Fig. 6.

SRSF10-mediated mIL1RAP upregulates the expression of CD47 via promoting IL-1β-induced NF-κB activation. a, b Identification of SRSF10 as an enhancer of IL-1β-induced NF-κB activation by luciferase reporter assay. Ms751 and SiHa cells were infected with either retrovirus expressing SRSF10 or empty-vector control (vector), then the cells were further transfected with NF-κB luciferase reporter. A total of 20 h after transfection, cells were treated with IL-1β (10 ng/mL) or left untreated for 8 h before reporter assays. c Western blot analysis of p-IκBα, IκBα, p-NF-κB p65, NF-κB p65, and CD47 was performed in Ms751 and SiHa cells indicated in a, b. Cells were treated with IL-1β (10 ng/mL) for 8 h. d, e Luciferase reporter assay to detect the effect of SRSF10-mediated mIL1RAP on IL-1β-induced NF-κB activation. Ms751 and SiHa cells were infected with either retrovirus expressing mIL1RAP or control vector, then the cells were further transfected with SRSF10-RNAi (siSRSF10-1, siSRSF10-2) or control siRNA for 36 h before NF-κB luciferase reporter transfection. Cells were treated with IL-1β indicated in a. f Western blot analysis for NF-κB activation and CD47 expression was performed in Ms751 and SiHa cells indicated in Fig. 5g and Supplementary Fig. S6d. Cells were treated with IL-1β(10 ng/mL) for 8 h. g The sketch map of primers for CD47 promoter sequences. Ten primer sets with a 300-bp partition were designed for PCR to test the direct binding of p65 to the CD47 promoter and the primer pairs produced 10 fragments of 300 bp. h The chromatin DNA of Ms751 and SiHa cells was chromatinimmunoprecipitated (ChIP) with p65 antibody. Sonicated input DNA and IgG was used as control. Amplification of the CD47 promoter sequence from ChIP DNA validated the binding of p65 to CD47 promoter site 4 and 8. i Western blot analysis was performed to detect the regulation of p65-knockdown on CD47 expression. Ms751 and SiHa cells were transfected with p65-RNAi or control siRNA. j NF-κB activation upregulated the expression of CD47 as detected by western blot. IL-1β was used as a stimulator of NF-κB. Ms751 and SiHa cells were treated with IL-1β as indicated in a. k IHC staining of SRSF10 and CD47 in tumors from Ms751 cell xenograft nude mice indicated in Fig. 5g (scale bar: 200 µm). *P < 0.05; ** P < 0.01; ***P < 0.001