Palmitoylation in Osteoblast Figure 6. PAT inhibitor decreased BMP2-induced osteoblast differentiation, which was likely mediated by Osx. A. 2BP diminished the ALP expression induced by BMP2. Primary osteoblasts were cultured in the presence or absence of 2BP for 1 day, and then were further cultured in the presence of 50 ng/ml of BMP2 for 4 days. The plates were then stained for ALP. B. 2BP down-regulated BMP2- induced Osx expression. Primary osteoblasts were cultured in the absence or presence of different concentrations of 2BP for a day, and then further cultured in the presence of 50 ng/ml of BMP2 for 16 hrs. The cells were collected and total RNA was isolated, which was used to perform RT-PCR to determine the mRNA levels of Osx and Runx2. The value of control (lane 1) was set at 1.00. doi:10.1371/journal.pone.0004135.g006 0.1% collagenase type V & 0.05% trypsin-EDTA for 15 min at 37oC five times. The supernatant from the first digestion was discarded and supernatants from the last four digestions were pooled. The cells were washed and plated onto 6 well plates and grown in MEM alpha medium supplemented with 15% FCS (Research Sera) and glutamine until confluent. The osteoblast cultures were amplified to passage 3 before use in further experiments. The mice were kept at the Biological Resources Center (BRC) of Singapore and were used for the experiments following protocols approved by the Institute of Molecular and Cell Biology and BRC. A palmitate analog inhibitor, 2-bromohexadecanoic acid (Merck, Darmstadt, Germany) was dissolved in 100% ethanol at a stock concentration of 100 mM and added to cells at the respective concentrations with ethanol as the control. Alkaline phosphatase (ALP) staining and quantitation Calvarial cells were seeded on 12-well plate at a density of 1.56105 cells in differentiation medium (growth medium supple- mented with 50 mg/ml ascorbic acid and 10 mM b-glycerophos- phate) and incubated at 37uC for 4 or 7 days. For ALP staining, the differentiation medium was removed and the cells were fixed with fixative solution [60% v/v acetone and 0.8% v/v citrate concentrate (Sigma-Aldrich, St. Louis, MO)] for 30 seconds before rinsing with deionized water for 45 seconds. For the biochemical assay, cell layer was rinsed twice with Tris buffered saline [20 mM Figure 7. Inhibition of protein palmitoylation compromised p38 MAPK. A. 2BP showed an inhibitory effect on p38 MAPK activation in differentiation assays, without a significant effect on Erk1/2 activation or Smad1/5/8 activation. Primary osteoblasts were cultured in differentiation medium with or without 2BP for different periods of time. Cells were collected for Western blot analysis of the activation of p38, Erk1/2, and Smad1/5/8. B. 2BP also compromised p38 MAPK activation in response to BMP2. Primary osteoblasts were pretreated with PAT inhibitor overnight, and then stimulated with 100 ng/ml of BMP2. Cells were collected at different time points for analysis of p38 and Erk1/2 activation. doi:10.1371/journal.pone.0004135.g007 Tris in 0.9% NaCl, pH 7.4] before harvesting in 500 ml of 50 mM Tris, pH 7.4 buffer. The cell suspension was sonicated for 20 seconds. The ALP activity was assayed in 221 assay buffer [0.1 M 221 buffer (Sigma-Aldrich), 10 mM MgCl2, pH 10.3] with 10 mM p-nitrophenylphosphate (Sigma-Aldrich) as the substrate. The reaction was stopped upon the addition of 0.3 N NaOH and the absorbance was read at 405 nm. The enzyme activity was then normalized with the protein concentration. Bone nodule formation Calvarial cells were seeded as for ALP staining but were incubated for 14 and 21 days. Cells were rinsed twice with Tris buffered saline and followed by fixing with 4% formalin for 5 to 10 minutes. The fixed cells were then rinsed twice with ddH2O. Mineral deposition was stained with 5% silver nitrate and exposed under UV until desired intensity was achieved before washing with ddH2O. RNA isolation and Quantitative RT-PCR Total RNA was extracted from cells after rinsing twice with ice- cold PBS and purified with TRIzolH reagent (Invitrogen) according to manufacturer’s protocol. cDNAs were synthesized from 1 mg of total RNA using Reverse-ItTM RTase Blend (Abgene, Epsom, UK) with Oligo(dT)15. The detection and PLoS ONE | www.plosone.org 7 January 2009 | Volume 4 | Issue 1 | e4135