Mechanical Regulation of NCSCs with respective no-strain control by using log-transformed one-sample t-test, n$3. (B) Immunoblotting analyses showing CNN1 protein expression. The level of total actin was used as equal loading control. (C) Immunofluorescent staining of CNN1. (D) Effect of mechanical strain on NCSC differentiation into Schwann cells. After 2 days’ cyclic strain treatment, NCSCs were cultured in Schwann differentiation medium for 1 week, and stained for Schwann cell markers S100b and GFAP. Scale bars = 100 mm. doi:10.1371/journal.pone.0026029.g004 organization in vivo and to investigate anisotropic mechanosen- sing in stem cells and other cell types [12]. Here we show that uniaxial strain promotes the proliferation and differentiation of NCSCs on micropatterned surfaces. The underlying mechanisms are not clear, but it is likely that mechanical strain activates multiple signaling pathways that resulted in changes in different aspects of cell functions. For example, mechanical strain increased Figure 4. Effects of mechanical strain on gene and protein expression. Cyclic uniaxial strain (5%, 1 Hz, in parallel to micro- grooves) was applied to NCSCs for 1 day (for qPCR and immunoflu- orescent staining) or 2 days (for protein expression analysis). (A) qPCR showing relative gene expression changes after 1 day. CNN1: calponin 1; SMTN: smoothelin; MHC: myosin heavy chain; TUJ1: class-III b-tubulin; Col-II: collagen type II; PPARG: peroxisome proliferator-activated receptor-c; and cbfa1: core binding factor a-1. * p,0.05 in comparison Figure 5. Long-term effects of mechanical strain on NCSC differentiation. Cyclic uniaxial strain (5%, 1 Hz, in parallel to microgrooves) was applied to NCSCs for 5 days. (A) Immunoblotting analysis showing SMA, CNN1 and MHC protein expression. The level of tubulin was used as equal loading control. (B) Immunofluorescent staining of SMA, CNN1 and MHC. Nuclei were stained by DAPI in blue. Scale bar = 100 mm. doi:10.1371/journal.pone.0026029.g005 PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26029