Fig. 2. Transcription factor E2F1 is indispensable for ESRP1-mediated breast carcinogenesis.

A Schematic representation of the ESRP1-472/+110 luciferase reporter construct. The E2F1 (−472/+110 bp) sequences for both wild-type and mutated constructs are shown; mutated nucleotides are represented by lowercase letters and underlined. B Wild-type or mutant E2F1 luciferase reporter constructs were co-transfected with the Renilla luciferase vector in MCF7 cells, and the luciferase activity was measured after 24 h of transfection. The relative luciferase values are shown as mean ± SD. C Immunoblots for E2F1 and ESRP1 in E2F1 knockout MCF7 and HCC1806 cells. D, E Densitometric analysis of representative blots. F ChIP qRT-PCR on ESRP1 promoter using E2F1 antibody in HCC1806 cells. Fold enrichment (E2F1/IgG) was normalized to 5% input. G MCF7 cells were co-transfected with ESRP1 (−472/+110 bp) promoter construct along with pCMV-3Tag-1A-E2F1 plasmid or pCMV-3Tag-1A as a control. The luciferase activities were measured and the luciferase values are shown. H Immunoblots for E2F1 and ESRP1 in E2F1 overexpression cells (MCF7 and HCC1806). I, J Densitometric analysis of representative blots. K Immunoblot of E2F1 in normal versus breast cancer tissue (n = 4). L Relative cell proliferation was analyzed through MTT assay (n = 3) in MCF7 cells. M Colony-formation assay of MCF7 cells transfected with the indicated expression vectors were seeded on 6-well plates and after 2 weeks, the colonies were stained with crystal violet. Error bars show mean values ± SD (n = 3 unless otherwise specified) calculated using two-tailed Student’s t test, *P < 0.05, **P < 0.01 and ***P < 0.001.