Figure 1

PADI2 expression is highest in MCF10DCIS.com cells in the MCF10AT model of breast cancer progression. (a) The MCF10AT model is a series of cell lines that recapitulates the transition from normal epithelium to malignant carcinoma. (b and c) PADI2 mRNA and protein expression is increased in transformed cells of the MCF10AT model, with very high levels seen in MCF10DCIS cells. Total RNA was isolated and PADI2 mRNA levels were determined by qRT-PCR (TaqMan) using MCF10A cells as a reference and GAPDH normalization. Data were analyzed using the 2 ^{-ΔΔ C(t)} method and are expressed as the mean ± SD from three independent experiments (* p < 0.001). PADI2 expression levels were evaluated by subjecting whole cell lysates to SDS-PAGE and immunoblot analysis using an anti-PADI2 antibody. HER2/ERBB2 expression levels, detected using an anti-HER2 antibody, are also upregulated in the transformed cell lines when compared to the MCF10A levels. The blot was stripped and equal protein loading was determined by probing with a β-actin antibody. (d) Citrulline levels in the cell lines were determined by citrullination enzymatic assays, with the highest level of activity measured in the MCF10DCIS cells. Briefly, cell lysates were incubated with PADI substrate BAEE, and the reaction stopped with perchloric acid. The perchloric acid-soluble fraction was subjected to a colorimetric reaction with citrulline used as a standard and absorbance measured at 464 nm.