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. 2022 Jun 21;18(10):4071–4087. doi: 10.7150/ijbs.69495

Figure 5.

Figure 5

E2F6 inhibited E2F1 transcription and interacted with CENPU. (A) Luciferase activity was measured after Huh-7 cells were cotransfected with the full-length fragment of the E2F1 promoter (-2000~+100) and si-CENPU. (B) Venn diagram analysis of predicted proteins that could simultaneously interact with CENPU and regulate E2F1 transcription. (C) Western blotting analysis of E2F1 following E2F6 knockdown or overexpression in HCC cells. (D) Schematic representation of the E2F6 binding site on the E2F1 promoter region. (E) ChIP-PCR and ChIP-qPCR results showing E2F6 binding to the E2F1 promoter at the -1632/-1620 site in Huh-7 cells. (F) Luciferase activity was measured after Huh-7 cells were cotransfected with the E2F1 promoter fragment (WT or MUT) and E2F6-overexpression plasmid. (G) EMSA was conducted to validate the binding of E2F6 to the E2F1 promoter sequences. (H) Typical IF imaging of the colocalization of CENPU and E2F6 in Huh-7 and HCCLM3 cells. (I) Exogenous and endogenous co-IP assays confirmed the interaction between CENPU and E2F6. ns: no significance; ***p < 0.001.