FIGURE 6.

MN1 knock-out osteoblasts show decreased differentiation and mineralization. A, WT and MN1 KO osteoblasts were cultured for 28 days in differentiation media and stained in situ for alkaline phosphatase (ALP) enzyme activity. Representative images of alkaline phosphatase stained cultures are shown. Color images are shown in supplemental Fig. 1. B, MN1 WT or KO osteoblasts were cultured under osteogenic conditions for the indicated times. Alkaline phosphatase activity was determined with a colorimetric assay as described under “Experimental Procedures.” C, Northern analysis of alkaline phosphatase transcript levels in differentiating cultures of WT and MN1 KO calvarial osteoblasts. D and E, WT and MN1 KO osteoblasts were cultured for 31 days in differentiation media and stained for mineralized nodules with Alizarin Red S. D, representative images of Alizarin Red S stained cultures. Darkened areas are mineralized nodules. Color images are shown in supplemental Fig. 1. E, mineralized nodules were quantitated and compared (unfilled bars). The Alizarin Red S stain was extracted with 100 mm cetylpyridinium chloride, and the absorbance at 500 nm (absorbance_{500 nm}) was measured (filled bars). Data represent the mean ± S.D. (n = 3 in each group).