Fig 1: The involvement of IFI27 in PAAD.A Summary of the expression landscape of IFI27 proteins in multiple cancer types. B Relative expression levels of IFI27 in pancreatic cancer analyzed individually using large-scale RNA-Seq datasets of PAAD from the TCGA database (n = 350). C, D Association between the expression of IFI27, and tumor stage and grade using large-scale RNA-Seq datasets in multiple cancer types from the TCGA database. E Association between the expression of IFI27 and tumor stage using large-scale RNA-Seq datasets of pancreatic cancer from the TCGA database. F Overall survival (OS) of patients with pancreatic cancer with high or low expression of IFI27 from the TCGA database. G Bioinformatic analysis of the correlation between IFI27 and immune effector molecules using datasets from the TCGA database. TPM transcript per million.
Fig 2: The tumor angiogenesis suppressive effects of RCAN1.4 in PDAC cells by VEGFA pathway.A The representative images and immunohistochemical staining quantification analysis of CD31 were performed, respectively, in RCAN1.4 overexpression BxPC-3 and WT-BxPC-3, RCAN1.4 knockdown SW1990, and WT-SW1990 orthotopic tumors in nude mice. Scale bar = 100 µm. B The association between RCAN1.4 expression and MVD in human PDAC tissues. Representative images and quantifications of RCAN1.4-high expression and RCAN1.4-low expression pancreatic tissues were individually subjected to CD31 staining. C The protein levels of VEGFA decreased in RCAN1.4 overexpression PANC-1 and BxPC-3 cells and increased in RCAN1.4 knockdown MIA PaCa-2 and SW1990 cells using western blotting. D NFAT1 increasingly bound four potential binding sites in the IFI27 promotor site by ChIP assay. E, F Proliferation of HUVECs were analyzed after incubating with CM or CM with VEGFA neutralizing antibodies by CCK-8 assay. G, H Representative images and quantification of migration of HUVCE after incubating with CM or CM with VEGFA neutralizing antibodies. Scale bar = 100 µM. I, J Representative images and quantification of tube formation induced after incubating with CM or CM with VEGFA neutralizing antibodies. Scale bar = 100 µM. Results are presented as mean ± SD from one representative experiment. Error bars, ±SD (determined using a two-tailed t-test, ns no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Fig 3: IFI27 is a direct transcriptional target of NFAT1 and essential for RCAN1.4-mediated PDAC progression.A, B Heatmap and volcano plot visualization of differentially expressed genes between RCAN1.4 overexpression BxPC-3 and WT-BxPC-3 cells from the RNA-seq data. C GO enrichment analysis was performed for differentially expressed genes between RCAN1.4 overexpression BxPC-3 and WT-BxPC-3 cells from the RNA-seq data. D The protein levels of IFI27 decreased in RCAN1.4 overexpression PANC-1 and BxPC-3 cells and increased in RCAN1.4 knockdown MIA PaCa-2 and SW1990 cells using western blotting. E NFAT1 increasingly bound three potential binding sites in the IFI27 promotor by ChIP assay. F, G The representative images and immunohistochemical staining quantification analysis of IFI27 were performed respectively in RCAN1.4 overexpression BxPC-3 and WT-BxPC-3, RCAN1.4 knockdown SW1990 and WT-SW1990 orthotopic tumors in nude mice. H, I Quantification of CCK-8 in IFI27-knockdown PANC-1 and BxPC-3 cells, and IFI27 overexpression SW1990 and MIA PaCa-2 cells to detect cell viability. J, K Representative images of migration and invasion of IFI27-knockdown PANC-1 and BxPC-3 cells. Scale bar = 100 μm. L Representative images of migration and invasion of IFI27 overexpression MIA PaCa-2 cells. Scale bar = 100 μm. M, N Representative images of migration and invasion of IFI27-knockdown of RCAN1.4 KD-SW1990 and WT-SW1990 cells. Scale bar = 100 μm. O, P The quantification of migration and invasion of IFI27-knockdown PANC-1 and BxPC-3 cells, and IFI27 overexpression MIA PaCa-2 cells. Q, R The quantification of migration and invasion of IFI27-knockdown of RCAN1.4 KD-SW1990 and WT-SW1990 cells. Results are presented as mean ± SD from one representative experiment. Error bars, ±SD (determined using a two-tailed t-test, ns no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Fig 4: Mechanism map showing a proposed molecular pathway of RCAN1.4-regulated PDAC progression and metastasis.The loss of RCAN1.4 leads to an activation of calcineurin–NFAT1 signaling pathway, which promotes IFI27 and VEGFA expression to promote the malignancy of PDAC.
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