Fig 1: FGL-LAG-3 checkpoint is involved in DCDC2-induced immune evasion. A Correlation analysis was conducted on the immune signaling pathways enriched by GSEA in the high DCDC2 expression group with the immune signaling pathways enriched by the high expression groups of FGL1 or ENO1 in the TCGA-CHOL databases. B The TCGA-CHOL dataset was divided into high and low expression groups based on the median values of DCDC2/ENO1/FGL1 expression. Genes that were differentially expressed within the high DCDC2/ENO1/FGL1 expression group compared to the low DCDC2/ENO1/FGL1 group were subjected to GSEA for immune-related pathways. C Tumor growth curves of humanized mice inoculated with the vehicle or DCDC2 overexpressed HuCC-T1 cells treated with or without α-LAG-3. D Image (left) and tumor weight (right) of subcutaneous xenograft tumor of humanized mice inoculated with the vehicle or DCDC2 overexpressed HuCC-T1 cells treated with or without α-LAG-3. E The expressions of granzyme B and IFN-γ in CD8+ T cells in xenograft tumors of humanized mice were assessed by flow cytometry. F Tumor growth curves of subcutaneous tumors of syngeneic model. G&H Image (left) and tumor weight (right) of subcutaneous tumors of syngeneic model. I The expressions of granzyme B and IFN-γ in CD8+ T cells in subcutaneous tumors of syngeneic model were assessed by flow cytometry. ***p < 0.001
Fig 2: DCDC2 promotes ICC immune evasion and upregulates FGL1 expression. A Schematic diagram of humanized mouse xenograft model. B Overexpression of DCDC2 significantly promoted tumor weight of humanized mice. C Tumor growth curves of humanized mice showed overexpression of DCDC2 significantly promoted tumor growth. D Overexpression of Dcdc2 significantly promoted tumor weight of SB-1 derived syngeneic tumor. E Tumor growth curves of syngeneic model showed overexpression of Dcdc2 significantly promoted tumor growth. F Analysis of GSEA scores for immune-related pathways in the high DCDC2 expression group versus the low DCDC2 expression group using TCGA-CHOL and xenograft RNA-seq data. G Expression analysis of immune checkpoint ligands in tumor cells with high DCDC2 expression in the HCCC-9810 RNA-seq and xenograft RNA-seq databases. H Correlation analysis between DCDC2 and immune checkpoint ligands in the TCGA-CHOL database. I The expression of DCDC2 and FGL1 was positively correlated in human ICC tumors. J The relative expressions of FGL1 mRNA in ICC cells were assessed by qPCR after DCDC2 overexpression. K The protein levels of FGL1 in ICC cells were assessed by western blot after DCDC2 overexpression. L The protein levels of FGL1 in supernatant of ICC were assessed by ELISA after DCDC2 overexpression. M The relative expressions of GZMA, GZMB, TNF, IFNG of CD8+ T cells in scRNA-seq data. N The expressions of granzyme B and IFN-γ in CD8+ T cells in xenograft tumors of humanized mice were assessed by flow cytometry. O The expressions of granzyme B and IFN-γ in CD8+ T cells in allograft tumor of syngeneic model were assessed by flow cytometry. ***p < 0.001
Fig 3: ENO1 promotes the transcription of FGL1. A The relative expressions of FGL1 mRNA in ICC cells were assessed by qPCR after ENO1 knockdown. B The protein levels of ENO1 were assessed by western blot after ENO1 knockdown. C The protein levels of FGL1 in supernatant of ICC were assessed by ELISA after ENO1 knockdown. D The two predicted binding sites and the corresponding mutants of ENO1 in FGL1 promoter region and relative luciferase activity in dual-luciferase reporter assay. E ENO1- or DCDC2-associated chromatin complexes were immunoprecipitated from HuCC-T1-Vector and HuCC-T1-DCDC2-OE cells. Precipitated DNA was analyzed for FGL1 promoter enrichment by qPCR (DCDC2-OE vs. Vector; IgG control). F The expressions of DCDC2 and ENO1 in cytoplasm and nucleus of ICC cells after DCDC2 overexpression were assessed by WB. G The relative expressions of FGL1 mRNA in DCDC2 overexpressed and ENO1 knocked-out cells were assessed by qPCR. H The protein levels of FGL1 in DCDC2 overexpressed and ENO1 knocked-out cells were assessed by western blot. I The protein levels of FGL1 in supernatant of DCDC2 overexpressed and ENO1 knocked-out cells were assessed by ELISA. ***p < 0.001
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