Fig. 8

TCF7L2-L transcriptionally upregulates RELB to activate NF-κB signaling. a Diagram showing the process for RNA-seq analysis of ccRCC cells with control plasmid, TCF7L2-L variant, or TCF7L2-S variant overexpression. b Heatmap showing the genes upregulated in cells transfected with TCF7L2-L but with unchanged expression in cells transfected with TCF7L2-S compared to control cells. c Comparative analysis of the TCF7L2-S and TCF7L2-L groups revealing that the NF-κB pathway was the predominant pathway enriched in the differentially expressed proteins. d Five essential upregulated genes (RELB, CCL2, IL4I1, PTX3, and TNFAIP3) were identified by overlapping the genes upregulated by overexpression of TCF7L2-L with those upregulated by overexpression of TPM3P9. e qRT-PCR was performed to validate the upregulation of RELB and TNFAIP3 mRNA expression after the overexpression of TPM3P9 in ACHN and 786-O cells. *p < 0.05; **p < 0.01; ***p < 0.001; ns, nonsignificant. f Western blot analysis confirmed that the overexpression of the TPM3P9 protein, but not the corresponding lncRNA, upregulated RELB protein expression in ccRCC cells. g Western blot analysis confirmed that ectopic expression of the TCF7L2-L variant but not the TCF7L2-S variant noticeably induced the expression of RELB in ccRCC cells. h Co-IP using an anti-HA antibody followed by MS was performed to identify the coregulatory factors of TCF7L2-L. The diagram was edited using Adobe Illustrator. i Coomassie blue staining showing the proteins that specifically bind to TCF7L2-L; specific bands are highlighted with black arrows. j The Venn diagram shows that 27 proteins that bound specifically to TCF7L2-L but not to TCF7L2-S. k Co-IP assays were performed using an anti-HA antibody to verify the interaction between the HA-TCF7L2-L variant and SAM68 in ACHN and 786-O cells. The HA-TCF7L2-S variant and the empty vector were used as controls. l Co-IP assays were performed using an anti-SAM68 antibody to detect the interaction between TCF7L2-L and SAM68 in ccRCC cells. m Western blot analysis confirmed that silencing TCF7L2-L or SAM68 noticeably attenuated the induction of RELB expression in ccRCC cells overexpressing TPM3P9. n The predicted sequence motifs for TCF7L2 binding DNA. Two potential sequences in the RELB promoter were predicted to bind to TCF7L2. o Dual luciferase reporter assays revealed that the activity of motif No.1 but not that of motif No.2 was increased by TPM3P9 overexpression, and this increase was strongly attenuated by treatment with siRNA targeting TCF7L2-L or SAM68. ***p < 0.001; ns, nonsignificant. p CCK-8 assays were performed to test the growth ability of ccRCC cells transfected with the indicated constructs. **p < 0.01. q–u Immunohistochemical and correlation analyses were performed in the SYSUCC cohort, comprising 385 clinical samples, to analyze the expression correlations of TPM3P9 with TCF7L2-L (q), RELB with TPM3P9 (r), SAM68 with TPM3P9 (s), RELB with TCF7L2-L (t), and RELB with SAM68 (u)