Materials and Reagents
LiCl (L9650), NaCl (S3014), and actinomycin D (A1410) were obtained from Sigma-Aldrich (Germany), meBIO (361556) and BIO (361550) from Calbiochem (San Diego, CA), anti-cadherin-11 (5B2H5) from Invitrogen (32-1700, Carlsbad, CA), anti-β-catenin antibody from BD Biosciences (610154, San Jose, CA), Dicer antibody from Abcam (ab14601, Cambridge, MA), anti-GSK3β antibody from Cell Signaling Technologies (9315, Boston, MA), anti-FLAG (M2) was from Sigma (F3165, Germany), anti-GAPDH was from Research Diagnostics Inc (TRK5G4-6C5, Flanders, NJ), anti-Snail (H-130) (sc-28199) antibody from Santa Cruz. The anti-eIF6 antibody S13 was a generous gift from Dr. Biffo and generated against a C-terminus peptide of eIF6 
. Small interfering RNA (siRNA) reagent (SMART pool) for human CTNNB1 (M-003482-00), Dicer (M-003483-00), and GSK3β (M-003010-03), were purchased from Dharmacon (Lafayette, CO). Non-specific (Scramble) siRNA was generated using forward: (5′-AAGCTCCTATAGCGTATGGTGCCTGTCTC-3′
) and reverse:
(5′-CACCATACGCTATAGGAGCTTCCTGTCTC-3′) primers and the Silencer siRNA Construction kit (AM1620, Ambion, Austin, TX).
Plasmid DNA encoding wild type (WT) β-catenin was used as previously described and DNA encoding wild type Snail was a kind gift from Dr. Mien-Chie Hung 
. Dominant negative (DN) Snail was generated by PCR from the wild type Snail construct using primers described in Yamasaki et al.
) and reverse: (5′-CGCTCGAGGCGGGGACATCCTGAGCA-3′
) and cloned into pCMV-Tag2 (Stratagene, La Jolla, CA) using BamHI
restriction sites 
. Cadherin-11 3′-UTR fragment was cloned from genomic DNA obtained from MDA-MB-231 cells using Expand High Fidelity PCR System (11732641001, Roche, Indianapolis, IN) according to the manufacturer's protocol. For amplification of the CDH11 3′UTR NCBI forward:
- (5′-TGCTAGCTAAGTAAGTAACAATAACGATACAAATTT-3′) and reverse:
- (5′-CCGGATCCACGCGTGAATCTTGTCTGAAAAAACATTTG-3′) primers were used. For amplification of the CDH11 3′UTR Ensembl forward:
- (5′-TGCTAGCTAAGTAAGTAACAATAACGATACAAATTT-3′) and reverse:
- (5′-GGATCCACGCGTGTTCCACCTGACATACAGAGCC-3′) primers were used. These PCR products were ligated in place of the SV40 3′UTR of pGL3-Promoter (Promega, Madison, WI).
Cell Culture and Transfection
MDA-MB-231, Hs578T, BT549 breast cancer cells, PC-3 prostate cancer cells, and HEK293 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% fetal bovine serum (FBS) in 5% CO2 incubator at 37°C. All transient transfections of plasmid DNA and siRNA in MDA-MB-231 and PC-3 cells were performed with Amaxa electroporation system (Amaxa, Inc, Geithersburg, MD) according to the manufacturer's protocol. Transient transfections for immunofluorescence were preformed using Lipofectamine 2000 Transfection Reagent (11668-019, Invitrogen, Carlsbad, CA) and luciferase analysis using Lipofectamine 2000 (Invitrogen) for PC-3 cells, ProFection Mammalian Transfection System—Calcium Phosphate (E1200, Promega, Madison, WI) for MDA-MB-231 cells, and FuGENE 6 Transfection Reagent (11814443001, Roche, Switzerland) for HEK293 cells.
RNA isolation (mRNA)
MDA-MB-231 or PC-3 cells were incubated in the presence or absence of LiCl (20mM) or BIO (1 μM) for 24 h. RNA was isolated using Trizol (15596-018, Invitrogen) combined with RNAeasy (74106, Qiagen, Valencia, CA) according to the manufacturer's instructions.
Total RNA isolation (microRNA)
MDA-MB-231 cells incubated in the presence or absence of 20 mM LiCl for 24 hours. Total RNA was isolated using the miRVana RNA isolation kit (AM1562, Ambion).
Real time quantitative PCR
Relative quantitation was used to evaluate the raw data obtained from real-time PCR (7900 HT real time PCR system, Applied Biosystems, Foster City, CA). Single-stranded cDNA was prepared using TaqMan Reverse Transcription Reagents (N808-0234, Applied Biosystems) following the manufacturer's protocol. TaqMan Universal PCR Master Mix (4304437, Applied Biosystems) was used for all reactions. All primer/probe mixes (CDH11 Hs00156438_m1, GAPDH Hs99999905_m1) were obtained from Applied Biosystems and performed in triplicate. The samples were analyzed using the delta-delta Ct method of analysis 
. The final value obtained was a measure of the fold change in gene expression for the particular gene of interest between the treated sample and the untreated sample. Experiments were run in triplicate, and for all analyses a p-value of <0.05 was considered to be statistically significant.
cDNA was generated by TaqMan MicroRNA Reverse Transcription kit (4366596, Applied Bioscience) following manufacturer's protocol. Primer/probe mixes specific for each microRNA (RNU6B, 4373381; hsa-miR-19a, 4373099; hsa-miR-27a, 4373287; hsa-miR-33, 4373048; hsa-miR-101, 4373159; hsa-miR-133b, 4373172; hsa-miR-337, 4373044; hsa-miR-424, 4373201) were obtained from Applied Biosystems (Foster City, CA). TaqMan Universal Master PCR Mix (4304437, Applied Biosystems) was used for all reactions. All experiments were performed in triplicate. The average value of the triplicate readings for each unknown was normalized to the corresponding value for U6 RNA. The samples were analyzed using the delta-delta Ct method of analysis 
. The final value obtained was a measure of the fold change in gene expression for the particular gene of interest between the treated sample and the untreated sample. For all analyses a P-value of <0.05 was considered to be statistically significant.
Chromatin immunoprecipitation (ChIP) analysis
One 15 cm tissue culture dishes were plated with 95% confluent MDA-MB-231 cells for each condition. Cells were treated with 5 μg/ml actinomycin D for 30 minutes in serum-free DMEM. As specified, cells were incubated with 20 mM NaCl or LiCl for an additional 24 hours. 37% formaldehyde solution was added to each plate for a final concentration of 1.5% and incubated at 37°C for 15 minutes. Plates were washed one time with PBS containing 0.125 M glycine and Complete Mini Protease Inhibitor Cocktail (11836153001, Roche), and then a second time with PBS plus inhibitor. Cells were collected and spun for at 2000 rpm, 4°C for 5 minutes. The pellet was resuspended in 1 ml SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) with protease inhibitors. Cells were then sonicated using a 15 second on, 45 second off program 4 times consecutively; then diluted 1:10 in ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH .1, 167 mM NaCl) plus protease inhibitor. The samples were then precleared overnight at 4°C with 75 μl Protein A/G Plus-agarose beads (sc-2003, Santa Cruz) supplemented with 3 μl 10 mg/ml Sonicated Salmon Sperm DNA (201190, Stratagene, La Jolla, CA) and 13 μl 1 mg/ml BSA. Samples were then incubated with 10 μg RNA Polymerase II (N-20) (sc-899, Santa Cruz) overnight, tumbling at 4°C. Add 40 μl Protein A/G Plus-agarose beads for 2 hours rotating at 4°C. Samples were spun at 1000 rpm for 1 minute, then washed once with each: Low Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), High Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), LiCl Immune Complex Wash Buffer (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and TE buffer. Samples were eluted twice in 100 μl in elution buffer (1% SDS, 0.1 M NaHCO3) vortexing for 15 minutes at room temperature. 12 μl 5 M NaCl was added to eluate and incubated at 65°C overnight. Then 4 μl 0.5 M EDTA, 8 μl 1M Tris-HCl, pH 6.5 and 2 μl of 10 mg/ml proteinase K was added and incubated at 45°C for 1 hour. The samples were cleaned up using the QIAGEN PCR Clean up kit (QIAGEN). PCR was performed using TaKaRa Premix Ex Taq kit (RR039, TaKaRa, Otsu, Shiga, Japan). The final reaction contains: 1× Premix Ex Taq, 2.5 μM of each primer, 8% DMSO, and 20% processed DNA (or 20% of a 1:10 dilution of input). For amplification of GAPDH, forward: (5′-TACTAGCGGTTTTACGGGCG-3′) reverse: (5′-TCGAACAGGAGGAGCAGAGAGCGA-3′). For amplification of CDH11,
reverse: (5′-AGGTACAAACCCCCTCTGCT-3′) primers were used.
Cells were treated for the indicated times. Cells were rinsed once with PBS and lysed with sample buffer (2% SDS, 10% glycerol, 10 mM Tris, pH 7.5) containing 1 mM sodium orthovanadate, 0.05 M sodium fluoride, and Complete mini protease inhibitors (11836153001, Roche Applied Science, Indianapolis, IN). Cell lysates were boiled for 10 minutes. Protein concentration was determined with a Bio-Rad DC Protein Assay (500-0116, Bio-Rad, Hercules, CA). After SDS-poly acrylamide gel electrophoresis, proteins were transferred to Protran BA 83 Nitrocellulose (10402495, Germany). Membranes were blocked with 5% milk in Tris-Buffered Saline containing 0.1% Tween-20, and incubated with primary antibody overnight at 4°C and subsequently with HRP-labeled secondary antibody. Proteins were visualized with ECL chemiluminescent reagents (RPN2106, Amersham Biosciences, Piscataway, NJ) or SuperSignal West Femto (34095, Pierce biotechnology Inc., Rockford, IL) using X-ray films (Denville Scientific Inc., Metuchen, NJ).
Luciferase Assay Analysis
Cells were plated at 2×105 in a 12-well plate in growth medium. Cells were transfected in triplicate with 1.8 ug of luciferase plasmid DNA and 0.2 ug of pCMV-Renilla (Promega). For cells treated with anti-Dicer siRNA, 100 nM siRNA was transfected together with Luciferase and Renilla using Amaxa nucleofection and collected and analyzed 48 hours later. Cells at varying densities were transfected using Amaxa nuclefection and 1.67×105 cells (low) or 6.25×105 cells (high) were plated in triplicate in a 12-well plate. Cells treated with meBIO or BIO (1 μM) were transfected in triplicate using Fugene6 (Roche). 24 hours after transfection, cells were treated with either BIO or meBIO. 48 hours after treatment cells were collected. In all cases, cells were lysed using passive lysis buffer provided with the Dual-Luciferase Reporter Assay System (Promega). 20 μl of each sample was loaded in duplicate in a 96-well plate and analyzed using the Dual-Luciferase Reporter Assay System reagents (E1960, Promega). The plate was read on a Berthold MicroLumat Plus LB 96V (Germany) using WinGlow software (Berthold).