Silencing of ELF5 induces EMT in T47D cells and increases migratory potential
(a, b) Quantitative RT-PCR analysis of ELF5 and EMT-related genes in T47D cells treated with control or ELF5 siRNA at low (a) or high (b) cellular density. Real time PCR values were normalized to the housekeeping gene GAPDH. Experiments were performed three times, each with qRT-PCR in technical duplicate, and data presented as the mean ± SD. * p < 0.05 by Student’s t-test. (c) Western blot analysis of ELF5 and EMT markers in T47D cells treated with control or ELF5 siRNA. Uncropped images of blots are shown in Supplementary Fig. S9. (d) Phase contrast and immunofluoresence images of control or ELF5-knockdown T47D cells stained for E-CADHERIN, β-CATENIN, and F-ACTIN. Loss of F-actin circumferential belts (arrows) and relocalization of β-CATENIN from adherens junctions of the membrane (arrows) to cytoplasm are highlighted with arrowheads. Size bar = 100 μm for brightfield images and β-CATENIN, and 20 μm for E-CADHERIN and 18 μm for F-actin. See Supplementary Fig. S3a which shows the unchanged morphology of T47D cells after mock transfection. (e) Transwell migration assay of T47D cells with or without ELF5 knockdown. The data represented are shown as mean ± SD collected from 6 fields of 3 independent experiments. Student’s t-tests were performed to assess statistical significance.