Niltubacin HDAC inhibitor nuclear b-catenin was seen extensively in the basal layers of the tumors, but was also seen in supra-basal cells. In ten independent tumors examined in detail, an average of 43% of tumor cells exhibited clear nuclear expression of b-catenin. Nuclear labeling of tumor cells was abolished when normal rabbit IgG was substituted for the specific b-catenin antibody. To confirm this finding, mRNA was prepared from normal mammary glands of young adult virgin females, ATF3-induced mammary tumors, and MMTV.neu-induced mammary tumors, and analyzed by quantitative polymerase chain reaction. b-catenin mRNA was detected at similar levels in normal mammary tissue from non-transgenic mice and BK5.ATF3 heterozygous mice. Seven independent ATF3-induced tumors exhibited an average of about three-fold higher levels of bcatenin mRNA as compared to normal mammary glands from BK5.ATF3 animals. As expected, MMTV.neu induced tumors had no such increase in b-catenin mRNA. mammary glands of TOPGAL mice that did or did not carry the BK5.ATF3 transgene. These findings strongly indicate that the Wnt/b-catenin pathway is activated in these tumors, but not in normal mammary glands of BK5.ATF3 transgenic mice. Analysis of bacterial b-galactosidase expression by IHC was complicated by background cross-reactivity in ATF3-tumors induced in non-TOPGAL mice. It can be seen that modest but fairly uniform Torin 1 staining was seen in the supra-basal tumor cells but not in the basal cell layer of the tumors. The antibody used for IHC is known to cross-react with mouse b-galactosidase, suggesting that the mouse protein may be present in the supra-basal layers of these tumors. In contrast, tumors arising in BK5.ATF3Tg/0;TOPGALTg/0 mice demonstrated uniform staining throughout both the basal and supra-basal layers of the tumor. We conclude that activation of the Wnt/b-catenin pathway occurs at least in the basal cell layers of these tumors, coincident with nuclear expression of ATF3. As a further test of Wnt/b-catenin pathway activation, we used qPCR and protein immunoblotting to monitor activity of known downstream transcriptional targets of this pathway. Wnt/bcatenin signaling is usually associated with increased cell proliferation, and correlated with this, Ccnd1 and Jun have both been shown to be transcriptional targets of the Wnt/b-catenin pathway. As shown in Figure 2B, the Ccnd1 gene was upregulated about four-fold in ATF3-induced tumors compared to normal mammary glands, and Jun expression was up about two-fold. Furthermore, at the protein level, robust up-regulation of both cyclin D1 and Jun proteins was seen in immunoblots.