Fig. 7. CircSLC38A1 recruits ILF3 to initiate TGF-β2 expression.

A Top: Heatmap of CUT&Tag-seq peaks associated with the ILF3 in T24 cell lines transfected with si-circSLC38A1#2 or si-NC, signals are displayed from -2.0 kb to +2.0 kb surrounding the TSS. Bottom: the average intensity curves for ILF3 signals at TSSs in a region comprising ±2 Kb in T24 cell lines transfected with si-circSLC38A1 or si-NC. B Heatmap of the differentially expressed RNAs in si-circSLC38A1 and si-NC cells by RNA-seq analysis. Red, upregulated RNAs; blue, downregulated RNAs. C Functional enrichment analysis of overlapped downregulated genes that were uniquely aligned to the genome using the online DAVID software. The results show that these genes are significantly associated with functional modules, such as regulation of epithelial cell proliferation and regulation of epithelial to mesenchymal transition. D Right: the IGV shows the CUT&Tag signals of ILF3 at the TGF-β2 gene loci. Left: quantitative diagram of the degree of enrichment of ILF3 signal on the TGF-β2 promoter. E, F Relative expression levels of TGF-β2 in T24 cells treated with ILF3 siRNA or corresponding negative control were measured by qRT-PCR (E) or western blot (F). G, H Relative expression levels of TGF-β2 in T24 cells transfected with vector control or ILF3 overexpression plasmid were measured by qRT-PCR (G) or western blot (H). I, J Relative expression levels of TGF-β2 in T24 cells treated with circSLC38A1 siRNA or corresponding negative control were measured by qRT-PCR (I) or western blot (J). K, L The expression of TGF-β2 in T24 cells with circSLC38A1 deficiency could be effectively reversed by overexpression of ILF3. M, N The expression of TGF-β2 that was upregulated with circSLC38A1 overexpression could be reduced by knocking down the expression of ILF3. Data represent mean ± SD from three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.