Figure S3 MicroRNA mimics and antisense inhibitors are readily taken up by HEK293, A431 and HaCaT cells. Cells were transfected with either 0 (mock), 10, 30 or 100 nM of Cy3-labeled Pre-miR or Anti-miR oligonucleotides and analyzed by flow cytometry. Experiments were performed in triplicate. (A) For each cell line, dot plots of mock-transfected cells (left panel) indicate the populations subjected to fluorescence analysis. The fluorescence distributions of gated cells are plotted for a single replicate, both for Pre-miR (middle) and Anti-miR-transfected cells (right). The fluorescence thresholds for positive cells are indicated (M1). The efficiencies of transfection are represented as the mean fractions of fluorescent cells (‘‘M1 positive cells’’) (B) and the mean geometric means (C) within the gated populations 6 S.D. (TIF) Figure S4 Relative changes in expression of selected mRNAs and mature miRNAs in healthy skin and SCCs. For each group, five samples were analyzed by qRT-PCR, representing ten individuals. Experiments were performed in quadruplicates. Data are based on CT values normalized to ACTB and RNU6B for mRNAs and miRNAs, respectively. (A) For each of the indicated assays, the fold difference in expression with regard to the reference is plotted for each of the healthy skin samples. Horizontal bars represent means of all healthy skin samples. (B) For each assay and for each of the cutaneous squamous cell carcinoma samples the fold difference in expression to the average of the healthy skin samples is indicated. Horizontal bars represent means of all squamous cell carcinoma samples. (TIF) Figure S5 Effects of altered miR-361-5p levels on the proliferative and migratory properties of skin-derived cells. The effects of overexpression and inhibition of miR-361-5p on proliferation and migration of A431 and HaCaT cells were assessed with (A) 4-methylumbelliferone-based proliferation and (B) monolayer wound healing (‘‘scratch’’) assays. Cells were transfected with 50 nM of miR-361-5p mimics, antisense inhibitors, or controls, and made responsive by exposition to reduced serum concentrations (72 and 12 hours, respectively). (A) 72 hours after transfection, relative fluorescence intensities were measured in eight replicates after incubating cells with 4- methylumbelliferyl heptanoate. (B) Cell monolayers were scratched and imaged after 0 and 18 hours. Relative differences in open wound areas (in %) were automatically quantified using TScratch [96]. For all assays, three independent experiments were performed. Mean values 6 S.D. from a representative experiment are plotted. Two-tailed, unpaired t-tests were used to calculate P values (one and two asterisks denote P values,0.05 and,0.01, respectively). (TIF) Table S1 MicroRNAs predicted to target VEGFA. A comprehensive list indicating the miRNAs predicted to have recognition elements in the VEGFA 39-UTR by any of the algorithms used in this study are indicated in the first sheet. For each MRE, the genomic coordinates, the 39-UTR region as defined in the main text, the relative position to the start site of the References VEGFA and miR-361-5p Expression in Human SCC 39-UTR, the particular algorithms and the total number of algorithms predicting the MRE, as well as the total number of algorithms for which target predictions for the corresponding miRNA were available in the accessed information, are indicated. For MREs previously subjected to experimental analysis, the corresponding references are indicated. An analysis of the density of predicted MREs in the whole 39-UTR as well as the defined 39- UTR regions is given in the second sheet. (XLSX) Table S2 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]. For each gene, the particular algorithms and the total number of algorithms predicting it are indicated. Genes acting in the VEGF pathway according to KEGG [100] are shown. (XLSX) Table S3 Gene set enrichment 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]. Putative targets were compared to a human reference gene list and analyzed for pathway enrichment using PANTHER [99]. Pathways significantly enriched among predicted miR-361-5p targets are given. For each pathway, the number of genes from the reference list, the number of genes from the putative target list, the number of genes expected by chance, and the P value are indicated. P values are not Bonferroni- corrected. The VEGF pathway is highlighted. (XLSX) Table S4 Overview of microRNA mimics and antisense inhibitors used in this study. Ordering information and, where applicable, identifiers and sequences of the mature miRNAs are indicated. (XLSX) Table S5 Overview of quantitative reverse transcription PCR assays used in this study. In addition to the ordering information, identifiers of the targeted genes and assayed transcripts are given. Where available, the binding regions of the primers and probes as well as the amplicon lengths are indicated. (XLSX) Acknowledgments We thank Michael Forrer for generating plasmids, Sinem Karaman for help with statistical analyses, and Prof. Jonathan Hall for helpful discussions. Author Contributions Conceived and designed the experiments: AK JI PJD AP AG GFLH APG MD. Performed the experiments: AK JI AP. Analyzed the data: AK JI AP APG MD. Contributed reagents/materials/analysis tools: AK JI PJD AG GFLH APG MD. Wrote the paper: AK PJD APG MD. 1. Detmar M (2000) The role of VEGF and thrombospondins in skin angiogenesis. J Dermatol Sci 24 Suppl 1: S78–84. 2. Carmeliet P (2005) Angiogenesis in life, disease and medicine. Nature 438: 932–936. 3. Lohmann CM, Solomon AR (2001) Clinicopathologic variants of cutaneous squamous cell carcinoma. Adv Anat Pathol 8: 27–36. 4. Hofbauer GFL, Bouwes Bavinck JN, Euvrard S (2010) Organ transplantation and skin cancer: basic problems and new perspectives. Exp Dermatol 19: 473– 482. 5. Schwartz RA, Bridges TM, Butani AK, Ehrlich A (2008) Actinic keratosis: an occupational and environmental disorder. J Eur Acad Dermatol Venereol 22: 606–615. PLOS ONE | www.plosone.org 11 November 2012 | Volume 7 | Issue 11 | e49568