human breast tumor vasculature, there is the potential that tacrolimus could bind to FKBP12 and inhibit angiogenesis in human breast tumors. Beta catenin is not required for SFRP2 induced endothelial tube formation To show whether beta catenin is involved in SFRP2-stimulated angiogenesis, beta catenin was silenced in 2H11 endothelial cells with siRNA and their ability to undergo tube formation was compared to sham-transfected cells. Western blot analyses of 2H11 cells transfected with siRNA to beta catenin demonstrated 80% knockdown of beta catenin (Fig. 3A, Figure S2). Some studies Tacrolimus Inhibits Breast Cancer Growth In Vivo have shown that SFRP2 is an inhibitor of beta catenin [6]; if the mechanism through which SFRP2 stimulated tube formation is through inhibition of beta catenin signaling, then we would expect that silencing beta catenin in endothelial cells would increase tube formation. However, we found no difference in de novo tube formation between sham transfected cells and siRNA beta catenin transfected cells (Fig. 3B). Other studies have shown that SFRP2 can increase nuclear beta catenin levels [7–10]; if the mechanism through which SFRP2 stimulated tube formation was through activation of beta catenin signaling, then we would expect that silencing beta catenin in endothelial cells would block SFRP2 induced tube formation. However, there was no difference in tube formation between sham-transfected cells stimulated with SFRP2 Figure 4. NFATc3 was required for SFRP2 stimulated tube formation. A) ShRNA to SFRP2 in 2H11 endothelial cells showed 69% reduction in NFATc3 level by Western blot. The loading control was TATA binding protein TBP antibodies (a nuclear marker). B) Sham transfected 2H11 cells increased tube formation in response to SFRP2 (7 nM) (n = 3 for all groups, p,0.01), which was not seen in shRNA to NFATc3 transfected cells. Full-length blots/gels are presented in Supplemental Figure S2B. Pictures of sham transfected cells and shRNA to NFATc3 transfected cells (both stimulated with SFRP2 (7 nM)) are in Figure S3. doi:10.1371/journal.pone.0020412.g004 Figure 5. Tacrolimus decreased SFRP2 induced nuclear NFATc3 in 2H11 endothelial cells. A) 2H11 cells were treated with control (1.5% DMSO), mouse recombinant SFRP2 (7 nM)+1.5% DMSO; or SFRP2 7(nM)+tacrolimus 10 mM in 1.5% DMSO for 1 hour, and nuclear protein lysates were collected and analyzed by Western blot analyses probing for NFATc3 as described in ‘‘Material and Methods’’. The loading control was TATA binding protein TBP antibodies (a nuclear marker). SFRP2 increased nuclear NFATc3 compared to control cells (p = 0.003). Full- length blots/gels are presented in Figure S2C. B) The experiment was repeated as above except that whole cell lysates rather than nuclear lysates were extracted and analyzed by Western blot analyses probing for NFATc3. The loading control was beta-tubulin. Tacrolimus did not inhibit total NFATc3 protein. Taken together this shows that tacrolimus inhibits SFRP2 induced NFATc3 nuclear translocation but not total protein levels. doi:10.1371/journal.pone.0020412.g005 PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e20412