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. Author manuscript; available in PMC: 2015 Oct 1.
Published in final edited form as: Bone. 2014 Jul 9;67:81–94. doi: 10.1016/j.bone.2014.06.040

Figure 8. TNAP deficient calvarial cell mineralization and osteoblastic gene expression.

Figure 8

(A,B) MC3T3E1(C4) calvarial cells stably expressing TNAP specific or non-target shRNA were cultured with or without ascorbate to induce osteoblast differentiation. TNAP enzyme activity was visualized by incubation of cells with a colorimetric substrate and quantified by densitometry. Results are shown as means +/− standard deviations of triplicate experiments. (C,D) Cells were cultured with ascorbate and β-glycerophosphate or NaPO4 to induce mineralized nodule formation. Mineralized nodules were stained by Von Kossa and quantified by densitometry. (E-J) Cells were cultured with ascorbate to induce osteoblast differentiation. RNA was isolated at indicated time points. Tissue non-specific alkaline phosphatase (TNAP), osteocalcin (OCN), bone sialoprotein (BSP), col1a1 and osteopontin (OPN) mRNA levels were measured by real time PCR. Results are presented as normalized to GAPDH. Black lines = non-target shRNA, grey lines = TNAP shRNA. *p<.05 vs. non-target shRNA cells.