Mice Intestinal Tumor Analysis At four months of age, W/APC and Tg/APC mice were euthanized by cervical dislocation to minimize suffering and their small and large intestines were removed and flushed with phosphate buffered saline solution. Intestines were dissected and analyzed for number, location, and size of tumors with the help of a stereoscopic microscope. Some tumors were subjected to RNA and protein analysis and the others were further histologically analyzed by the Hematoxylin-Eosin staining procedure. Reagents Primary antibodies for Western blot analysis are anti-ERK5 antibody:ab40809 (Abcam, Cambridge, UK), anti-c-Myc anti- body: ab11917(Abcam), anti-p68 antibody: ab21696 (Abcam), anti-p72 antibody: sc-130650 (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), anti-cyclinD1 antibody: sc-450 (Santa Cruz Biotechnology, Inc.), anti-c-jun antibody: 2315 (Cell Signaling Technology Inc., Danvers, MA, USA), anti-GAPDH antibody: AM4300 (Ambion, Austin, TX,USA), and anti-HA antibody: 12CA5 (F. Hoffmann-La Roche Ltd., Basel, Switzer- land), and second antibodies are Protein A-Horseradish Peroxi- dase: NA9120V (GE Healthcare UK Ltd., Buckinghamshire, UK) and Goat F(ab’)2 Anti-mouse Ig’s HRP conjugated : AMI4404 (Life Technologies Co., Carlsbad, CA, USA). siRNAs for human c-myc (#1:HS01-00222676, #2:HS01-00222677, #3:HS02- 00466635) and human ERK5 (HS01-00226859), and MISSION siRNA Universal Negative Control were purchased from Sigma- Aldrich Co. (St. Louis, MO, USA). miRNA mimics of miR-143, miR-145, miR-34a and miR-26a were purchased from Qiagen GmbH (Hilden, Germany), and miR-206 mimic was obtained from Ambion. DNA Construction For making the CAG/miR-143, ,300 bp human pri-miR-143 fragment was subcloned into the EcoRI site of pCAGGS vector. To construct CAG/EGFP, the insert fragment of pMXs-puro- EGFP -miR-145/miR-143 [39] was subcloned into XhoI site of pCAGGS vector, and pri-miR-145 fragment was excised by ClaI and NotI. The ends were blunted and self-ligated. A SalI and HindIII fragment of CAG/miR-143 was purified from agarose gels with ELUTIP-D (GE Healthcare UK Ltd.), and injected into eggs. For luciferase reporter assay, the 39UTR fragments of the mouse p68 and p72 containing possible target sites for miR-145, miR-26a, miR-34a or miR-206 were amplified from genomic DNA of a C57BL/6 mouse by PCR using specific primers containing an XhoI site at the 59end. Each fragment was subcloned into XhoI site of the firefly luciferase pGL3-Promoter vector (Promega, Madison, WI, USA), and clones harboring inserts in the forward direction were selected by DNA sequencing. Primers for construction are shown in Table S1. No new sequence data have not been generated in this study. All experiments were authorized by the Institutional Recombinant DNA Experiment Committee of Chubu University (approval no. 06–8, 08–07). Cell Culture and DNA/RNA Transfection DLD-1 cells and Lovo cells were grown in RPMI-1640 medium (#189-02025, Wako, Osaka, Japan) and Ham’s F-12 medium (#087–08335, Wako) with 10% fetal calf serum, respectively. HEK293 cells were cultured in DMEM medium (#D5796, Sigma) with 10% fetal calf serum. Transfection was performed according to the manufacture’s methods. Briefly, DNA plasmids, siRNA or miRNA was mixed with Lipofectamine 2000 (#11668, Invitrogen, Carlsbad, CA, USA) in Opti-MEMI Reduced Serum Repression of Tumors in miR-143 Transgenic Mice Medium (#31985, Gibco, Billings, MT, USA), then transfected into 30–40% confluent cells. Seventy two hours after the transfection, cells were subjected to analysis. DLD-1 cells and Lovo cells were purchased from Japan Health Science Research Resources Bank (HSRRB) and grown according to the HSRRB protocol and HEK293 cells were purchased from ATCC. Western Blot Analysis Whole cell extracts were prepared by lysing cells in RIPA buffer (10 mM Tris-HCl, pH7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate) containing 1 mM phenylmethylsulfonyl fluoride. Fifty,one hundred fifty mg protein was electrophoresed on SDS- polyacrylamide gels, transferred to PVDF membrane (Immobilon, Millipore Corporation, Billerica, MA, USA). The membrane was blocked by 5% skim milk in phosphate buffered saline solution with 0.05% Tween 20 (Sigma) for 30 minutes, stained with each antibody according to the manufactures’ methods and thereby subjected to ECL systems. The membrane was analyzed with Image Analyzer LAS-3000 (Fuji Film, Tokyo, Japan). Northern Blot Analysis Total RNA was extracted with Trizol (Invitrogen) following the manufacture’s manual and separated on 8 M Urea 15% polyacrylamide gels for microRNA analysis, then transferred to Hybond N+(GE Healthcare UK Ltd.). DNA oligonucleotide probes (Table S1) complementary to each miRNA were labeled with [c32P] ATP (MP biomedicals, Solon, OH, USA) by T4 nucleotide kinase (New England Biolabs, Inc., Ipswich, MA, USA). The membrane was hybridized with the probe at 42uC for 2 hours and washed 3 times with 0.2 x SSC containing 0.1% SDS at 42uC for 10 minutes. All probes were purified by MicroSpin G-25 Columns (GE Healthcare UK Ltd.) and hybridization was performed in Rapid-Hyb Buffer (GE Healthcare UK Ltd.). Data were analyzed by Typhoon Trio (GE Healthcare UK Ltd.). Densitometric analysis was performed by Multi Gauge 3.0 software (Fuji film). Reverse Transcription and Quantitative Real-time PCR (qRT-PCR) 100 n g of total RNA was subjected to the reverse transcription reaction using TaqMan Reverse Transcription Kit (Life Technologies Co.) and Taqman PCR was performed using Taqman Universal Master Mix II, No AmpErase UNG (Life Technologies Co.) on the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The expression of mature miR-143 and miR-145 was assayed with the Taqman MicroRNA Assays (Applied Biosystems) specific for hsa-miR-143 (P/N: 4395360) and hsa-miR-145 (P/N: 4395389), respectively. snoRNA202 (Life Technologies Co., P/N: 4380914) was used for normalization. For analysis of primary transcript of pri-miRNA, total RNA was extracted by RNAqueous-R-4PCR (Ambion P/N AM1914) and subjected to qRT-PCR using the standard Taqman protocol. b-actin was used for normalization. Evaluation of b-actin expression was performed by the SYBER Green PCR. Briefly, one mg of total RNA was subjected to the reverse transcription reaction using PrimeScript RT reagent Kit (Takara Bio Co., Shiga, Japan). Then, qRT-PCR was done using SYBR premix Ex Taq II (Takara Bio Co.). All values were corrected by each calibration curve and the relative expression level was measured using the DDCt method. Primers for PCR are shown in Table S1. PLoS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e42137