IFITM1 and PLSCR1 expression in endocrine-sensitive and AI-resistant breast cancer cells. (A) The cell extracts isolated from the indicated cell lines (endocrine sensitive-T47D/MCF-7, AI-resistant MCF-7:5C) were detected by Western blot analysis for PLSCR1 and IFITM1 protein and β-actin as a loading control. (B) Total RNA was extracted from each cell line and IFITM1 (upper panel) and PLSCR1 (lower panel) mRNA was determined by real-time PCR. Fold change was determined for each cell line relative to the internal control gene PUM1. Each value is a mean ± SD from three experiments. *P <0.05 (C) Relative intensity of the IFITM1 or PLSCR1 in T47D, MCF-7 and MCF-7:5C cells were determined by immunocytochemistry (ICC) staining. (D) Immunohistochemistry (IHC) staining for IFITM1 or PLSCR1 was performed on tissue microarrays generated from normal breast tissue (left panel), primary breast tumor tissue (middle panel) and recurrence breast tumor tissue (right panel). For immunohistochemical analysis, the scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining, giving rise to a Staining Index (SI) value for each sample. The proportion of positively stained tumor cells was graded as follows: 0 (<5% positively stained tumor cells), 1 (5% to 25% positive tumor cells), 2 (25% to 50% positive tumor cells), 3 (50% to 75% positive tumor cells) and 4 (>75% positive tumor cells). Representative photomicrographs were taken using a phase-contrast microscope (original magnification, ×200). AI, aromatase inhibitor; IFITM1, interferon induced transmembrane protein1; PLSCR1, phospholipid scramblase 1; SD, standard deviation.