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. 2009 Apr 2;284(23):15676–15684. doi: 10.1074/jbc.M809787200

FIGURE 1.

FIGURE 1.

miR-29b expression profile during MC3T3 osteoblast differentiation. A, MC3T3 preosteoblasts cells cultured in differentiation medium for 28 days. Histochemical staining of alkaline phosphatase (AP) activity and von Kossa (VK) for mineral deposition at 10, 14, 21, and 28 days is shown. B, Western blot for Runx2 protein which increases during osteoblast differentiation. β-Actin protein was used as control. C, quantitative mRNA normalized by GAPDH for osteoblastic markers Runx2, AlkP, and osteocalcin on selected days during MC3T3 osteoblast differentiation used in miR profiling studies. D, significantly changed microRNAs that putatively target extracellular cellular matrix genes. Total RNA of MC3T3 cells during differentiation time points (0, 7, 14, 21, and 28 days) was used for miRNA microarray analysis. Relative fold changes of the microRNAs were hierarchically clustered by using dChip software. E and F, representative Northern blot analysis of miR-29b using total RNA isolated from mouse (MC3T3) (E) and rat primary osteoblasts (F), which were induced to differentiate. U6 RNA was used as a loading control. G, densitometric quantitation of miR-29b in indicated osteoblasts normalized to U6. The average volumes from two different MC3T3 studies are shown, and one time course from primary osteoblasts is shown.