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. Author manuscript; available in PMC: 2016 May 18.
Published in final edited form as: Nat Mater. 2015 Oct 12;14(12):1262–1268. doi: 10.1038/nmat4444

Figure 5. Fibrillar ECM remodeling promotes focal adhesion (FA) formation and FAK phosphorylation to increase proliferation.

Figure 5

a, FA formation of representative hMSCs seeded on DexMA hydrogels of low and high stiffness as visualized by cytosol extraction, vinculin immunostaining, and subsequent image analysis to identify FAs (orange). The cell’s cytoplasm (green) and nucleus (blue) are also shown (scale bars, 50 µm). b, FA formation of representative hMSCs on DexMA fiber networks of low and high fiber stiffness 16 h after seeding. Composite images (left) showing FAs (orange), cytoplasm (green), nuclei (blue) and fibers (grey). Single channel images of vinculin (middle) and fibers (right, bottom) as well as identified FAs (right, top) (scale bars, 50 µm). Cell area (c), total FA area (d), total number of FAs (e), and average FA size (f); mean ± s.d., n = 12 cells, * P < 0.05. g, Merged images of representative hMSCs 16 h after seeding co-stained for vinculin (red) and phospho-FAK (green). Single channel images of phospho-FAK with cells outlined in magenta (scale bars, 50 µm). h, Quantification of phospho-FAK localization to FAs determined by fluorescence intensity; mean ± s.d., n = 10 cells, * P < 0.05. i, Effect of FAK phosporylation inhibition on proliferation of hMSCs over two days, as determined by EdU incorporation; mean ± s.d., n ≥ 9 ROI with totals of 450–1500 cells analyzed, * P < 0.05. j, To test fiber-fiber connectivity, a diamond sharpened blade was placed adjacent to individual fibers and reciprocated via micromanipulator. Soft networks as fabricated in all previous studies possess limited connectivity as demonstrated by free sliding of fibers (top row) in contrast to “welded” networks with high fiber-fiber connectivity (bottom row). k, Remodeling of fiber networks 16 h after MSC seeding. Fiber recruitment in 50 µm diameter circular regions centered on the cell nucleus. Fibers imaged by coupling with rhodamine methacrylate (cyan) and thresholded cell nuclei labeled with Hoechst 33342 (magenta) (scale bars, 100 µm). Fluorescence intensity was normalized to adjacent acellular areas. l, Effect of altering fiber-fiber connectivity on proliferation of hMSCs over two days as determined by EdU incorporation; mean ± s.d., n ≥ 9 ROI with totals of 450–1500 cells analyzed, * P < 0.05.