SIRT6 activates the NF-κB/p65, p38-MAPK and ERK signaling pathways and thus affects cellular mitochondrial energy metabolism in DMCs.

<p>(A) A western blot analysis of the <i>Sirt6</i> KO group showed enhanced activation of the NF-κB/p65, p38-MAPK, and ERK signaling pathways on phosphorylation of those signaling molecules. Quantitative analysis obtained the same relative results. (B) PCR array analysis showed a significant difference between the KO and WT group with respect to the mRNA expression levels of Atp4b and Cox6a2. They were reduced by 51% and 54% in the KO group, whose ATP levels in the DMCs were significantly lower than those of the WT group. The gene expression level was normalized to GAPDH (n = 3). *<i>p</i><0.05 versus CO. CO = control. GAPDH was used as a control.</p

Expression of SIRT6 in mouse dental pulp tissue.

<p>(A) The DMCs were either fusiform or triangular in shape; they were positive for vimentin, and negative for cytokeratin. (B) The higher magnification images of Immunohistochemial (IHC) staining showed that SIRT6 was expressed in the nuclei of mature odontoblasts in the WT group. The KO group in contrast showed SIRT6-negative expressions (bars 50μm). Cell nuclei were visualized with hematoxylin. (C) The mRNA and protein levels of <i>Sirt6</i> were almost undetectable in the KO mice group. The gene expression level was normalized to GAPDH (n = 6). An assessment ofH3K9ac and H3K56ac in both the WT and KO groups was carried out by western blot analysis. This analysis found that the KO groups experienced a significant increase in these histones. (n≥3) *<i>p</i><0.05; **<i>p</i><0.01versus CO. CO = control.</p

<i>Sirt6 r</i>egulates the osteogenic/chondrogenic/ adipogenesis differentiation of DMCs.

<p>(A) Alkaline Phosphatase Staining, (B) Alizarin Red Staining, (C) Von Kossa and (D) Toluidine Blue Staining, all of which analyze the differentiation of DMCs, indicate that osteogenic and chondrogenic differentiation is reduced in the <i>Sirt6</i> KO group. (E) Oil Red O staining revealed an enhanced ability for adipogenic differentiation in the <i>Sirt6</i> KO group.</p

<i>Sirt6 r</i>egulates the mRNA levels of the osteogenic/ adipogenic -related transcription factor in the differentiation of DMCs.

<p>qPCR revealed that the <i>Sirt6</i> KO group experienced a substantial down-regulation in the mRNA levels of the osteogenic-related transcription factor, whereas the levels of the adipogenic-related transcription factor were increased significantly. The gene expression level was normalized to GAPDH (n = 3 to 6), *<i>p</i><0.05; **<i>p</i><0.01versus CO; CO = control.</p

<i>Sirt6</i> deletion does not affect the development of the tooth germs.

<p>(A) Micro-Computed Tomography testing revealed that the KO group differed from the WT group in several ways. The size of the tooth and mesial and distal roots were reduced in the KO group, and the diameter of the apical foramen of their distal root canal was wider. (B) H&E staining confirmed that there was no significant difference between the two groups with respect to <i>in utero</i> tooth germ morphogenesis. However, at or after DPN14, the first mandibular molar of the <i>Sirt6</i> KO group suffered an obvious growth retardation and developmental delay when compared to the WT group (E13.5-2W) (n = 6). Bars, as shown at the corner of the images.</p