before permeabilization with Triton X-100 and blocking with FBS. Cells were stained for one hour at room temperature with either 1:50 FITC-conjugated anti-tubulin, 1:200 Rhodamine Phalloidin , or 1 mg/mL rabbit anti-vinculin followed by 1:1000 goat anti-rabbit Alexa Flour 488 before sealing with Vectashield (Vector Labs) containing DAPI. For morphological visualization, cells were stained with crystal violet then rinsed extensively. All cells were visualized using either an inverted Nikon Eclipse Ti or Zeiss LSM 510 UV confocal microscope. Quantitative Image Analysis Quantitative image analysis was performed by a series of custom written MATLAB algorithms. For cell elongation, borders were manually drawn around cells before segmentation to extract cell major and minor axis. After normalization for background, stress fibers were segmented by a Laplacian filter, and then normalized to cell area extracted from a built-in MATLAB thresholding algorithm. Nuclei were analyzed using a semi-automated MA- TLAB algorithm that visualized each image before quantification to ensure only single nuclei were measured. Similarly, for actin and vinculin quantification, images were normalized then segmented for quantification. Migration Assays To quantify migration, 3 or 8 mm pore transwell inserts (Greiner Bio One) were added to a 24 well plate, rinsed in PBS then incubated overnight in serum free DMEM. The following day, the media in the well was replaced with 600 mL of CM or TCM and 10,000 serum-starved MSCs in 100 mL of CM or TCM were added to the top of each insert. After desired incubation time, non- migrated cells were gently removed from the top of each transwell using a cotton swab. The cells on the bottom of the inserts were then fixed in 4% formaldehyde with 0.01%Triton X-100 and stained with DAPI. For quantification, an automated image acquisition algorithm collected 9 fields of view at 106 magnifi- cation per transwell and nuclei were counted using standard functions in MetaMorph software. The average number of cells per image was reported. References 1. Balkwill F, Mantovani A (2001) Inflammation and cancer: back to Virchow? Lancet 357: 539–545. 2. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646–674. 3. Covas DT, Panepucci RA, Fontes AM, Silva WA, Orellana MD, et al. (2008) Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivas- cular cells and fibroblasts. Exp Hematol 36: 642–654. 4. Haniffa MA, Wang X-N, Holtick U, Rae M, Isaacs JD, et al. (2007) Adult Human Fibroblasts Are Potent Immunoregulatory Cells and Functionally Equivalent to Mesenchymal Stem Cells. The Journal of Immunology 179: 1595–1604. 5. 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Hall B, Andreeff M, Marini F (2007) The Participation of Mesenchymal Stem Cells in Tumor Stroma Formation and Their Application as Targeted-Gene Delivery Vehicles. In: Kauser K, Zeiher A-M, eds. Bone Marrow-Derived Progenitors Springer Berlin Heidelberg. pp 263–283. Mechanical Changes of MSCs and Fibroblasts Gene Expression Analysis Total RNA was isolated using TRIzol reagent (Invitrogen) per manufacturer’s protocol before reverse transcription using the iScript cDNA synthesis kit (BioRad). DNA concentration and quality were verified by spectroscopy. To verify differentiation, PCR was run per previous literature [34]. For qRT-PCR on the small Rho GTPases, Cdc42, RhoA, and Rac1, as well as an endogenous GAPDH control, the real-time PCR reaction was run using SsoAdvanced SYBR Green Supermix (BioRad) in an AB Step One Plus thermocycler (n = 5). Primer sequences are reported in Information S2. 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PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e33248