Western blot analyses for NFATc3 in tacrolimus treated endothelial cells 2H11 mouse endothelial cells were grown to 80–90% confluence in DMEM with 10% FBS. The 2H11 cells were serum starved in DMEM with 2% FBS overnight. The following day the media was changed to DMEM with 5% FBS and supplements. Control cells received 1.5% DMSO; SFRP2-treated cells received mouse recombinant SFRP2 7 nM with DMSO 1.5%; and tacrolimus treated cells received tacrolimus (10 uM), in 1.5% DMSO with and without mouse recombinant SFRP2 (7 nM). Cells were incubated for one hour with the SFRP2/ tacrolimus treatment. Nuclear and cytoplasmic proteins were extracted by using NE-PER nuclear and cytoplasmic extraction reagent. For experiments extracting whole-cell lysates, the M-PER Mammalian Protein Extract reagent (Pierce Biotechnology) was used as described in the manufacturer’s manual. Western blot was performed as described above. Endothelial tube formation assay in vitro A Matrigel tube formation assay was used to evaluate whether silencing beta catenin or NFATC3 in endothelial cells augments de novo or SFRP2 induced angiogenesis. ECMatrix (Chemicon, Tacrolimus Inhibits Breast Cancer Growth In Vivo Temecula, CA) was thawed, diluted, and solidified in a 96 well plate according to the manufacturer’s instructions. 2H11 endo- thelial cells, sham transfected 2H11 cells, siRNA beta catenin transfected or shRNA NFATc3 transfected cells were kept in appropriate media. 48 hrs post-transfection the media was changed to DMEM with 2% FBS. 72 hrs post-transfection the cells were seeded onto the matrix at 5,000 cells/well in 150 ml of DMEM with 5% FBS and supplements and incubated with and without mouse recombinant SFRP2 (R&D Systems, Inc., Minneapolis, MN) (7 nM) at 37uC, 5% CO2 for 6 hours. Wells were photographed and tube formation was quantified by counting the number of branch points. To evaluate whether tacrolimus inhibits endothelial tube formation in vitro, we evaluated the effect of tacrolimus on SFRP2 and VEGF induced tube formation. ECMatrix was thawed, diluted, and solidified in a 96 well plate. 2H11 endothelial cells were serum starved in DMEM with 2% FBS overnight, and then seeded onto the matrix at 5,000 cells/well in 150 ml of DMEM with 5% FBS and supplements. Control cells received 1.5% DMSO; SFRP2-treated cells received mouse recombinant SFRP2 7 nM with DMSO 1.5%; and tacrolimus treated cells received mouse recombinant SFRP2 7 nM with tacrolimus (LC Laboratories, Manassas, VA) (10 uM, 1 uM, and Figure 2. Tacrolimus reduces tumor vascularity. MMTVneu tumors from 4 control 20% intralipid mice and 4 tacrolimus treated mice were resected and embedded in paraffin. Immunohistochemistry with antibody to factor VIII was performed as described in ‘‘Material and Methods’’. Microvessel density was determined by counting the number of microvessels in 3 high-power fields at 2006. A, D) The mean number of microvessels per high power field in control tumors was 104613. B, D) The mean number of microvessels per high power field in tacrolimus treated tumors was 2061 (p,0.001). This shows that tacrolimus reduces angiogenesis in vivo. C) Negative control human breast tumor without primary antibody shows no staining. doi:10.1371/journal.pone.0020412.g002 PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20412