The differentiation stages of murine MC3T3-E1 preosteoblasts used for profiling studies. (A) Staining for alkaline phosphatase (ALP) activity (upper panel) and mineralization (Von Kossa, lower panel) in MC3T3-E1 cells during three stages of differentiation: proliferation (day 0), matrix deposition (days 9 to 21), and mineralization (day 28). (B) Protein (left panel) and mRNA levels (right panel) of Runx2 during osteoblastic differentiation of MC3T3-E1 cells. Histone H3 (H3) was used as loading control for western blotting. (C) Expression profile of osteoblast-related markers Osx/Sp7, Col1a1 (left panel), Akp2/Alpl (alkaline phosphatase liver/bone/kidney), Bsp/Ibsp, and Ocn/Bglap2 (right panel) in MC3T3-E1 cells during differentiation. Relative mRNA levels (versus day 0) were determined by quantitative RT-PCR (reverse transcription PCR), normalized by Hprt1 mRNA levels and plotted as mean values ± SEM (standard error of mean) from three independent biological replicates. The expression levels of the genes in (B,C) at days 9, 21, and 28 were significantly upregulated when compared to those at day 0 (P < 0.05, t-test).