Decreased ECM collagens and fibronectin in KC keratocyte cultures. Representative images of Sirius red and fast green stained cultures (20x) in (A) 2 separate DN and (B) 2 separate KC cultures, scale bar: 50 μm. (C) Cell associated hydroxyproline quantification for total collagenous protein, (D) media associated hydroxyproline. Each point representing means of three measurements from individual cultures, n = 6. Statistical significance was assessed using the Student's t-test; *P < 0.05. (E) Cell immunoblot of COL1A1 and GAPDH from 14-day cultures. (F) Corresponding Immunoblot of COL1A1 released into the media over 3 days (days 11–14) with total protein for normalization. Quantitative PCR of (G) COL1A1 and (H) COL5A1 transcription relative to POLR2A. Points represent mean of individual donor cultures and shown as mean ± SEM; statistical significance was assessed using the Student's t-test; *P < 0.05, **P < 0.01. (I, J) Immunohistochemical localization of COL1A1, COL5A1 and FN1 in DN cultures and KC cultures. Green: protein of interest, Red: F-actin, Blue: DAPI. Scale bar: 50 μm.