Fig 2: 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 3: Smad3 constitutes an important effector in both proneural and mesenchymal GSCs.A Heatmap showing the molecular subtype marker expression in proneural GSCs (PN12, PN16, and PN19), and mesenchymal GSCs (ME23, ME27, and ME29) with or without TGF-β1 treatment for 4 or 8 h. Proneural markers: DLL3, OLIG2, ASCL1, CD133, and SOX2. Mesenchymal markers: CD44, CHI3L1, TIMP1, BCL3, COL3A1, MGP, and POSTN. Z-scores were calculated from the △Ct values obtained in the quantitative polymerase chain reaction analysis. B Western blotting analysis of SMAD2, p-SMAD2, SMAD3, and p-SMAD3 expression in proneural GSCs (PN12, PN16, and PN19) and mesenchymal GSCs (ME23, ME27, and ME29) with or without TGF-β1 treatment for 8 h. C Kaplan–Meier survival curves for patients with mesenchymal GBM and a high Smad3 gene signature (n = 44) and those with a low Smad3 gene signature (n = 33). D Protein–protein interaction networks indicated by STRING and Cytoscape software show overlapping peaks in both proneural and mesenchymal GSCs and also identify those that are unique to a particular subtype of GSCs or shared between the proneural and mesenchymal GSCs. E Lysates from proneural GSCs and mesenchymal GSCs were subjected to immunoprecipitation using anti-Smad3 antibody and then immunoblotted with anti-E2F4 and anti-EP300 antibodies. GSCs glioblastoma stem cells.

Fig 4: Overexpression of GAS5 prevents H3K27 trimethylation and upregulates CDKN1C expression to accelerate oxidative stress in melanoma by inhibiting EZH2. a Correlation analysis of lncRNA GAS5 expression and EZH2 expression. b RIP assay of the recruitment of transcription factor E2F4 by lncRNA GAS5. c ChIP assay of the enrichment of E2F4 in EZH2 promoter region. d RNA pull-down assay of the direct interaction of lncRNA GAS5 and E2F4. A375 cells were treated with oe-GAS5 alone or in the presence of oe-EZH2. e RT-qPCR assay of lncRNA GAS5 expression and mRNA expression of EZH2 and CDKN1C. f Western blot assay of the protein expression of E2F4, EZH2, H3K27me3 and CDKN1C. g Western blot assay of the protein expression of MDA5, IRE1α and SOD-1. h ELISA analysis of the content of ROS. i CCK-8 assay of cell viability. j Flow cytometric analysis of cell apoptosis. *p < 0.05, compared with the IgG group, cells transduced with oe-NC, Bio-probe NC group, cells transduced with oe-G-NC or cells transduced with oe-GAS5 + oe-E-NC. The above measurement data are expressed as mean ± standard deviation. The unpaired t-test is used for comparison of the data between two groups. Data among multiple groups are analyzed by one-way ANOVA, followed by Tukey’s post hoc test. The statistical analysis concerning time-based measurements within each group is realized using ANOVA of repeated measurements, followed by a Bonferroni’s post hoc test. Pearson correlation analysis is performed for correlation analysis

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.