Fig. 6. The Gli1-NICD interaction targets Dvl2 and modulates NEK7/NLRP3 function in MSC-mediated immune regulation.

(A) Experimental design of Gli1 ChIP-seq analysis. BMMs were collected and fixed after co-culture with MSCs. Following chromatin shearing and Gli1 antibody selection, the precipitated DNA fragments bound by Gli1-containing protein complexes were used for sequencing. (B) Localization of Gli1-binding sites on the mouse Dvl2 gene. The 15 exons, 14 introns, 3' untranslated region (UTR), 5’ UTR, and transcription start sites (TSS) of the mouse Dvl2 gene on chromosome 11 are shown. (C) ChIP-PCR analysis of Gli1 and NICD binding to the Dvl2 promoter. Protein-bound chromatin was prepared from BMMs and immunoprecipitated with Gli1 or NICD antibodies. For sequential ChIP, the protein-bound chromatin was first immunoprecipitated with the Gli1 antibody followed by elution with a second immunoprecipitation using NICD antibody, and then the immunoprecipitated DNA was analyzed by PCR. The normal IgG was used as a negative control. (D) RNA in situ hybridization for Dvl2 transcripts in LPS-stimulated macrophages after co-culture with MSCs or CD47-deficient MSCs (n=3-4 samples/group). (E) Western blot analysis and relative density ratio of SMO, Gli1, NICD, Dvl2, NRX, NEK7, NLRP3, ASC, and cleaved caspase-1 in macrophages after co-culture with MSCs or CD47-deficient MSCs. (F) qRT-PCR analysis of IL-1β, TNF-α, IL-6, CXCL-2, and CXCL-10 in LPS-stimulated macrophages (n=3-4 samples/group). All Western blots represent three experiments, and the data represent the mean±SD. *p<0.05, **p<0.01, ***p<0.001.