Supplementary table 2

Filtered differentially expressed 38 predicted targets of differentially expressed miRNA at 3 and 7 weeks

Supplementary table 3

Filtered differentially expressed mRNA targets of differentially expressed miRNAs (3 weeks time point

Supplementary table 1

Differentially expressed mRNAs at both 3 and 7 weeks of exposure to arsenit

Fibroblast-derived ECM alter Lung Cancer Cell Line Morphology.

<p>(A) Phase contrast microscopy photos of A549, H358, and HPL1D cells on FN, WI38 ECM, IMR90, and HDF ECM. (B) Immunofluorescent confocal microscopy of A549 cells on WI38-derived ECM stained with Phallodin and DAPI. (C) The circularity of A549 on FN and 3 different fibroblast-derived ECM was calculated. n = 10. *, p ≤ 0.05.</p

Identifying mRNA, MicroRNA and Protein Profiles of Melanoma Exosomes

<div><h3>Background</h3><p>Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and in various disease conditions. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth. Molecular profiling may increase our understanding of the role of exosomes in melanoma progression and may lead to discovery of useful biomarkers.</p> <h3>Methodology/Principal Findings</h3><p>In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. We also used proteomic analysis and discovered differentially expressed melanoma exosomal proteins, including HAPLN1, GRP78, syntenin-1, annexin A1, and annexin A2. Importantly, normal melanocytes acquired invasion ability through molecules transported in melanoma cell-derived exosomes.</p> <h3>Conclusions/Significance</h3><p>Our results indicate that melanoma-derived exosomes have unique gene expression signatures, miRNA and proteomics profiles compared to exosomes from normal melanocytes. To the best of our knowledge, this is the first in-depth screening of the whole transcriptome/miRNome/proteome expression in melanoma exosomes. These results provide a starting point for future more in-depth studies of tumor-derived melanoma exosomes, which will aid our understanding of melanoma biogenesis and new drug-targets that may be translated into clinical applications, or as non-invasive biomarkers for melanoma.</p> </div

Fibroblast-derived ECM alters mRNA profile of A549 and H358 Cells.

<p>Heat map significantly changed genes from microarray (fold-change >1.5 and p-value ≤ 0.05). Columns represent individual gene probes; rows are the different samples and each row represents one of the biological triplicates ran on the microarray. (B) Validation of microarray data. Representative genes were chosen for quantitative real-time qRT-PCR analysis. New biological triplicates were prepared, RNA extracted and converted to cDNA, and real-time qRT-PCR was performed. All samples tested were validated and all genes were changed in the same direction as the microarray, however some amplitudes of change were slightly different.</p

Fibroblast-derived ECM Protects Lung Cancer Cell Lines from Serum Deprivation.

<p>(A) A549, H358, and HPL1D cells were grown on fibronectin (FN) or on WI38 ECM in serum-free media for 48 hours and then photographed. (B) Relative cell numbers were quantified for A549 cells (B), H358 cells (C) and HPL1D cells (D) using Alamar Blue every 24 hours after cells were put into serum free media. By the fourth day in serum free media, basically all cells were dead.</p

Correlation of miRNA signals between cells and exosomes.

<p>Affymetrix miRNA 1.0 arrays were used to analyze miRNA signals in HEMa-LP melanocytes and A375 melanoma cells, as well as exosomes from the two cell lines. Two different arrays were performed from two different RNA preparations for each sample, except for only one RNA preparation for HEMa-LP exosomal miRNA array. Scatterplots of miRNA signals in HEMa-LP exosomes compared with their originating cells (<b>A</b>), A375 exosomes compared with their originating cells (<b>B</b>), and A375 exosomes compared with HEMa-LP exosomes (<b>C</b>). Regression analysis showed that miRNA signals in HEMa-LP cells versus HEMa-LP exosomes, and A375 cells versus A375 exosomes were correlated, whereas miRNA signals in A375 exosomes versus HEMa-LP exosomes were not well correlated.</p

Fibroblast-derived ECM decreases Lung Cancer Cell Growth.

<p>(A) A549, H358 and HPL1D cells were grown on fibroblast derived matrices and every day one well of cells was trypsinized and manually counted in triplicate using trypan blue. (B) A549, H358 and HPL1D cells were grown on fibroblast derived matrices and every day Alamar Blue was added to wells of cells in triplicate and relative conversion of Alamar Blue was determined. n = 3. *, p-value ≤ 0.05.</p

Characterization of fibroblasts and their ECM.

<p>A. Microscopic analysis of fibroblasts and de-cellularized matrix. Left colum- 5X phase contrast imaging of confluent fibroblasts just prior to de-cellularazation. Middle colum- 40X phase contrast microscopy of ECM following decellularization. Right column- Immunofluorescent confocal microscopy of fibroblast-derived ECM using an NHS-ester probe conjugated to Alexa Fluor 488. (B) Protein content of ECM was quantitated after scraping of the ECM from decellularized plates. Protein amount is indicated a μg/cm², N = 3. (C) SDS-PAGE and colloidal blue stain of fibroblast-derived ECM. n = 3.</p