<p>Micrographs showing immunostaining of primary rat (7-day-old) Sertoli cells and peritubular cells. The cells were seeded at an initial density of 10<sup>6</sup> cells/cm<sup>2</sup> in DMEM and cultured for one week. Cells were fixed at day 7. Peritubular cells are marked for α-smooth muscle actin (brown precipitate). Nuclei were stained blue with hematoxylin. 1A–D) Cells cultured on flat PDMS substrate (1A), and substrates carrying nanogratings of 200 nm (1B), 350 nm (1C) and 5 um (1D) dimensions. Red arrows indicate the direction of nanogratings. 1E) Quantitatitive analysis of directional changes in peritubular cells on different substrates. All nanogratings evoked changes in the orientation of peritubular cells. No visible influence on Sertoli cells was noted. 1F–H) Cells cultured on 350 nm PDMS substrate after inhomogeneous seeding creating diversity in plating density. Areas of high cell density (1F) revealed a random distribution of Sertoli cells in contrast to an aligned orientation towards nanogratings in low cell density zones (1G) on the same substrate in the same well. Peritubular cells were always oriented in accordance with the direction of the nanogratings. Quantification of cellular orientation in Sertoli cells (1H). Sertoli cell density was determined in randomly selected microscopic frames (as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060054#pone-0060054-g001" target="_blank">Figs. 1F–G</a>) and the predominant cellular characteristic (either randomly shaped or spindle shaped (aligned)) was recorded for each frame. Seven recordings were performed per experiment and seven independent experiments were analyzed. We established that the threshold to respond to the nanogratings occurred at a density of approximately 1000 cells/mm<sup>2</sup>. Scale bar = 50 um.</p
