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. 2008 Dec 22;29(5):1291–1305. doi: 10.1128/MCB.01566-08

FIG. 8.

FIG. 8.

Effects of inhibition of microtubule assembly on osteoblast differentiation. (A) Effects of TN16 on ALP activity. 2T3 cells were incubated with TN16 at the indicated doses for 48 h, and ALP activity of cell lysates was measured using a Sigma ALP kit and normalized to total cellular proteins. #, P < 0.05; *, P < 0.01 versus results for control. (B) Effects of TN16 on osteocalcin expression. 2T3 cells were treated with TN16 at 1.0 μM for 24 h. Reverse transcription-PCR of mouse osteocalcin mRNA was performed with GAPDH as an internal control. (C) Effects of TN16 on cell proliferation. 2T3 cells were treated with TN16 at the indicated doses for 24 to 48 h. Cell proliferation was measured spectrophotometricaally at 450 nm using a Promega MTS kit. (D) Time course of TN16 stimulation of ALP activity. 2T3 cells were seeded in the 48-well plate at a cell density of 3 × 103 cells per well and incubated with TN16 at 1 μM for 5 days. After a wash to remove TN16 with fresh medium, the cells were continuously cultured for up to 21 days. The ALP activity of the cell lysates were then measured as described above. *, P < 0.05 versus results for day zero. (E) BMP-2 mRNA expression in Gli2-deficient osteoblasts. Primary osteoblast cells were isolated from calvariae of Gli2+/+ or Gli2−/− embryos (E20.5). Total RNA was extracted from the cell lysate, and the BMP-2 mRNA level was quantitated by real-time reverse transcription-PCR with mouse BMP-2 primer. Data presented are normalized to GAPDH. *, P < 0.05 versus results for Gli2+/+ cells. (F) Effects of TN16 on ALP activity in osteoblasts null for Gli2 (left panel) or BMP-2 (right panel). Calvarial osteoblastic cells were isolated from Gli2+/+ and Gli2−/− littermates at E20.5, and bone marrow mesenchymal stem cells (BMSCs) were flushed from the conditional BMP-2 knockout (BMP2−/−) or wild-type (BMP2+/+) mice (2 months old). These primary osteoblastic cells were cultured in α-MEM in the presence of TN16 at 1.0 μM for 24 h, and ALP activity of cell lysates was determined. *, P < 0.01 versus results with vehicle; #, P < 0.05 versus results with Gli2+/+ or BMP-2+/+ cells; **, P < 0.01 versus results for Gli2+/+ or BMP-2+/+ cells with TN16 treatment.