Fig 1: Ectopic ADAMTS16 expression promotes GC cell invasion and migration via the NF-??/IFI27 axis. (A) Heat map analysis showed altered genes in AGS-vector and AGS-ADAMTS16 cells. (B) Quantitative real-time polymerase chain reaction was performed to detect IFI27 mRNA expression in stably ADAMTS16-expressing cell lines. (C) Gene set enrichment analysis showed enrichment of ADAMTS16-associated genes in the HALLMARK_TNF_SIGNALING–VIA_NFKB pathway. (D) Western blot and quantitation of phospho-I?Ba, I?Ba, phospho-P65, P65, and IFI27 in stably transfected GC cell lines. (E) Western blot and quantitation were performed to analyze the expression of IFI27 in stably transfected HGC27 and AGS cells treated with or without 10 µM BAY11-7082 (NF-?B inhibitor) for 24 h. (F) Western blot and quantitation showed that ADAMTS16 overexpression promoted the phosphorylation of P65 in the nucleus. (G,H) Co-immunoprecipitation assays revealed that ADAMTS16 interacted with I?Ba in HGC27 and AGS. (I) Immunofluorescence assays revealed that ADAMTS16 was co-localized with I?Ba in HGC27 and AGS cell cytoplasm. (J) Schematic representation of IFI27 promoter organization and the corresponding luciferase reporter constructs pGL3-IFI27-WT, Mut1, and Mut2. Transcriptional start site, E1 exon1, and Luc luciferase. The blue and red bars indicate the binding sites of P65, including original and mutated sequences. (K) Dual-luciferase reporter assays were performed to analyze the activity of the pGL3-IFI27-WT, Mut1, and Mut2 constructs in HGC27 and AGS cells. Scale bar, 50 µm. The data are shown as means ± standard deviations. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.
Fig 2: IFI27 protein knockdown reverses ADAMTS16-induced GC cell promotion. (A) Western blot and quantitation analysis of IFI27 in stably ADAMTS16-transfected HGC27 and AGS cells and stably knockdown ADAMTS16–transfected MKN1 and SGC7901 cells. (B–E) Transwell assays were performed to assess cell migration and invasion in the indicated cell lines (original magnification: 200×). (F,G) colony formation assays were performed to assess cell colony formation and proliferation ability in the indicated cell lines. Scale bar, 50 µm. The data are shown as means ± standard deviations. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.
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