05). Primers for ALX/FPR2 PCR were designed using Primer3: ALX/FPR2 forward, 5��-TTCCGGATGACACACACAGT-3��; and reverse, 5��-CTTTAGGGTCGTTGGTCCAG-3��. Protein Extraction and Western Blot Analyses Lysates were harvested in RIPA lysis buffer containing 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, DAPT secretase order 150 mM NaCl, and 1 mM EDTA, supplemented with 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and a protease inhibitor cocktail (1.0 ��g/ml pepstatin, 1.0 ��g/ml leupeptin, 1.0 ��g/ml bestatin, and 1.0 ��g/ml aprotinin) (Sigma-Aldrich). Total protein was estimated using the Bradford assay.

For Western blot analysis, antibodies used included the following: ��-actin (1:20,000; Sigma-Aldrich), CDH1 (1:1,000; BD Biosciences, Oxford, UK), CDH2 (1:1,000; BD Biosciences), JAG1 (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA), HMGA2 (1:500; Santa Cruz Biotechnology), FN1 (1:2,000; BD Biosciences), TGF��R1 (1:1000; Santa Cruz Biotechnology), TGF��R2 (1:1000; Abcam, Cambridge, MA), and THBS1 (1:1,000; Abcam, Cambridge, UK). miRNA Microarray miRNAs were extracted using the miRNeasy Mini Kit (Qiagen) and the miRNA microarray was performed using Micro Paraflo microfluidic chips (LC Sciences, Houston, TX). Small RNA fraction was labeled (Cy3, Cy5) and hybridized to the LC Sciences human miRNA microarray (MRA-1001, miRBase V.14, 894 miRNA probes). Data were analyzed by normalizing the signals using a LOWESS filter (locally weighted regression). Normalized microarray expression data are detailed in Supplemental Table 5.

The ratio of the two sets of detected signals (log2 transformed) and P values of the t test were calculated (P��0.05). Bioinformatic Analyses of miRNAs: Target Prediction TargetScan, miRanda, and PicTar were used to assess predicted targets sites for miRNAs. miRNA sequence was downloaded from the miRNA database (http://www.mirbase.org/). Transcription factor binding site prediction within the putative let-7c promoter was performed using both the UCSC genome browser TFBS Conserved track and the Footer algorithm,45 using default parameters (weighted average P<0.001). RNA Hybrid46 was used to predict the secondary structure of the RNA/miRNA duplex. Pathway targeting by miRNAs was performed using DIANA miRPath (http://diana.cslab.ece.ntua.gr/).47 siRNA/miRNA Mimic/Anti-miR: Transfections siGENOME SMARTpool ALX/FPR2 siRNA and siGENOME RISC Free control siRNA were purchased from Dharmacon.

siRNAs were transfected into HK-2 cells at 60% confluence using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) at a final concentration of 20 nM for 24 hours. Let-7c miScript miRNA mimic (20 nM) and All Stars negative control siRNA (20 nM) (Qiagen), and let-7c anti-miR (40 nM) and control anti-miR (40 nM) (Applied Biosystems) were Brefeldin_A transfected into HK-2 cells using Lipofectamine 2000 (Invitrogen). Cells were then stimulated with LXA4 and/or TGF-��1 as previously described.