Fig 2: E2F4 Regulation of DSCC1 Transcription and Expression in GC. (A) Detection of differentially expressed genes (DEGs) within the major cluster as targets identified by enriched motifs/tracks associated with the E2F4 transcription factor. (B) Heatmap illustrating the expression of transcription factors regulating DSCC1 in GC and normal samples. (C) Analysis of expression correlations between E2F4 and PLAUR based on TCGA-seq data. (D) Representative confocal microscopy image demonstrating the co-localization of E2F4 protein (green) and DSCC1 (red) in AGS and NCI-N87 cell lines. (E-F) Positive regulatory effect of E2F4 on DSCC1 expression in GC cells. Evaluation of E2F4 silencing on DSCC1 mRNA and protein expression in GC cells using RT-qPCR (E) and Western blot (F), respectively. (G) Prediction of the E2F4 binding motif within the DSCC1 promoter through the JASPAR dataset. (H) ChIP-qPCR analysis indicating E2F4 binding to the DSCC1 promoter in AGS and NCI-N87 cell lines, with the first position being the most significant binding site. (I) Luciferase activity assessment following mutation of the first E2F4 site in the DSCC1 promoter in 293T cell lines. The luciferase activity of the wild-type DSCC1 promoter was notably higher than that of the mutant DSCC1 promoter. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001) - Significance levels indicated by asterisks.

Fig 3: Up-regulation of E2F4 in GC tissues and cells. (A) Comparison of F2F4 expression between tumor tissues and adjacent tissues from the TCGA database. (B) Comparison of E2F4 expression levels between high and low expression groups from the Kaplan-Meier Plotter. (C) Detection of E2F4 mRNA expression in 16 pairs of GC and corresponding adjacent tissues using RT-qPCR. (D) Detection of E2F4 protein expression in 16 pairs of GC and corresponding adjacent tissues using Western blot. (E) Analysis of E2F4 mRNA and protein expression in GES-1 and GC cell lines by RT-qPCR. (F) Analysis of E2F4 mRNA and protein expression in GES-1 and GC cell lines by Western blot. (G) Detection of E2F4 expression in 80 paired GC and adjacent non-tumor tissues by IHC. (H) Survival analysis of GC patients with varying levels of E2F4 expression. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001) - Significance levels denoted by asterisks.

Fig 4: TBX3 promoter analysis.(A) Potential TF binding sites at the TBX3 locus obtained from the UCSC genome browser (hg19). Binding sites for the TFs STAT5, GATA and E2F are highlighted. (B) RQ-PCR analysis of TBX3 after siRNA-mediated knockdown of STAT5A, STAT5B, GATA3, and E2F4. The data indicated that E2F4 activated TBX3 while STAT5A, STAT5B and GATA3 showed no transcriptional impact. (C) RNA-seq data show expression levels of E2F4 in 100 leukemia/lymphoma cell lines using dataset E-MTAB-7721. Western blot analysis of E2F4 protein in HL cell lines HDLM-2, KM-H2 and L-428, demonstrating high levels in KM-H2. TUBA served as loading control (insert). (D) RQ-PCR analysis of E2F4 expression in HL cell lines, confirming elevated transcript levels in KM-H2. (E) Genomic profiling data for chromosome 16 of HL cell line KM-H2, demonstrating amplification of E2F4 at 16q22. (F) ChIP data from ENCODE for E2F4 in HELA-S3 cells demonstrating binding at the TBX3 promoter.

Fig 5: Gene regulatory network around TBX3 in HL.This diagram summarizes the results of this study, placing TBX3 at the centre of an aberrant gene regulatory network. TBX3 is directly targeted by genomic amplification just like its activators E2F4, KDM2B and FOXC1. TBX3 inhibits expression of CDKN2A, NFKBIB and CD19, thereby impacting proliferation, NFkB-signaling and B-cell differentiation, respectively.