Figure 6

PADI2 is expressed in MCF10DCIS xenograft tumors and localizes to the luminal epithelium. (a) MCF10DCIS cells (1 × 10⁶) were injected subcutaneously into female nude (nu/nu) mice (Charles River) and comedo-DCIS tumors formed after 2 weeks. Tumor sections were probed with an (I, II) anti-PADI2 (1:100) or (III, IV) anti-ERBB2 antibody (1:100), and counterstained with hematoxylin. The black arrows indicate the luminal epithelium of the duct-like structures in DCIS tumors (I, III – 20X, scale bar = 200 μm; II, IV – 100X, scale bar = 50 μm). While the HER2/ERBB2 staining is predominantly cytoplasmic (IV), there are some nuclei staining positively for PADI2 (II). (b) Tumors were collected; two sections from each mouse were sampled and total RNA was isolated. PADI2 mRNA levels were determined by qRT-PCR (TaqMan) using MCF10A cells as a reference and GAPDH normalization. PADI2 levels were higher in the tumor samples than normally found in the MCF10DCIS cells. Data were analyzed using the 2 ^{-ΔΔ C(t)} method, and are expressed as the mean ± SD from three independent experiments (* p < 0.005). (c) Immunofluorescence staining (40X) shows that PADI2 is expressed in the luminal but not myoepithelium cells in MCF10DCIS tumors. Tumors were probed with anti-PADI2 (II, III, V, VI – green fluorescent signal), anti-cytokeratin (I, III – luminal marker – red fluorescent signal), and anti-p63 (IV, VI – myoepithelial marker – red fluorescent signal). Nuclei were stained with DAPI (blue fluorescent signal). The dashed arrows delineate the luminal epithelium layer, while the dashed straight line delineates the myoepithelium layer, which is adjacent to the basement membrane. In the merged images, the co-localization of cytokeratin and PADI2 can be seen in the luminal epithelium (III), while in contrast, PADI2 is absent from the p63 stained myoepithelium (VI).