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Additional Supporting information may be found in the online version of this article.

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JBMR_267_sm_SuppFig1.tif5544KSupplemental Figure 1. (A) Northern blots of Nell-1, Ocn and Gapdh using total RNA of whole head tissues from newborn mice with the indicated Runx2 genotypes. Notably, a single band of 3.5 kb Nell-1 or 0.7 kb Ocn transcript was detectable in Runx2+/+ and Runx2+/− tissues, but not in Runx2−/− tissue, respectively. (B) Runx2+/−/CMV-Nell-1 P21 mice demonstrating significantly increased calvarial bone formation relative to the Runx2+/− animal on microCT (arrows in green). (C) Three major MAPK pathways including ERK, JNK and p38 were tested for their activation status upon rNell-1 stimulation in Runx2+/+ NMCCs. The phosphorylation pattern of both ERK1/2 and JNK1 with rNell-1 stimulation was similar to that seen in Runx2+/− cells. Unexpectedly, the level of p38 and p-p38 in Runx2+/+ cells was low while high levels of p-38 and p-p38 were observed in Runx2−/− and Runx2+/− cells under the current study condition in Figure 4i. (D) Graph showing dose dependent rNell-1 stimulation of 6OSE activity in both Runx2+/− and Runx2−/− cells with significant increase from 100 ng/ml to 1600 ng/ml rNell-1 (* p < 0.05 and ** p < 0.01). (E) Cell proliferation assay using MTT at different time points after MAPK inhibitors treatment (* p < 0.05, ** p < 0.01 and NS: no significant difference).
JBMR_267_sm_SuppFig2.tif7322KSupplemental Figure 2. Flow cytometry analysis of apoptosis with Annexin V and PI staining on NMCCs treated with MAPK inhibitors. (A) Distribution of apoptotic and non-apoptotic cells. (B) Relative percentage of early and late apoptotic cells among different treatment groups. FAS: Fas-induced apoptosis as positive control; NT: no treatment control; Ap: apoptosis.

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