50 μM). Lysates were subjected to IP using anti-Flag. Bound proteins were analyzed by Western blotting using anti-Flag, anti-HA, anti-GFP, or anti-GAPDH. (G) Effects of ISD and Act D on THP-1 cells. THP-1 cells were treated with ISD or Act D for 16 h. Transcription of SP110, IRGM, and p21 was determined by qRT-PCR. The values were normalized to the mean of those in the ISD− or DMSO group (+SD). The statistical significance of differences in relative mRNA expression was assessed by two-way ANOVA (**, P < 0.01). (H) Effect of ISD on THP-1 cells. THP-1 cells were treated with ISD for 24 h. The proteins were determined by Western blotting using anti-SP110, anti-LRG47, and anti-GAPDH. (I) Model of Ipr1 functions in signal transduction from ISD to Irgm1. First, ISD is transfected into cells and arrested by cGAS. Then, the signals are transferred through cGAS-STING-TBK1 to IRF3, resulting in Ipr1 transcriptional activation. Afterward, IPR1 increases the release of P53 by promoting the interaction of RPL11 and MDM2. The released P53 regulates Irgm1 expression through the p53 motif in the Irgm1 promoter.