Aldrich, St. Louis, MO). Nicotine was added to the sucrose solution starting at an initial concentration of 25 mg/ml to the treatment group mice. This was increased to 50 mg/ml on days 3 to 4, 100 mg/ml after day 5. The control mice were given only sucrose solution as a drinking water. The pregnant mice were sacrificed at noon on gestational day 15. The embryo were immediately weighed, and the legs were immediately removed and fixed in 4% paraformaldehyde phosphate buffer solution (Wako) for 24 h. Then, the legs were dehydrated through alcohols, embedded in paraffin, and sections were stained with Hematoxylin and Eosin for histological analysis. Maternal nicotine exposure in alpha7 nAChR-disrupted mice B6.129S7-Chrna7,tm1Bay./J, the alpha7 nAChR +/2 mice were obtained from Charles River Laboratories Japan. Ten- to 12- week old alpha7 nAChR +/2 mice were mated, and pregnant mice were given sucrose solution with or without nicotine. The fetuses were obtained and analyzed as in the case of wild-type C57BL/6J mice, as described above. The alpha7 nAChR genotype was determined by means of PCR reaction with the specific primers (Table S3). Statistics The results of the quantative assays were expressed as mean6S.D. Significance was determined with Student’s t test and ANOVA. All experiments were replicated twice. Supporting Information Figure S1 In vivo chondrocytic proliferation assay. A: Paraffin section of the femur of E15.5 C57BL/6J mice immunohistochem- ically stained with antibody to PCNA. Proliferative chondrocytes extensively stained positive for PCNA regardless of maternal nicotine exposure. B: Percentage of PCNA-positive cells in chondrocytes of the proliferative zone. There is no significant difference between the two groups. Hind legs of E15.5 C57BL/6J References 1. Davies DP, Abernethy M (1976) Cigarette smoking in pregnancy: Associations with maternal weight gain and fetal growth. Lancet 1: 385–387. 2. 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(1995) Do pregnant smokers eat differently from pregnant non-smokers? Paediatr Perinat Epidemiol 9: 307–319. 9. Haddon W Jr, Nesbitt RE, Garcia R (1961) Smoking and pregnancy: carbon monoxide in blood during gestation and at term. Obstet Gynecol 18: 262–267. 10. Mochizuki M, Maruo T, Masuko K (1985) Mechanism of foetal growth retardation caused by smoking during pregnancy. Acta Physiol Hung 65: 295–304. 11. Nordstrom ML, Cnattingius S (1996) Effects on birthweights of maternal education, socio-economic status, and work-related characteristics. Scand J Soc Med 24: 55–61. 12. Mau G (1976) Letter: Smoking and the fetus. Lancet 1: 972. 13. Nelson E, Jodscheit K, Guo Y (1999) Maternal passive smoking during pregnancy and fetal developmental toxicity. Part 1: gross morphological effects. Hum Exp Toxicol 18: 252–256. Nicotine Delays Bone Growth mice were prepared, fixed in 4% paraformaldehyde phosphate buffer solution (Wako) overnight at 4uC, and embedded in paraffin. After deparaffinization, slides were autoclaved in 0.01 M citrate buffer (pH 6.0) for 10 min, incubated with primary antibody to PCNA (DAKO: PC10) diluted 1:400 in PBS containing 1% BSA at room temperature for 3 h, and incubated with polyclonal rabbit anti-mouse immunoglobrins/HRP (DAKO: P260) at room temperature for 1 h. Staining was undertaken using a solution containing diaminobenzidine and 0.01% H2O2 in 0.05 M Tris-HCl buffer at pH 6.7, followed by counterstaining with hematoxylin. Found at: doi:10.1371/journal.pone.0003945.s001 (0.06 MB PDF) Table S1 Primers for nAChR subunit genes Found at: doi:10.1371/journal.pone.0003945.s002 (0.03 PDF) Table S2 Primers for chondrocyte specific genes Found at: doi:10.1371/journal.pone.0003945.s003 (0.01 PDF) Table S3 Primers for genotypying alpha7 nAChR gene Found at: doi:10.1371/journal.pone.0003945.s004 (0.01 PDF) MB MB MB Acknowledgments We would like to express our sincere thanks to M. Nasu, C-H. Cui, H. Akutsu, K. Miyado, and M. 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