reporters. We used RKO cells that stably express BAR and Renilla [24]. In LOF studies, cells were seeded to 30% confluency before siRNAs were transfected using Lipofectamine RNAimax (invitro- gen) at a concentration of 20 mM. 24 h later the cells were treated with L-cell media or WNT3A media (or transfected with WNT1 (0.1 ng) and TOPFLASH (1 ng)/Renilla luciferase(10 ng)). Lysis and luminescence (using Promega dual-luciferase assay kit) was carried out 24 h after WNT treatment. For GOF experiments, cells were seeded to 50% confluency after which cDNA (GFP (50– 100 ng) or Bili-FLAG (25–100 ng), TOPFLASH, Renilla luciferase, WNT1 (0.1 ng), DNLRP6 (5 ng), DVL (5 ng), b-catenin (1 ng) was transfected. BIO (0.5 mM) was also added to the cells at this point. Cells were lysed and luminescence measured 24 h hours later. Bili siRNAs were purchased from Ambion; siRNA ID# 332724 that targeted Exon 8 (sense 59ggcgugcacgucaucgauatt39, antisense 59uaucgaugacgugcacgcctt39) and siRNA ID# 45515 that targeted Exon 7, 8. Control siRNA and Axin 1 & 2 siRNAs have been described [24]. Drosophila in situ hybridization and immuno-staining In situ hybridization and immuno-staining in the fly embryos and larval imaginal discs were performed using standard protocols described in the laboratory manual ‘‘Drosophila Protocols’’ by William Sullivan et al., CHSL press. qRT-PCR in Drosophila embryos RNA was isolated from Drosophila embryos using Trizol and was further purified using the RNA ‘‘clean-up’’ protocol with the Qiagen RNAeasy kit. This was followed by reverse transcription (Applied Biosystems - High Capacity cDNA Reverse Transcrip- tion Kit) and quantitative PCR (SYBR green) using Applied Biosystems 7300 Real-Time PCR Machine. PCR primers used were as follows: Actin: Act Forward 59 – GAA GAT CTC CCA ATC GGC GAA CAA TTC ATA CC – 39; Act Reverse 59 - GAA GAT CTT TGA ACG CGA CTT GAG AGC GG – 39; Armadillo: Arm Forward 59 – CAA CGT TCC TCG ATA GCC AC – 39; Arm Reverse 59 – CGA ATG AGT GCC CGG TTG TG – 39; C11848/dBili: CG11848 Forward 59 – CTT GGC TGT GGC TAC CGA TC - 39; CG11848 Reverse 59 – GCG CCA TAG AAT GGC AGA GC – 39. Constructs MGC clone for human Bili (MHS1010-9204707) was pur- chased (Open biosystems). Bili was sub-cloned into pCS2+ using the EcoR1 and Xho1 sites. Forward primer sequence 59 GTAGTAAAGCTTGCCACCATGGCCCTGAGGATG- GACGGG 39 and reverse primer sequence 59 TACTACGTC- GACTCAGCCCTGCTCCAGGCTGT 39. Zebrafish Bili was cloned out of a mixed embryonic library (6 hpf-24 hpf) using forward primer 59 TCTCCAGCTCAGGATTTGTTGGTG 39 and reverse primer 59 TACTACCTCGAGTCAACTTTCAGT- CAC 39 based on an ENSEMBLE BLAST prediction using Bili. Since the ATG was missing, a 59 RACE kit (Invitrogen) was used with a nested primer 59 GCCACTGACGACACAGCTTGTAA 39 that yielded the full-length clone. Co-immunoprecipitation HEK293T cells were made to stably express LRP6-EGFP by transfecting pCS2+LRP-EGFP and pPUR (Clontech) into cells and treating with 2 mg/ml of puroMYCin. Stable cells were seeded into 6-well plates at 50% confluency and transfected with AXIN-MYC (300 ng), GSK3B (50 ng) and Bili-FLAG (200 ng). Cells were lysed the next day in a buffer containing 1% Triton-X, Bili Inhibits Wnt Signaling 50 mM Tris-HCL pH 7, 150 mM NaCl, protease and phospha- tase inhibitors. LRP6-EGFP was immunoprecipitated using polyclonal GFP and protein G beads for 2 hours. The beads were washed three times with the lysis buffer that contained 0.6 M NaCl instead of 150 mM NaCl. Proteins that bound were eluted using SDS gel loading buffer and western blot analysis was used to detect GFP, MYC, FLAG and GSK3B. Quantification was done using NIH image. Drosophila S2R+ cells were used for coIP. S2R+ cells were co- transfected with HA-tagged dBili and Myc-tagged,DNLRP6 IP was performed with Rabbit anti-Myc antibody (1:50, Upstate-Cell Signaling Solutions, Cat # 06-549) and IB with both anti-Myc (1:500) and anti-HA (1:1000, Roche Cat # 1 867 423). Standard IP protocol was used with mild lysis buffer and Protein A/G sepharose. Zebrafish in situ hybridization and microinjection In situ hybridization and microinjections were carried out as previously described on zebrafish online resource, ZFIN. ATG blocking morpholino sequences (Gene Tools) GGAAGTCGCCAT- CATCTCCCTCCAT (bilimo) and GAGACACTCTCCTTC- GATTCAGAAG (bilimo2). A 10 mM stock was diluted to 1.5 mM. bilimo, bilimo2, or control morpholino at this concentration were then coinjected into 1-cell embryos at three doses of wnt8 RNA (stock concentrations 2 ng/ml, 4 ng/ml and 8 ng/ml). The same wnt doses were used for zfBili overexpression coinjection experiments with Renilla as a control at 50 ng/ml stock concentration. Injection drop size 0.5–1 nl. QT-RTPCR RNA was isolated following treatment using Qiagen RNAeasy kit. This was followed by reverse transcription (Invitrogen ThermoScript or SuperScript) and quantitative PCR (sybr green) using Roche Light cycler 2.0. PCR primers Bili forward 59CCAAGCAGGCC- GAACT39, Bili reverse 59CCTTGCCGTCCTCCACGTA39, GAPDH forward 59CCA CCC ATG GCA AAT TCC ATG GCA39, GAPDH reverse 59TCTAGACGGCAGGTCAGGTC- CACC39. Axin 2 forward 59CTCCCCACCTTGAATGAAGA39 Axin 2 reverse 59 TGGCTGGTGCAAAGACATAG39 Gene accession numbers. NM_143085 (Drosophila), XM_682750.2 (Zebrafish), NM_031904 (Human). Supporting Information Table S1 Table showing dsRNA targeting eighteen genes with known human orthologs that increased reporter activation two- fold or greater. Found at: doi:10.1371/journal.pone.0006129.s001 (0.01 MB DOC) Figure S1 (A) Sequence alignment of Bili protein shows it is well conserved phylogenically. (B) Human Multi Tissue Northern blot show mRNA expression. (C) Illustration of Bili protein structure and domains. Found at: doi:10.1371/journal.pone.0006129.s002 (2.14 MB EPS) Figure S2 (A) qRT-PCR analysis of dBili knockdown. Expres- sion of short-hairpin (shRNA) dbili (#3M) under the control of prd-GAL4 in the Drosophila embryo results in .75% knockdown in message level (Red bar) as compared to prd-GAL4 control embryos (Blue bar). As a control, Armadillo mRNA was not affected in embryos expressing dBili shRNA. (B) Embryos expressing dBili shRNA under the control of even skipped stripe 3/7-GAL4: These embryos display a partial lack/disruption of the 2nd or 3rd denticle belt which coincides with the eve-stripe 3 PLoS ONE | www.plosone.org 8 July 2009 | Volume 4 | Issue 7 | e6129