Palmitoylation in Osteoblast Figure 2. Inhibition of protein palmitoylation impeded osteoblast differentiation. A. The effect of 2BP on ALP staining. Primary osteoblasts were treated with increasing amounts of 2BP for 4 days in differentiation medium and then stained for ALP. B. Quantitation of ALP activities that were normalized to the protein levels of the cells. C. The effect of 2BP on the expression of several osteoblast differentiation markers. The experiments were carried out like Fig. 2A and total RNA were isolated from these cells. RT-PCR was carried out to determine the mRNA levels of these markers. The value of control (lane 1) was set at 1.00. D. The effect of 2BP on bone nodule formation. The experiments were carried out like Fig. 2A. After 21 days in culture, the plates were stained using a Von Kossa method. doi:10.1371/journal.pone.0004135.g002 of the canonical BMP-Smad pathway as well as the non-canonical BMP-MAPK pathway. We found that 2BP had little effect on Smad1/5/8 phosphorylation in the in vitro differentiation assays (Fig. 7A). No significant change was observed in the activation of Erk1/2 either. However, PAT inhibitor seemed to inhibit the activation of p38 MAPK (Fig. 7A). Since BMPs are the driving force of osteoblast differentiation and they activate MAPKs, these results suggest that protein palmitoylation is necessary for the activation of the p38 MAPK pathway, but not the canonical BMP- Smad pathway. This conclusion was supported by the finding that 2BP also compromised BMP2-induced activation of both Erk1/2 and p38 MAPKs (Fig. 7B). The failure of 2BP to affect Erk1/2 activation in differentiation assays (Fig. 7A) suggest that other signaling pathways might have made compensation. The corre- lation between compromised p38 MAPK activation and the down- regulation of Osx suggests that protein palmitoylation is involved in BMPs-induced p38 MAPK activation, Osx expression and osteoblast differentiation. PLoS ONE | www.plosone.org 4 January 2009 | Volume 4 | Issue 1 | e4135