Fig. 4

Identification of transcription factors regulating HOMER3 expression in glioma. (A) Prediction of transcription factor binding sites in the HOMER3 promoter using JASPAR, highlighting ELK4, SP1, and CREB1 as potential regulators. (B) Association of expression levels of SP1, CREB1, and ELK4 with the overall survival of glioma patients from TCGA datasets. (C) Expression patterns of SP1 and ELK4 in LGG and GBM tissues compared to normal tissues. (D) ELK4 binding site predictions on the HOMER3 promoter illustrated. (E) Chromatin immunoprecipitation (ChIP) analysis confirming ELK4 binding to the HOMER3 promoter. (F) Luciferase reporter assays demonstrating increased luciferase activity in the presence of wild-type ELK4 binding sites in U251 and U87 glioma cells. (G) Elevated expression levels of ELK4 in glioma cell lines compared to normal cells as assessed by RT-PCR and western blot analysis. (H) Knockdown of ELK4 via shRNAs, which was confirmed by RT-PCR and western bot. (I and J) The effect of ELK4 knockdown on glioma cell proliferation assessed by CCK-8 and clonogenic assays. (K and L) In vivo analysis of ELK4 knockdown effects on tumor growth. Each group contained n = 6 mice, and the experiment was repeated three times independently. (M) Western blot for the expression of CDK4 and CDK6 after ELK4 knockdown. (N) The effects of ELK4 knockdown on the migration and invasion of U251 and U87 cells. (O) The effects of ELK4 knockdown on the expression of EMT-related proteins by western blot. All experiments were performed in three independent biological replicates (n = 3) to ensure the reproducibility and reliability of the results