biological specimens. To evaluate whether the binding protein of tacrolimus, FKBP12, is present in the vasculature of human breast tumors, we stained paraffin-embedded human breast tumors and mouse MMTV-neu breast carcinomas (on an IACUC approved protocol) with a polyclonal antibody to FKBP12. IHC was performed as described above, accept the primary antibody was FKBP12 antibody (100 ml–200 ml at 1:200 dilution). A negative control without primary antibody was performed for all experiments. Slides were evaluated for the presence of FKBP12 staining in tumor endothelium. Cell culture 2H11 mouse endothelial cells (American Type Culture Collection ATCCH, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose (Sigma- Aldrich, St. Louis, MO) with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO). MMTV-neu breast tumor cells [21] (a gift from Dr. John Serody, UNC-Chapel Hill) were cultured in GIBCOH RPMI 1640 media (Invitrogen, Carlsbad, CA) 20%FBS, 2 mM L-glutamine, 12 mM, HEPES, 0.1 mM NEAA, 1 mM Sodium pyruvate, 1% Pen Strep, 50 uM 2-ME, 0.2 U/ml Regular insulin. siRNA to beta catenin in endothelial cells To prove that SFRP2 does not stimulate tube formation via the beta catenin pathway, we silenced beta catenin in endothelial cells and evaluated their ability to respond to SFRP2 treatment in a tube formation assay. 2H11 mouse endothelial cells were transfected with siRNA to beta catenin and control nonsilencing siRNA. The siRNA for beta catenin, which is a pool of 4 target- specific 21 nt siRNAs (beta catenin siRNA (m): sc-29210, Santa Cruz Biotechnology, Santa Cruz, CA), and nonsilencing siRNA (sc-36869, Santa Cruz Biotechnology) were used in 100 pmol/ml (100 nM) concentration. The 2H11 cells were maintained in DMEM with 10% FBS and were transfected with siRNA to beta catenin or the control siRNA using LipofectamineTM RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The transfection efficiency was deter- mined using siGLO Green Transfection indicator (Dharmacon, Inc. cat# D0016300105) according to the manufacturer’s instruction. To verify the knockdown of beta catenin, cells were harvested 72 hrs post transfection for protein analysis by Western blot. Nuclear extracts using NE-PER nuclear and cytoplasmic extrac- tion reagent were prepared as described in the manufacturer’s manual (Pierce Biotechnology, Rockford, IL). Nuclear fractions were confirmed on Western blot using the loading control, TATA binding protein TBP antibodies, which is a nuclear marker. Protein concentration was measured using Bio-Rad Protein Assay at OD595 (Bio-Rad Laboratories). Equal amounts of protein (20 mg) were loaded onto SDS-PAGE gels. Proteins were transferred to Polyvinylidene Difluoride membrane (PVDF), and Western blotting was carried out using a primary antibody to beta catenin, with horseradish peroxidase (HRP)-conjugated IgG as the secondary antibody. The ECL Advance substrate was used for visualization (GE Healthcare Bio-Sciences). shRNA to NFATc3 in endothelial cells To evaluate the functional results of silencing of NFATc3 in SFRP2 stimulated endothelial cell tube formation, we used shRNAmir to NFATc3 (Open Biosystems/Thermo Scientific). The sh-RNA plasmids were supplied in E.coli cells from Open Biosystems. They were grown overnight at 37uC with shaking in 100 ug/ml ampicillin/LB broth. The culture was spun down and Tacrolimus Inhibits Breast Cancer Growth In Vivo plasmids were extracted to be used in transfecting the 2H11 cells. Three different shRNAmir constructs to NFATc3 from Open Biosystems were tested: a) RMM4431-99213393 Mouse GIPZ lentiviral shRNAmir individual clone V2LMM_110117, b) RMM4431-98727337 Mouse GIPZ lentiviral shRNAmir individ- ual clone V2LMM_110113 (resulting in the best down regulation of NFATc3), c) RMM4431-98750613 Mouse GIPZ lentiviral shRNAmir individual clone V2LMM_110112, RHS4430- 98525659 Human GIPZ lentiviral shRNAmir individual clone V2LHS_161372. Control shRNAmir constructs were GADPH- pGIPZ and non silencing- pGIPZ construct purchased from Open Biosystems. In order to establish stable cell lines expressing the shRNA to NFATc3 we first generated a puromycin kill curve for the 2H11 endothelial cells. The minimum concentration of puromycin required to kill non-transfected 2H11 cells was 4 ug/ml. The 2H11 cells were then seeded in 6 well plates 24 hours prior to transfection using LipofectamineTM LTX Transfection reagent (Invitrogen) according to the manufacturer’s protocol. Forty-eight hours post transduction the DMEM 10% FBS media was replaced with full growth selective media (containing 4 ug/ml Puromycin) (Mediatech, Inc., Manassas, VA) into the appropriate wells. The selective media was changed every two-three days. After selection the cells were kept in full growth media containing 2 ug/ml puromycin. Western blot analysis on nuclear extracts using NE- PER nuclear and cytoplasmic extraction reagent (Pierce Biotech- nology) was done to evaluate the degree of NFATc3 silencing. This indicated the best results for knock down of NFATc3 was obtained with the RMM4431-98727337 Mouse GIPZ lentiviral shRNAmir construct. Figure 1. Tacrolimus inhibited the growth rate of MMTV-neu transgenic mouse tumors. MMTV-neu transgenic mice were treated with tacrolimus 3 mg/kg/day i.p., no treatment control, 20% intralipid i.p. control beginning when tumors became palpable, and was continued for 21 days. Tumor volumes were measured on day 7, 14 and 21, and the growth rate (percent change in tumor volume per day) was compared between control (no treatment), control (20% intralipid) and tacrolimus treated groups. There was no statistically significant difference between control (untreated) and control (20% intralipid) groups, and therefore the controls were combined for analyses. At 21 days there was a 73% reduction in the growth rate for tacrolimus treated mice compared to no treatment (n = 19 tacrolimus treated, n = 15 control, *p = 0.003). Tacrolimus treated mice were significantly different from control (no treatment, p = 0.01, and control 20% intralipid, p = 0.01). There was no weight loss or lethargy in the tacrolimus-treated mice or the 20% intralipid treated mice. doi:10.1371/journal.pone.0020412.g001 PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20412