Fig. 5: GINS2 regulates ECT2 transcription levels through TF EGR1.

A A transcription factor regulatory network of upregulated genes in SHG44-GINS2 cells was constructed. Venn diagram showed the intersection of transcription factors (purple) and differentially up-regulated genes (yellow) in regulatory network. B Western blotting to detect the regulatory effect of GINS2 on EGR1. C Enrichment analysis of GINS2-binding proteins. Protein and protein interaction network of GINS2-bind RNA metabolism-related proteins. D mRNA stability assay to assess the effect of GINS2 on the mRNA stability of EGR1. E Western blotting was performed to detect changes in GINS2, EGR1 and ECT2 protein expression after the transfer of EGR1 plasmids in GINS2 knockout glioma cells. F Database prediction of EGR1 binding sites in the ECT2 promoter region. G ChIP assay to explore the enrichment of EGR1 in the promoter region of ECT2 gene in GINS2 knockout cells overexpressing EGR1 and control cells. H Schematic diagram of the dual luciferase reporter gene truncator construct. Dual luciferase reporter gene experiments further confirmed the positive regulation of EGR1 as a transcription factor on ECT2. Act-D Actinomycin D, TSS Transcription start site, E1 EGR1 binding site 1, E2 EGR1 binding site 2, P1 Promoter 1, P2 Promoter 2, NS No significance. *P < 0.05, **P < 0.01, ***P < 0.001. n = 3 independent experiments. Two-tailed t test assuming equal variances. Error bars represent the mean ± standard deviation of the mean.