anti-rat IgG antibody (Sigma; A 5795) at a dilution of 1:3000 in TBS containing 0.05% Tween-20. After rinsing, the membrane was immersed in ECL solution (GE Healthcare, Buckinghamshire, UK). Then, the blots were visualized by LAS-1000plus IDX2, the luminescent image analyzer (Fuji Photo Film, Japan). Immunocytochemical and immunohistochemical analysis Immunocytochemical analysis was performed as previously described [26]. Briefly, dishes were incubated with antibody to alpha7 subunit of nAChR in PBS containing 1% BSA. As a methodological control, the primary antibody was omitted. After washing in PBS, dishes were incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-rat IgG antibody. Staining was developed by using a solution containing diamino- benzidine and 0.01% H2O2 in 0.05 M Tris-HCl buffer, pH 6.7. For immunohistochemical analysis, hind legs of E15.5 C57BL/ 6J mice were prepared, fixed in 4% paraformaldehyde phosphate buffer solution (Wako) overnight at 4uC, and embedded in paraffin. Immunohistochemical analysis was performed as previ- ously described [27]. Briefly, slides were treated with 0.4% pepsin (DAKO) at 37uC for 30 min, incubated with primary antibody to alpha7 subunit of nAChR(Sigma, product number: N 8158) diluted 1:2000 in PBS containing 1% BSA at room temperature for 3 h, and incubated with simple mouse stain MAX-PO (RAT), a second antibody, at room temperature for 1 h. Staining was developed by using a solution containing diaminobenzidine and 0.01% H2O2 in 0.05 M Tris-HCl buffer, pH 6.7. Finally, slides were counterstained with hematoxylin. Agarose gel cultures Chondrocytes were cultured in agarose-stabilized suspension using a modified method as previously described [28]. Primary monolayer cultures were trypsinized, re-suspended in agarose gel medium : DMEM/F-12 containing 10% FBS, 100 units/ml penicillin G, 100 mg/ml streptomycin, and 50 mg/ml ascorbate, to a concentration of 26104 cells/ml, then mixed with equal volume of 1% low-temperature melting agarose (Sigma-Aldrich, Steinheim, Germany) in agarose gel medium, giving a final concentration of 16104 cells/ml suspended in 0.5% low- temperature melting agarose in agarose gel medium (suspension agarose). Three milliliters of suspension agarose were added to 60 mm culture plates that were precoated with 2 ml of 1% autoclaved standard agarose (Bio-Rad, Hercules, CA). The gel was allowed to solidify at 4uC before addition of agarose gel medium. Then, culture plates were placed in a 37uC, 5% CO2 humidified incubator for 21 days, and medium containing indicated concentration of nicotine was replaced once at the beginning of the week. After 21 days, suspension agarose was transferred to a glass slide, and placed on a plate warmer at 50uC with a covering of positively-charged nylon membranes (Roche, Mannheim, Germany). The slides were completely dried in a incubator at 42uC overnight, and fixed in 4% paraformaldehyde for 15 min, and stained with ALB to identify colonies producing glycosami- noglycans and to observe histologically. Colonies were defined as a cluster of cells with a diameter greater than 50 mm. ALP activity was determined in non-fixed agarose slide by Histofine, ALP substrate kit (Nichirei, Tokyo, Japan) following the manufacturer’s product information. Type 10 collagen expression was also determined in the agarose slide using specific monoclonal antibody (Sigma; product number: C7974). The slide was fixed in acetone (Nacalai Tesque, Kyoto, Japan) at room temperature for 20 min. Non-specific binding was blocked with 2.5% normal rabbit serum (DakoCytomation, Glostrup, Denmark) in PBS containing 1% BSA and 1% Triton X-100. Slides were incubated for 6 h at room Nicotine Delays Bone Growth temperature with primary antibody, diluted 1:2000 in PBS containing 1% BSA. Bound antibody was detected by HRP- conjugated polyclonal rabbit anti-mouse IgM antibody (Dako, Glostrup, Denmark; product number: P 0260) diluted 1:100 in PBS at room temperature for 30 min. Peroxidase activity was visualized with diaminobenzidine tetrahidrochloride plus 0.03% H2O2, and slide was counterstained with hematoxylin. Alginate bead cultures Chondrocytes were cultured in alginate beads following the method described by De Ceuninck et al. Primary monolayer cultures were trypsinized, washed, and centrifuged. The isolated chondrocytes were suspended at a concentration of 26106 cells/ ml in a 1.25% alginate in 0.15 M NaCl. The cell suspension was slowly expressed through a 21 gauge needle and dropped into a 102 mM CaCl2 solution. The beads with approximately 25,000 cells/bead were allowed to polymerize for 10 min and washed three times with 0.15 M NaCl, followed by two washes in DMEM/F12. The beads were then transferred to medium (200 beads/10 ml/60 mm culture dish): DMEM/F-12 containing 10% FBS, 50 mg/ml ascorbate, 100 units/ml penicillin G, 100 mg/ml streptomycin. The beads were cultured at 37uC in a 5% CO2 humidified incubator for four months, and medium with or without nicotine was replaced twice weekly. The beads were transferred to new dishes every other week to avoid the formation of monolayer cultures on the bottom of the dish by chondrocytes escaping from the beads. For histological analysis, the beads were fixed in 4% paraformaldehyde, 0.1 M cacodylate buffer, pH 7.4, containing 10 mM CaCl2 for 4 h at room temperature, and then washed overnight at 4uC in 0.1 M cacodylate buffer, pH 7.4, containing 50 mM BaCl2. The beads were dehydrated through alcohols and embedded in paraffin. The sections were routinely stained with ALB and safranin-O. For RT-PCR analysis, chondrocytes were separated from the beads by incubating the beads in dissolution solution (at a ratio of 200 ml/bead), containing 55 mM EDTA, for 5 min and centri- fuged. Total RNA was isolated by using RNeasy (Qiagen) following manufacturer’s instructions, and was converted to cDNA by same method as described above. The sequences of PCR primers of human chondrocyte-related gene are listed in Table S2. PCR was performed in a final volume of 50 ml containing 2 ml of the single strand cDNA product (10 ng/ml), 10 mM Tris-HCl (pH8.3), 50 mM KCl, 1.5 mM MgCl2, 200 mM dNTPs, 1.25 U Taq (Takara), and 20 pmol of each forward (59) and reverse (39) primers. Calcium imaging Primary monolayer cultures in 35 mm glass-bottomed plates were prepared. At near confluence, measurement was done by using Fluo-4 NW calcium assay kit (Molecular Probes, product number: F36206) following the manufacturer’s product informa- tion. In short, the cells were incubated in dye loading solution containing 2.5 mM probenecid at 37uC for 30 min, then at room temperature for an additional 30 min before nicotine stimulation. The fluorescence was measured in LSM 510 (Carl Zeiss) with the settings appropriate for argon laser. Nicotine and its antagonists were prepared as a solution in assay buffer. If antagonists were used, they were added 30 min prior to nicotine stimulation. Maternal nicotine exposure in wild-type mice Three-month-old pregnant mice were purchased at day 1 of pregnancy from Sankyo Laboratories (Tokyo, Japan). The mice were given drinking water containing 2% sucrose (Wako, Osaka, Japan) with or without nicotine (hydrogen tartrate salt; Sigma- PLoS ONE | www.plosone.org 9 December 2008 | Volume 3 | Issue 12 | e3945