Negative relationship between Slug and ERα in human breast cancer cells. ( a , b ) Analysis of Slug and ERα expression levels in MCF-7, T47D, SKBR3, MDA-MB-231 and BT549 by western blot and RT–PCR. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of protein and mRNA levels as indicated in MCF-7 cells transfected with increasing concentrations (2, 4 and 8 μg, respectively) of Slug. ( e , f ) Western blot ( e ) and RT–PCR ( f ) analysis of protein and mRNA levels in MDA-MB-231 cells transfected with two different interference sequences of Slug (40 n m , separately). ( g ) Western blot analysis of Slug knockdown level in stable cell lines of MDA-MB231shSlug. ( h ) Immunofluorescence micrographs of Slug (red) and ERα (green) expression in MDA-MB-231shNC cells (upper) and MDA-MB-231shSlug (lower). DAPI is for nuclei (blue). Magnification, × 400. Protein and mRNA expression was normalized to β-actin. In figure 2d, NC means transfecting with control pcDNA3.1. In figure 2e and f, siNC is scrambled control siRNA used for transient transfection. ShNC without shRNA is used as a control by infecting pLKO.1 lentivirus in MDA-MB-231 cell line. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P