Role for PAD2 in Gene Regulation Figure 1. PAD2 is expressed in human endometrial, kidney, breast ductal and luminal epithelial cells. (A) Endometrial, (B) kidney, (C) mammary duct, and (D) mammary alveolar epithelial cells express PAD2, while staining is absent (or weak) in adjacent tissue. Tissue sections were probed with anti-PAD2 antibody or equal concentration of rabbit IgG as a control and counterstained with hematoxylin. The black arrows indicate epithelial cells with varying PAD2 staining in the nucleus between different tissues. doi:10.1371/journal.pone.0041242.g001 (4352934E). Data was analyzed by the delta/delta Ct method in which all target gene Ct values are adjusted to corresponding reference gene Ct levels. Stable MCF-7 shRNA scrambled control Ct values were normalized to 1 and all values are expressed as the mean 6 SEM. Means were separated using Student’s T-Test with * indicating means significantly different (P,0.05) from shRNA control. For microarray qPCR validation experiments, cDNA was generated as described above and subject to qPCR using the primers listed in Table S1. Data was analyzed using the ABI Data Assist Software v3.0 and P value set to P,0.05. Gene Expression Microarray Total RNA from shRNA control and PAD2 depleted MCF- 7 cells was prepared using RNeasy mini kit (Qiagen) from four independent biological replicates. Microarray studies and data analysis was performed by the Cornell University Life Sciences Core Laboratories Center. Briefly, RNA was assessed for quality using an Agilent Bioanalyzer 2100. Labeling was carried out using the Amino Allyl MessageAmpTM II ARNA amplication kit from Ambion following the manufacture’s protocol. Hybridization followed a one-color microarray-based gene expression analysis, low input quick amp labeling protocol from Agilent (Santa Clara, CA). Slides were scanned immediately after washing using an Agilent DNA Microarray Scanner (G2505B), and the scanned images were analyzed with Feature Extraction Software 9.1 (Agilent). Data was analyzed with Ingenuity Systems IPA version 8.8 and significant genes determined using P,0.001 with a q value (FDR) of ,0.01. Microarray experiments were compiled using MIAME guidelines and the resulting dataset has been deposited into the NCBI GEO database (GSE37087). Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed essen- tially as described previously [22]. Briefly, stable FLAG-tagged PAD2 overexpressing, shRNA control, and PAD2 depleted MCF- 7 cells were grown to ,80 to 90% confluence, cross-linked with Figure 2. PAD2 is detected in the cytoplasm and nucleus of human breast luminal epithelial cells. (A) PAD2 is expressed in luminal but not myoepithelial human breast cells. Normal human breast tissue sections were probed with anti-PAD2 (green fluorescent signal), anti-cytokeratin (luminal marker - red fluorescent signal), and anti-p63 (myoepithelial marker - red fluorescent signal) antibodies or equal concentration of rabbit and mouse IgG as a control and nuclei are stained with DAPI. (B) A portion of PAD2 staining in human breast epithelial cells is detected in the nucleus. Normal human breast tissue sections were probed with anti-PAD2 (green fluorescent signal) and nuclei are stained with DAPI. The white arrow illustrates a breast epithelial cell with robust PAD2 staining in the nucleus. doi:10.1371/journal.pone.0041242.g002 PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e41242