Canine Mammary PAD2 Expression Figure 3. Treatment of the canine mammary tumor (CMT25) cell line with EGF results in an increase in PAD2 expression levels. (A) Western blot illustrating that only EGF treatment increases PAD2 protein levels with b-actin shown as a loading control. Densitometry measurements quantified treatment groups compared to vehicle treated control in three independent experiments and bars represent the means 6 SEM. * indicates a statistically significant difference from other treatments (P,0.01) (B) Quantitative PCR analysis revealed elevated PAD2 transcript levels in CMT25 cells treated with EGF compared to vehicle treated control and * indicates a statistically difference from the other treatments (P,0.05). doi:10.1371/journal.pone.0011768.g003 QPCR analysis of mammary tissue samples from anestrus, estrus/early diestrus and late diestrus indicate that expression of PAD2 increases from estrus/early diestrus through late diestrus suggesting that hormonal changes during the estrus cycle may be involved in regulation of PAD2 expression. Thus, the ovarian sex steroids E2 and P4 would be appear to be the most likely candidates to regulate PAD2 expression in vivo, however our results using the CMT25 cell line indicate that E2 and P4 are likely not directly involved in regulation of PAD2 in vitro. Although the CMT cell lines are derived from canine mammary epithelial cells, their ability to respond to E2 and P4 is currently unknown and can therefore not be conclusively ruled out in contributing to PAD2 expression. In contrast, EGF can directly increase PAD2 expression in vitro. Growth factors are believed to establish auto-, para- and juxtacrine loops that are thought to augment and amplify initial steroid hormone signals. Therefore, the regulation of PAD2 expression in vivo is likely multifactoral. Interestingly, EGF receptor levels in canine mammary tissue increase from PLoS ONE | www.plosone.org 6 July 2010 | Volume 5 | Issue 7 | e11768