After 4 days in culture, immunostaining for CollI (green) was performed on substrates with square islands with side lengths of (A) 100 m, (B) 80 m, (C) 60 m, (D) 40 m, and (E) 20 m, and (F) homogeneous EDS surfaces (unconstrained). Ethidium homodimer was used as a nuclear counterstain (red). (Scale bar = 100 m.) (G) A three-dimensional plot of the percentages of single cells producing CollI are shown versus projected area and time. Cell exposure to patterned adhesive islands between 1,600 and 3,600 m2 resulted in the highest CollI expression levels. (H) A dual ordinate plot of NSI (area/height), a metric of nuclear spreading, and CollI synthesis indicate an inverse relationship.

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