Nicotine Delays Bone Growth Figure 1. Detection and localization of nAChR subunits in growth plate chondrocytes. A: The expression of each subunit of nAChR. Total RNA was isolated from primary culture of human growth plate chondrocytes. The primers for each subunit are listed in Tables S1–S3. RT-PCR amplified products of alpha5, alpha7, beta1 and epsilon subunits of nAChR and GAPDH. B: Western blot analysis of alpha7 subunit of nAChR in primary chondrocyte cultures. NC: negative control (adipocyte), PC: positive control (PC-12 cell), GPC1,2: human growth plate chondrocyte derived from extra fingers of two individuals. C: Immunocytochemical analysis of alpha7 nAChR subunit in human growth plate chondrocytes. Primary chondrocytes were stained with alpha7 nAChR subunit-specific antibody. D: Immunohistochemical analysis of alpha7 nAChR subunit in tibia of E15.5 fetuses. Alpha7 nAChR are detected at resting, proliferating and pre-hypertrophic chondrocytes of murine growth plate. doi:10.1371/journal.pone.0003945.g001 of maternal nicotine exposure on skeletal development of murine fetuses in which the alpha7 nAChR gene is disrupted. Maternal genotype is alpha7 nAChR +/2 in this experiment (Figure 6), unlike the experiment using wild type mice (Figure 5, maternal genotype: alpha7 nAChR +/+), and littermate fetuses (alpha7 nAChR 2/2 and alpha7 nAChR +/+) were compared to exclude the effect of nicotine on maternal bodies. Nicotine significantly reduced FL and HL/FL in alpha7 nAChR +/+ fetuses but not in alpha7 nAChR 2/2 fetuses (Figure 6A, B). However, nicotine did not significantly affect body weight (BW) in both genotypes PLoS ONE | www.plosone.org 3 December 2008 | Volume 3 | Issue 12 | e3945