manufacturer’s recommendations. After subtraction of blank values, triplicates were averaged and quantified using a standard curve prepared from serial dilutions of purified VEGFA. Data were normalized by cell number. Flow Cytometry 125,000 A431, HaCaT or HEK293 cells were reverse transfected with 10, 30 or 100 nM Cy3 dye-labeled Pre- or Anti-miR Negative Control #1 (Applied Biosystems, Table S4), or mock-transfected, using siPORT NeoFX (Applied Biosystems) according to the manufacturer’s recommendations. Transfections were performed in triplicate in 24-well plates. After 24 hours, cells were de-attached with 0.05% trypsin-EDTA (Invitrogen, Cat. No. 25300-054) and washed with PBS. Dye-labeled molecules were excited with a blue laser (excitation wavelength = 488 nm) and analyzed with a FACScan flow cytometer (Beckon Dickinson). At least 5000 events were recorded for each sample. Data were analyzed with WinMDI 2.8. Quantitative Reverse Transcription PCR For each reaction, cDNA was prepared from 10 ng total RNA using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) for miRNA detection, or the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) for mRNA detection. MicroRNA and gene expression assays were purchased from Applied Biosystems (Table S5). Quantitative PCR reactions were performed in quadruplicates, using FastStart TaqMan Probe Master (Rox; Roche) in an AB 7900 HT Fast Real-Time PCR System (Applied Biosystems). Quantification was performed using the 22DDCT method [94], with RNU6B and ACTB as references for the normalization of miRNA and mRNA expression levels, respectively. Based on the manufacturer’s assertions, equal amplification efficiencies of close to 100% were assumed for all assays, thus justifying the application of the 22DDCT analysis method and the comparisons between different microRNA and gene expression assays. Monolayer Wound Healing and Proliferation Assay For proliferation assays, 5,000 A431 or HaCaT cells were reverse transfected with 50 nM Pre2/Anti-miR-361-5p or Pre-/ Anti-miR Negative Control #1 (Applied Biosystems, Table S4) by using siPORT NeoFX (Applied Biosystems) according to the manufacturer’s recommendations. Transfections were performed in eight replicates per condition in black, clear-bottom 96-well plates (Corning, Cat. No. CLS3603). Cells were incubated in DMEM supplemented with 1% fetal bovine serum. 72 hours after transfection, cells were incubated with 4-methylumbelliferyl heptanoate (Sigma-Aldrich, Cat. No. M2514) as previously described [95]. Fluorescence intensities were measured with a SpectraMax Gemini EM microplate reader (Molecular Devices). For monolayer wound healing assays, 125,000 A431 or HaCaT cells were reverse transfected with 50 nM Pre-/Anti-miR-361-5p or Pre-/Anti-miR Negative Control #1 (Applied Biosystems, Table S4) by using siPORT NeoFX (Applied Biosystems) according to the manufacturer’s recommendations. Transfections were performed in triplicate in 24-well plates. Monolayer wound healing assays were performed as previously described [96]. Briefly, 14 hours after transfection, the medium was replaced with DMEM supplemented with 1% fetal bovine serum, and cells were incubated for 12 hours. Cell monolayers were scratched crosswise with a sterile 200 mL pipette tip and imaged at 56 magnification using an Axiovert 200 M microscope with an AxioCam MRm camera (Carl Zeiss AG) immediately after scratching, as well as VEGFA and miR-361-5p Expression in Human SCC after 18 hours of incubation. Open wound areas were quantified using the TScratch software [96]. MicroRNA Target Gene Prediction and Pathway Analysis Predictions of human miRNA targets were downloaded from microRNA.org [54] (August 2010 release, http://www.microrna. org/, accessed: August 12th, 2011; all predictions with mirSVR of less than -0.1 were considered), TargetScan [55] (Release 5.2; http://www.targetscan.org/, accessed: August 12th, 2011; all predicted miRNA recognition elements were considered, regard- less of conservation), DIANA-microT v3.0 [56] (Release 3.0, http://diana.cslab.ece.ntua.gr/microT/, accessed: August 12th, 2011), miRDB [57] (Release 3.0, http://mirdb.org/miRDB/, accessed: August 12th, 2011), and MicroCosm [58] (Release 5, www.ebi.ac.uk/enright-srv/microcosm/, accessed: August 12th, 2011). The miR-361-5p sequence was obtained from miRBase [97] (Release 17, http://www.mirbase.org/, accessed: May 5th, 2011). RNAhybrid [59] (http://bibiserv.techfak.uni-bielefeld.de/ rnahybrid/) was used online, with default settings and the following sequences: UUAUCAGAAUCUCCAGGGGUAC (miR-361-5p, microRNA) and UGUAUAUAUGTGAUUCU- GAUAAA (VEGFA 39-UTR fragment containing the putative miR-361-5p MRE, target RNA). For the pathway analysis, predicted targets for miR-361-5p were converted to Entrez identifiers using DAVID [98] (http://david.abcc.ncifcrf.gov/), if not present in the respective outputs. Results were pooled and filtered for unique records, and subjected to gene set enrichment analysis with PANTHER [99] (http://www.pantherdb.org/tools/ compareToRefListForm.jsp). For VEGFA pathway analysis, the KEGG PATHWAY database [100] (http://www.genome.jp/ kegg/pathway.html) was consulted. All services were used with default settings. Supporting Information Figure S1 Overview of the human VEGFA 39-UTR, hsa- mir-361 and their predicted hybrid structure. (A) UCSC genome browser [101] view of the genomic locus encoding the human VEGFA 39-UTR. Indicated are the terminal exon of the VEGFA gene, PhyloP and PhastCons conservation scores, GC content, the 39-UTR fragment common to all isoforms, and the 39- UTR region subcloned downstream of the luciferase reporter used in this study. Moreover, the relative locations of the recognition elements for miRNAs demonstrated to be able to regulate either human or mouse VEGFA (see introduction for details) are highlighted. The miR-361-5p binding site analyzed in this study is indicated in red. Please note that predicted MREs whose activities have not been unambiguously identified by mutational analysis are listed separately. (B) Secondary structure of the hybrid between miR-361-5p and the putative miRNA recognition element within the VEGFA 39-UTR, as predicted by RNAhybrid [59]. The calculated free energy of the interaction is indicated. (TIF) Figure S2 Pathway analysis of predicted miR-361-5p targets. Target predictions were obtained from the following web services: microRNA.org [54], TargetScan [55], DIANA-microT [56], miRDB [57], and MicroCosm [58]. Results were pooled and converted to uniform gene identifiers using the DAVID web service [98]. A manually curated representation of the VEGF signaling pathway available at KEGG [100] was color-coded according to the number of algorithms that predict an individual gene to be targeted by miR-361-5p. (TIF) PLOS ONE | www.plosone.org 10 November 2012 | Volume 7 | Issue 11 | e49568