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Supplemental Materials: This section contains:

- 7 Supplemental Figures - 1 Supplemental Table - Experimental Procedures

Miranda-Carboni_Fig S1

S1 Wnt10b promotes expression of stem cells markers in mammary tumor cells and in mammary cell lines. A: Independent cell lines were established from primary mammary tumors arising in ErbB2TG or Wnt10bTG. Cells were processed for immunofluorescence and labeled with anti-sera against the stem cell marker Sca1 (green) (i-ii) and then counter stained with DAPI (blue). B: The mammary epithelial cell line Eph4-Wnt10b (stable clone expressing Wnt10b) also showed enhanced Sca1 expression when compared with the parental cell line (i-ii). Images were collected with a 40x objective.

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Miranda-Carboni_Fig S2

S2 Hierarchical analysis of microarray data from Wnt10bTG tumors reveals enhanced Cul4A mRNA expression. A: RNA expression profiles (Affymetrix GeneChip Mouse Genome 430 2.0) were collected from the following mammary tissues: virgin (7wk, mammary gland #3), 12d-pregnant (7wk, mammary gland #3), Wnt10bTG primary mammary tumor, or ErbB2TG primary mammary tumor. Each sample value represents a statistical average of 3 independent tissue samples. Analysis of cohorts conforms to MIAME structure http://www.mged.org. Hierarchical condition trees were constructed using GeneSpring software (Agilent Technologies, GX 7.3.1). Probe set ID and gene symbol/name are listed for several genes induced by at least 1.6 fold in the Wnt10bTG data set. B: Validation of the array data was carried out using QT-PCR on cDNA generated from the primary tissue (and additional data presented in Figure 1). Panel B represents a further conformation using RNA generated from the NMG and NMG-Wnt10b cells. Wnt-responsive induction of Cyclin D1 and c-Myc mRNA was observed at 0h, 12h and 16h after release from synchronization. Samples were normalized to actin and experiments were conducted as triplicates. Error bars represent S.D.M. QT-PCR data for Cul4A expression is presented in Figure 6D.

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Miranda-Carboni_Fig S3

S3 The levels of p27Kip1 mRNA are increased in Wnt10b-expressing cell lines, but Wnt signaling promotes turnover of p27KIP1 protein in a dose dependent manner. A: NMG and NMG stably expressing Wnt10b (NMG-Wnt10b) cells were synchronized in G1-phase and released by re-plating. QT-PCR was carried out on first strand cDNA at the indicated time points for expression of p27 mRNA and normalized to L32. Error bars represent S.D.M. for triplicate samples. B: Randomly cycling human HEK-293T fibroblasts, known to respond to wnt signals, were transiently transfected with plasmid vectors directing expression of wild-type human p27KIP1 containing an N-terminal FLAG epitope (1 ug) alone (Lane 1), or with increasing amounts of pBA-Wnt10b expression vector (0.5 ug, 1 ug, 1.5 ug, 2.5 ug and 5 ug), Lanes 2-6. 24h post-transfection, cells were lysed and whole cell extracts were analyzed by SDS-PAGE and immunoblotted with anti-sera raised against the FLAG epitope (M2, Sigma) or ß-actin. FLAG blots are over-exposed to illustrate that p27KIP1 is down-regulated, but not extinguished, by Wnt10b expression. The failure to achieve complete loss is related to the continued expression of FLAG-p27KIP1 mRNA from the strong CMV promoter associated with the FLAG epitope vector.

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Miranda-Carboni_Fig S4

S4 Wnt10b relieves G2-arrest in Skp2-/- embryonic fibroblasts and promotes their entry into M-phase following synchronization. Schematic diagram illustrates arrest and release protocol for panels A and B. For both A and B, cells were stained with DAPI and analyzed for G2/M progression using a nuclear morphology protocol established for the LSCTM. Data are plotted as DAPI Integral (DNA Content) vs. DAPI Max Pixel (chromatin density). A: Embryonic fibroblasts (MEFs) from Skp2+/+ and Skp2-/- mice were synchronized in early S-phase in the presence of aphidicolin (1 µg/ml) (I and III). Released from S-phase block and analyzed 24h post release (II and IV). B: Skp2-/- MEFs were stably transduced with lentivirus expressing GFP or Wnt10b. Subsequently Skp2-/- MEFs expressing GFP or Wnt10b were arrested in early S-phase (V and VII) by aphidicolin treatment. The cells were analyzed for M-phase entry 8h after release (VI and VIII). Experiments were conducted in triplicate and graphically illustrated in Figure 4 E and F.

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Miranda-Carboni_Fig S5

S5: p27KIP1-low phenotype of Wnt10bTG mammary tumors correlates with low SKP2 protein: Histological morphometry of immuno-histochemistry (IHC) reactions from Figure 5. Scoring of DAB reaction products present in tissue sections from MMTV-ErbB2, -Wnt10b, -TGF-β1 and -Myc primary tumors for: A: p27Kip and B: SKP2 protein levels. Scoring was conducted by counting DAB nuclear localization using high (hi), intermediate (med) and low/negative (lo/neg.) relative values. IHC percentage scoring is representative of a minimum of 1000 nuclei identified by counterstaining with nuclear fast red.

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Miranda-Carboni_Fig S6

S6 Cul4A and Cul4B are required for proliferation and p27KIP1 turnover in mammary epithelial cells expressing Wnt10b. A: NMG (siScrmbl) or NMG-Wnt10b (siScrmbl) controls were synchronized at G1-phase for two days along with NMG-Wnt10b-siCul4A and/or siCul4B silenced clones. Cells were synchronized, released for 12h (early S) and analyzed for BrdU incorporation by immunofluorescence microscopy. DAPI was used as a counter stain. Experiments were conducted in duplicate. Images were collected with an 10x objective. Similar results were observed at 20h (data not shown) B: Whole cell lysates from panel A were analyzed at 0h, 12h and 20h by immuno-blotting and probed with the CUL4 and p27 sera. C: NMG-Wnt10b cells silenced for Cul4A have decreased levels of Cul4A mRNA. shRNA-silencing vectors expressing a scrambled siRNA control (siScr) or Cul4A siRNA (siCul4A) were used to generate subclones of NMG-Wnt10b cells. QT-PCR was carried out to assess the degree of silencing in randomly cycling NMG-Wnt10b siRNA clones.

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Miranda-Carboni_Fig S7

S7 Wnt10b-expression in MCF7 cells induces elevated CUL4A protein and mRNA during S-phase and this correlates with S-phase turnover of p27KIP1 . Estradiol (E2) treated cells also turnover p27KIP1 but do not induce CUL4A. A: Immuno-fluorescence (IF) of endogenous β-catenin (red) was counter stained with DAPI (blue). Images were collected with a 63x objective. B: Arrest and release study showing full time course of the experiment shown in Figure 7. MCF7-neo (vector only) and MCF7-Wnt10b cells were synchronized at G1-phase of the cell cycle for 2-3 days at 100% confluency. Synchronized cells were released by re-plating and whole cell extracts were prepared at 2h, 4h, 8h, 12h, 16h, 20h and 24h. Lysates were analyzed by SDS-PAGE and immuno-blotting for p27KIP1, CUL4A, Cyclin E, CDK2, γH2AX and β-Actin. Lanes 9-16 are presented in Figure 8. C: MCF7 cells were synchronized by hormone deprivation (CDT media) for 3 days in the presence or absence of Wnt10b. Subsequently MCF7 cells were treated with E2 (10 nM) or vehicle alone (ETOH) in CDT media and harvested at the indicated time points. QT-PCR was carried out on first strand cDNA for expression of CUL4A mRNA. Error bars represent S.D.M data normalized to L32. D: To ensure specific-activation of E2-signaling, MCF7 cells were synchronized as in panel B. Treatment with E2 or vehicle alone for 12h and analyzed by QT-PCR for expression of pS2 (a known target of E2 in breast cancer) and NFATc1 (not a target of E2 in breast cancer). E: Whole cell extracts were prepared on MCF7 cells synchronized as in panel B in the presence or absence of LiCl (50 mM) or E2 (10 nM) in CDT media. Control was treated with both vehicle (ETOH) and 50 mM NaCl. Treatments were conducted for 16h and whole cell lysates analyzed by immunoblotting for CUL4A, p27KIP1 and ß-ACTIN expression.

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Miranda-Carboni_Supl. Table 1 Table 1 (Supplemental) Primers1 Plasmid SEQUENCE2 Orientation3

1. KR5 FLAG-p27wt 5'-TAACGGGAGCCCTAGCCTGGAGCGGA S 5'-TGCTCGAGTTACGTTTGACGTCTTCTGAGGCCAG AS 2. T187A FLAG-p27wt 5'-CTGTGGAGCAGGCGCCCAAGAAGCC S or pEYFP-p27wt 5'-GGCTTCTTGGGCGCCTGCTCCACAG AS 3. siClu4A pSupNeo 5'-TCGAgaagattaacacgtgctggcaggaattcgtgccagcacgtgttaatcttcTTTT S 5'-CTAGAAAAgaagattaacacgtgctggcacgaattcctgccagcacgtgttaatcttc AS 4. siCul4B pSupNeo 5'-TCGAgttaaacagcagcagtagcagggaattcgctgctactgctgctgtttaacTTTT S 5'-CTAGAAAAgttaaacagcagcagtagcagcgaattccctgctactgctgctgtttaac AS 5. siScramble pSupNeo 5'-TCGAtcagtcacgttaatggtcgttttcaagagaaacgaccattaacgtgactgaTTTT S 5'-CTAGAAAAtcagtcacgttaatggtcgtttctcttgaaaacgaccattaacgtgactga AS

QT-PCR Primers4 Template5 SEQUENCE2 Orientation3 6. p27(Kip1/Cdkn1b) cDNA 5'-TGGGTCTCAGGCAAACTCTGA S 5'-TCCGCTAACCCAGCCTGAT AS 7. Skp2 cDNA 5'-GCGCTAAAACAGGAGTCTGG S 5'-AGACACCATGCCTGATAGC AS 8. Cul4A cDNA 5'-CGACAGGATGGTGCAGAGTA S 5'-CAAACTCCGAAGCAGACTCC AS 9. Wnt10b cDNA 5'-ATGCGGATCCACAACAACAG S 5'-TGACGTTCCATGGCATTTG AS 10. Myc cDNA 5'-ACACGGAGGAAAACGACAAGAG S 5'-GCAAAAAAGCTGCGCTTCA AS 11. Cyclin D1(Ccnd1) cDNA 5'-TCGTGGCCTCTAAGATGAAGGA S 5'-TCGGGCCGGATAGAGTTGT AS 12. Cyclin A2 (CcnA2) cDNA 5'-ATGCCCTGGCTTTTAATGCA S 5'-TGCCATCCATTGGATAGTCAAG AS 13. Cyclin B1(CcnB1) cDNA 5'-ACATGGTGCATTTTGCTCCTT S 5'-TCCATTCACCGTTGTCAAGAAT AS 14. CUL4A cDNA 5'-TTCGGTAGTGGAAAACCAG S 5'-CAGCAGCTCGAATTTCTTCC AS 15. CYCD1 (CCND1) cDNA 5'-TGGAGGTCTGCGAGGAACAGAA S 5'-TGCAGGCGGCTCTTTTTCA AS 16. MYC (MYC) cDNA 5'-TTCGGTAGTGGAAAACCAG S 5'-CAGCAGCTCGAATTTCTTCC AS 17. pS2 cDNA 5'-TTGTGTTTTCCTGGTGTCA S 5'-CCGAGCTCGGGACTAATCA AS 18. NFATc1 cDNA 5'-AGAAAGCGAAGCCAGTACCA S 5'-CGGTCTCACTAACGGGACAT AS 19. Cul4A-Promoter ChIP 5'-GCCAGCCTGGTCACGAGT S 5'-GGTACAACCTGGAGGAGCTG AS 20. Chr. 12-intergenic ChIP 5'-GAGGGCACTGTTCGAATCTC S 5'-ACCCAGGAATTTGGGTCATT AS 1Primers were required to alter several plasmid based expression vectors. Site directed mutagenesis was conducted to generate human-p27 expression vectors containing T187A and KR5 mutations within the context of FLAG-p27 and pEYFP-p27 tagged vectors. Additional primers were designed as inserts to create shRNA vectors for the indicated gene using the pSuppressorNeo (pSupNeo) system (Imgenex). Italicized sequence represents the specific short hairpin generated. Other reagents were provided by collaborators, and are described in the text. 2The nucleotide sequence of each primer is listed. All reactions required primer pairs. 3The orientation of the listed primer: Sense (S) or Antisense (AS). 4Mouse (e.g. Cul4A) and human (e.g. CUL4A) gene targets are listed according to nomenclature standards, see Experimental Procedures. 5Both first strand cDNA (cDNA) and Chromatin (ChIP) were used as templates for Quantitative (QT)-PCR, as indicated.

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Miranda-Carboni et al. Experimental Procedures: Nomenclature and Mice: Mouse genes, RNA, and cDNA are lower case italicized (e.g. Cul4A, p27Kip1) and human genes are upper case and italicized (e.g. CUL4A, p27KIP1). Mouse and human proteins are listed as capitalized and non-italic (e.g. WNT10B, CUL4A, or p27KIP1). Naming follows published guidelines: Human: http://www.genenames.org/guidelines.html and mouse: http://www.informatics.jax.org/mgihome/nomen/gene.shtml). Alternative gene symbols exist for p27Kip1 (Cdkn1b) and the cyclins; these are provided as a table in supplemental data (S5). Unless otherwise indicated, all transgenic mice (e.g. Wnt10bTG, ErbB2TG) were derived in the FVB/N strain (Taconic), see individual references. Wnt10b-null mice were generated in 129/SvEv and backcrossed to FVB/N for at least 12 generations to create isogenic lines. Skp2-null embryonic fibroblasts were created in a C57BL/6 background as described (Nakayama et. al. 2000). Cell Culture and Synchronization: Mouse mammary epithelial cell lines (NMuMg (NMG) and EpH4), Skp2+/+ and Skp2-/- embryonic fibroblasts (MEF), human embryonic kidney fibroblasts (HEK-293T), and human mammary tumor cell lines (MCF7) were maintained in a humidified atmosphere with 5% CO2 in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 ug/ml streptomycin. NMG, EpH4 and MCF7 cell cultures were supplemented with insulin (1 ug/ml). NMG are a non-transformed subclone of NMuMG cells (ATCC: CRL-1636) originally isolated from NAMRU strain by Owens and are widely used because they preserve responses to TGFß. For cell cycle progression analysis, human and mouse mammary epithelial cell lines were synchronized at G1 by maintenance at 100% confluency for 2-3 days (contact inhibition) then released with trypsin and replated for the indicated time. HEK293T fibroblasts were synchronized at G1 by growth in 1% serum for 2-3 days, then released by replating. Skp2+/+ and Skp2-/- and NMG-Wnt10b were synchronized at the G1-S boundary with aphidicolin (1 mg/ml) for 14h, as described (Nakayama et. al. 2000). In some experiments, proteasome activity was blocked by pretreatement of cells with MG132 (10uM), or CRM1 dependent nuclear export machinery was inhibited by pretreatment with LMB (1ug/ml, Leptomycin B). MCF7 cells and estradiol (E2) treatment: MCF7 cells were synchronized by hormone-deprivation for three days in phenol red-free medium (Invitrogen Corporation) supplemented with 5% Charcoal-dextran treated FBS (CDT-FBS) (Omega Scientific) (Krum et al., 2008). Cells were treated with 10 nM Estradiol (E2), or ethanol as a vehicle control, for indicated time points and RNA was isolated as described below. Generation of Wnt10b-expressing cell lines: Mouse Wnt10b cDNA was obtained by PCR and cloned into the expression vector pcDNA3.1 (Invitrogen). The sequence was identical to the sequence of Wnt10b (Lane and Leder 1997). The vector was linearized with PvuI and transfected into either the mouse mammary gland cell line NMG or the human mammary gland cell line MCF7 with Fugene (Roche) to generate clones NMG-Wnt10b and MCF7-Wnt10b. Stable constructs were selected using G418 and Wnt10b expression was verified by QT-PCR and immuno-blotting. Clones expressing linearized vector alone were also established in both cell lines. Transient transfections: Transfection of HEK293T were carried out using a standard calcium chloride method. NMG and MCF7 cells were electroporated using an NucleofectorTM following the manufactures protocol (Amaxa). Plasmids are identified in the text and included, pcDNA3.1-FLAG-human p27KIP1, p27-KR5 and -p27-T187A were generously provided by Dr. M. Shirani. pcDNA3.1-YFP-human p27wt was generously provided by Dr. Joyce Slingerland, pcDNA-HA and -Cul4A were provided by Dr. Kristin T. Chun. We purchased pcDNA3-DN-hCUL4A-FLAG (15821) and pcDNA3-DN-hCUL4B-FLAG (15822) from Addgene. In addition, pBA-Wnt10b, pcDNA3.1-Wnt10b, and pcDNA3.1-YFP-p27-T187A were generated internally and reported here. The inserts of all plasmids were confirmed by direct sequencing. Plasmids used for virus production, gene knockdown, and protein production are described in other sections.

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WNT10B antibody production: WNT10B antigen was generated by using two distinct domains of the mouse WNT10B protein, corresponding to nucleotides 778-875 and nucleotides 1186-1302 (Lane and Leder 1997). GST-WNT10B peptide fusions were created (pGEX-2TK Gene Fusion System, Amersham) and antigen was purified from BL-21 bacteria using glutathione affinity chromatography. Antisera was generated using New Zealand White Rabbits per the manufacturer’s protocol (Sigma-Genosys). Immunohistochemistry (IHC): Tumor tissue and/or mammary gland tissue were fixed in 4% paraformaldehyde-PBS and embedded in paraffin. Sections were processed through standard deparafinization protocols followed by 3% H2O2 treatment. Antigen recovery was conducted using 0.1 M sodium citrate, pH 8.5, and 0.1 M citric acid, pH 6.0, and boiled twice for 5-10 minutes each. The tissue was incubated in blocking buffer (5% normal goat serum, 2.5% BSA in PBS pH 7.5) for 30-45' in a humidifier chamber. Primary antibodies p27KIP1 (C-19, Santa Cruz Biotechnology, Inc), ß-Catenin (C2206, Sigma), Cyclin D1 (Ab3, Labvision), SKP2 (Zymed) and WNT10B (see below) were incubated at 4°C overnight in a humidified chamber followed by a secondary biotinylated goat anti-mouse or rabbit antibody (Pierce). IHC staining was conducted using Vectastain ABC System (Vector Laboratories) and counterstained with Nuclear Fast Red (Vector Laboratories) per manufacturers’ protocol. Immunofluorescence (IF) and Laser Scanning Cytometer (LSC): Cells were fixed with 4% paraformaldehyde-PBS for 30', washed, incubated with blocking buffer (TBS pH 7.8, 3% BSA, 1% NGS, 1% triton X-100, 0.01%NaAzide). Primary antisera were purchased against: ß-catenin (H-102, Santa Cruz Biotechnology), p27KIP1 (C-19 from Santa Cruz Biotechnology), mouse Cul4 (pan-Cul4 (A and B cross reactive T-15, Santa Cruz);); Sca1 (E13-161.7-FITC, BD-Pharmingen); human CUL4A (Ab34897, Abcam). Secondary antisera included: highly cross absorbed goat anti-rabbit Alexa-633 goat anti-rabbit Alexa-488, or goat anti-mouse Alexa-633 (Molecular Probes). Cells were counterstained with 6-diamidino-2-phenylindole (DAPI, Molecular Probes) or propidium iodide (PI; Molecular Probes) at a concentration of 10-30 mg/ml to identify DNA and to distinguish cell cycle stages. Immuno-fluorescence photomicrographs were obtained using 10X, 40x and 63X objectives on a Leica DM IRBE fluorescent microscope equipped with a Hamamatsu C4742-95 digital camera (Leica Microsystems). Openlab 5.0 software (Improvision) was used for image processing. Cytometric analysis was performed on similarly prepared samples using a Laser Scanning Cytometer (LSCTM, CompuCyte Corp). The LSC was equipped with UV laser, used to contour DAPI using a minimum cell area of 10 um3, and an Argon and HeNe lasers used to measure immuno-fluorescence. Cytometric analysis was conducted with WinCyteTM software (CompuCyte). BrdU labeling for cell cycle analysis: Cells were plated in 8-well chamber slides (Nalge NUNC) and treated as indicated. Randomly cycling or G1 synchronized cells were pulsed with 10 mM BrdU (5-Bromo-2-Deoxyuridine, Calbiochem) for 20' prior to fixation, washed twice with PBS containing 1% BSA and fixed in place using cold 70% ethanol for 30'. Chromatin was made accessible by treatment with 2N HCl and 0.5% Triton X-100 for 30', followed by 0.1 mM Na2B4O7

.10H2O, pH 8.5 for 5’ at room temperature, and blocked in PBS containing 1% BSA for 30-45'. BrdU labeled nuclei were detected with Alexa594 conjugated anti-BrdU (monoclonal-PRB1, Molecular Probes) and counterstained with 30 mg/ml DAPI. Analysis of S-phase labeling was performed using the LSC and IF. Cell extraction and Immuno-blotting: Cells were lysed in low salt buffer EBC (20 mM HEPES, pH 7.4, 0.5% Nonidet P-40, and 100 mM NaCl) and or modified RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5mM EDTA, 1% Triton X-100, 0.1% SDS and 1% sodium deoxycholate) supplemented with phosphatase inhibitors (10 mM NaF, 100 mM Na3VO4) and a protease inhibitor mixture (CompleteTM without EDTA; Roche Applied Science) for 30' on ice. The samples were pelleted at 14,000 RPM for 10', 4ºC. Protein concentrations were determined using the BioRad Protein Assay (BioRad Labs, Hercules CA). 50-250ug of protein were loaded per lane and separated by SDS-PAGE 10% gels. After transfer, Immobilon-P (Millipore Corp., Bedford, MA) was immunoblotted using the following primary antibodies: ß-Tubulin (D-10), p27KIP1 (C-19), Cyclin E1 (M-

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20) and Cullin 4 (T-15) (Santa Cruz Biotechnology); Human CULLIN 4A (Abcam); Cullin 1(Ab1868-500) and Cdk2 (Ab7954-1) (Abcam); Skp2 (32-3400 Zymed) and anti-phospho p27Thr187 (71-7700) (Zymed Laboratories); FLAG M2 (Sigma); and WNT10B (see below). Immunoprecipitation (IP): Immunoprecipitation reactions were conducted on whole cell lysates from HEK293T transiently transfected with pcDNA-HA or pcDNA-HA-Cul4A (Li et al 2003) in the presence or absence of pBA-Wnt10b or MG132 (10 uM). IP’s were conducted with monoclonal p27KIP1 (BD-Pharmigen) on 200ug of total protein and blotted with previously described antibodies. RNA, Quantitative PCR (QT-PCR) and RT-PCR: Isolation of total RNA was performed using TRIzol (Invitrogen) according to manufacturer’s protocol. RNA was treated with DNA-free kit (Ambion) and converted to cDNA with iScript cDNA Synthesis Kit according to manufacturer’s protocol (BioRad). QT-PCR was performed on cDNA using the Applied Biosystems Power SYBR Green amplification system with the following conditions were: 95°C (3'), 40 cycles of 95°C (20s), 55°C (30s) and 72°C (20s). All QT-PCR reactions were performed with an iCycler thermocycler (BioRad). Primer pairs for each gene are provided in supplemental data (S5). RT-PCR reactions were carried out using a PTC-200 thermocycler (MJ Research) and products were analyzed on 1.5% agarose/EtBr gels. Reactions included cDNA, 1x Thermopol buffer (New England Biolabs), 0.5 units of Deep Vent (New England Biolabs), 250 uM dNTPs, and 1 µM each oligonucleotide. PCR conditions were as follows: 1 cycle of 95°C for 2'; 30 cycles of 95°C (20s), 50°–62°C (20s), 72°C (30s); and 1 cycle of 72°C for 5'. Primer pairs for each gene are provided in a supplemental table (Table 1, Supplemental). Chromatin immunoprecipitation (ChIP). Cells were grown to 100% confluency for 2-3 days and harvested at 0h and 8h post release. ChIP was performed as previously described (Krum et. al., 2008). Briefly, cross-linked chromatin was isolated from each plate and separate precipitation reactions were set up for each target protein complex using specific antisera. DNA was amplified by QT-PCR using the Applied Biosystems Power SYBR Green amplification system. Immuno-precipitated chromatin was amplified in triplicate for each target gene and run on an iCycler thermocycler, as described above. Normalization was conducted to input chromatin, and to an intergenic region on chromosome 12, located -6000 base pairs upstream of the Id2 minimal promoter. Antibodies used included RNA pol II (Abcam 8WG16), LEF1 and ß-catenin (Santa Cruz Biotechnology, Inc N-17 and H-102). Each experiment was repeated at least 3 times. Primer pairs for each gene are provided in a supplemental table (Table 1, Supplemental). Generation of epitope tagged human p27-expression vectors: To generate the p27KIP1 mutant expression vectors used previously published vectors pcDNA3.1-FLAG-p27wt (Ishida et al., 2002) and pEYFP-p27wt (Connor et al., 2003), as templates. To generate FLAG-T187A and pEYFP-T187A site-directed mutagenesis was conducted as follows: 100 ng of plasmid, 5 uM each primer, 0.4 uM dNTP and 1 unit of Deep Vent polymerase (New England Biolabs) in a final volume of 50 ul, run at 95°C for 30 seconds, 55°C for 1', and 68°C for 12' for 12 cycles. The amplified product was then treated with DpnI at 37°C for 2 hours, purified, transformed into E. coli and colonies were selected for growth. pGEX-6P3-p27-KR5 (GST-KR5, Shirane et al., 1999) was the generous gift of Dr. M Kitagawa. GST-KR5 was used as a PCR template and the insert was subcloned into pcDNA3.1-FLAG-p27wt with Sbf1 and XhoI to generate pcDNA3.1-FLAG-p27-KR5. All clones were sequence verified in both orientations to confirm identity to the desired sequence. Generation of shRNA-expressing cell lines. Short-hairpin RNA (shRNA) vectors were designed against various gene products and cloned into the pSuppressorNeo system (Imgenex). Terminology: shRNA is used to describe the plasmid vector used to generate short hairpin antisense sequences. siRNA (eg siCul4A) is used to describe the short inhibitory dsRNA product that is generated by shRNA vectors after the hairpin is processed in cells. To silence Skp2 we used a previously published pSuppressorNeo-shSKP2 vector originally designed to an identical sequence in human SKP2 (Katagiri et al., 2006). We generated additional pSuppressorNeo vectors targeting Cul4A and Cul4b. An siScramble (siControl, siScr) was used as a negative control (Gene Suppressor Manual,

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Imgenex). Sequences used for generation of shCul4A, shCul4B and shScr, are provided as part of a table in supplemental data (S5). To establish silenced subclones in mammary cell lines that over express Wnt10b, the pSuppressorNeo vector required co-transfection with pBABE-puro vector and selection of clones was conducted with both puromycin and neomycin. Lentiviral-expression vectors. We utilized the lentiviral construct pSIN18RhMLV-E-CPPT to generate a new Wnt10b-expression vector. This vector is a derivative of pSIN-18-Rh. with cDNA for the human immunodeficiency virus type 1 central polypyrimidine tract (cPPT) introduced into the XhoI site upstream of the Rh murine leukemia virus promoter. IRES and hrGFP sequences were generated by PCR from pIRES-hrGFP 1a (Stratagene). This fragment was cloned into pCR-Blunt TOPO (Invitrogen, Carlsbad, CA), sequenced and subsequently subcloned into AgeI and SalI sites of pSIN18RhMLV-E-CPPT replacing the original EGFP sequence. The Wnt10b sequence was excised from the plasmid pcDNA3.1-Wnt10b as a BamHI-EcoRV fragment and cloned into the BamHI-AgeI site of pSIN18RhMLV-E-CPPT-IRES-hrGFP to produce pSIN18.RhMLV-E-CPPT-Wnt10b-hrGFP vector. Construct pSIN18RhMLV-E-CPPT-IRES-hrGFP was used in the experiments as the negative control. Lentivirus Production. Lentivirus was produced in HEK293T cells by cotransfection of three plasmids: (i) pSIN18.RhMLV-E-CPPT-Wnt10b-hrGFP or the control plasmid, (ii) the vesicular stomatitis virus G(VSVG) expression plasmid pHCMVG, and (iii) the packaging plasmid pCMVΔR8.2DVPR. 5 x106 HEK293T human fibroblasts were plated on 100-mm2 dishes, then transfected with 5ug of pSIN18.RhMLV-E-CPPT-Wnt10b, 5ug of pCMVΔR8.2DVPR, and 2ug of pHCMVG, using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Culture supernatants that contained virus were collected on day 3 post-transfection, centrifuged to remove floating cells, passed through 0.45 um filters, and subjected to centrifugation at 17,000 rpm for 90 min at 4°C in an L8-M ultracentrifuge (Beckman) to concentrate the virus. The viral pellets were resuspended in 1x PBS overnight at 4°C. Concentrated virus was titrated on the HEK293T fibroblast cell line. Lentiviral transduction. Skp2+/+ and Skp2-/- MEFs were plated on 12-well plates at the density of 5x104 cells per well and then infected with 1x105 IU per well of Wnt10b-expressing virus or the control virus in 500 ul of standard tissue culture medium containing 1 ug/ml protamine sulfate. After two hours, the virus-containing medium was replaced with fresh medium. The cells were expanded and used in the experiments as described in the text. Transduction efficiencies were always greater than 95% as assessed by GFP fluorescence. RNA expression Microarrays. Analysis was conducted on the AffymetrixTM GeneChip Mouse Genome 430 2.0 arrays in triplicates for cDNA prepared from the indicated mammary tissue or mammary tumor tissues. Samples were processed by UCLA’s DNA Microarray Core Facility (http://www.genetics.ucla.edu/microarray/). The procedures for probe preparation, hybridization, washing, scanning and signal intensity normalization were directly from manufactures protocols using GeneChip Operating Software (GCOS) v1.1.1 Affymetrix (Santa Clara, CA). Bioinformatics. Analysis of AffymetrixTM array data conformed to MIAME structure (http://www.mged.org). Log of ratio normalized expression data was analyzed with a cross-gene error model turned on and normalized per manufacture protocol (GeneSpring GX 7.3 software (Agilent Corp.)). Calculations were done using Welch’s approximate t-test and 1-way-ANOVA, with p value cutoff of 0.05, and no assumption of equality of variance and Benjamini and Hochberg false discovery rate was used. Hierarchical clustering analysis was performed using the average-linkage method. Additional hypothesis-driven analyses were conducted using a conjoint Boolean consistency so noise ratio could be decreased. A cut off of 1.6-fold or greater (difference) was set for test samples when compared to samples isolated from virgin mammary gland.