Palmitoylation in Osteoblast Figure 3. PAT inhibitor show minimal effect on proliferation/survival of osteoblast cultures. A. Primary osteoblast cells were cultured in differentiation medium with different amounts of 2BP for 4 days and the cell numbers in each culture plates were determined by trypan blue exclusion methods. B. The experiments were done like Fig. 3A and the cells were collected and lysed in the same volume of lysis buffer and the protein concentrations were determined by the BioRad method. C. Recovery of 2BP treated cells in ALP expression. Primary osteoblasts were treated with different concentrations of 2BP for three days, washed off, and further cultured in differentiation medium for 4 more days before being stained for ALP (bottom panel), in comparison to the cells that were treated with 2BP all the time (upper panel). doi:10.1371/journal.pone.0004135.g003 PLoS ONE | www.plosone.org 5 January 2009 | Volume 4 | Issue 1 | e4135 Figure 4. Regulation of Osx by 2BP. Primary osteoblasts were cultured in differentiation medium with or without 100 mM of 2BP for different periods of time. These cells were then collected to isolate total RNA. RT-PCR was carried out to determine the mRNA levels of these proteins. The value of control (lane 1) was set at 1.00. doi:10.1371/journal.pone.0004135.g004 Discussion This study shows that most of the 24 PATs are expressed in primary osteoblasts, with some showing an up-regulation during differentiation. In addition some proteins are palmitoylated in osteoblasts and the modification can be inhibited by the substrate analog inhibitor, 2BP. It was also found that inhibition of PATs with the inhibitor impeded osteoblast differentiation and function, evidenced by a decrease in the expression of ALP, osteocalcin, Osx, and in bone nodule formation. Moreover, this inhibitor also compromised BMP2-induced osteoblast differentiation. However, treatment of the cells with the same concentration of palmitic acid did not show such effects. In addition, inhibition of PATs showed little effect on osteoblast proliferation and survival under differentiation assay conditions. These results suggest that protein palmitoylation plays an important role in osteoblast differentiation. How does protein palmitoylation regulate osteoblast differenti- ation? Our results suggest that Osx might mediate this effect. Firstly, treatment with PAT inhibitor down-regulated Osx expression, not Runx2 or Atf4. Secondly, BMP2-induced osteoblast differentiation was also hindered by the PAT inhibitor and this was accompanied by a decrease in the expression of Osx, but not Runx2. Thirdly, when Osx could not be repressed in p532/2 osteoblasts, which showed enhanced differentiation due to elevated Osx expression [16], osteoblast differentiation was refractory to the inhibitory effect of the PAT inhibitor. Thus it appears that protein palmitoylation is required for the basal