Pigment epithelium-derived factor expression in primary and recurrence breast tumor tissues. (a) Immunohistochemistry (IHC) staining for pigment epithelium-derived factor (PEDF) and estrogen receptor alpha (ERα) were performed on tissue microarrays generated from normal breast tissue (left panel), primary breast tumor tissue (n = 59; middle panel), and recurrence breast tumor tissue (n = 59; right panel). Cores used to generate tissue microarrays (TMAs) were 0.6 mm in diameter. Tumor tissues were also stained in the absence of a PEDF antibody to act as a negative control (left panel, inset). PEDF staining was quantified as an intensity score ranging from 0 to 255. A scale of 0 to 3 was used to score staining intensity of ERα. (b) Western blot analysis of normal tissue (N), primary breast tumor tissue (PT), or recurrence tumor tissue (RT) to assess PEDF, total ERα, and phosphorylated ERα (Ser118 and Ser167) protein level. β-actin was used as a loading control. (c) Representative quantitative real-time PCR analysis of PEDF and ERα mRNA expression in N, PT, or RT. For experiment, total RNA was extracted from paraffin-embedded tissues using Trizol and analyzed by real-time PCR as described in Materials and methods. PEDF and ERα mRNA were normalized to the internal control gene PUM1. All experiments were performed in triplicate. PEDF mRNA level was statistically significantly lower in the recurrence tumor tissue compared with the primary tumor tissue. *P < 0.001.
Jan et al. Breast Cancer Research 2012 14:R146 doi:10.1186/bcr3356