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

FIG. 1.

FIG. 1.

Effects of inhibition of microtubule assembly on BMP-2 transcription. (A) Effects of microtubule-targeting compounds on BMP-2 promoter activity. 2T3 cells transfected with the murine BMP-2 promoter reporter, −2712/+165-Luc (BMP-2-Luc), were incubated with TN16, colchicine (Col), or nocodazole (Noc) in a dose range from 0 to 20 μM (upper x axis) or with taxol at the doses of 0 to ∼100 nM (lower x axis) for 24 h. Luciferase activity of cell lysate was measured and normalized by activity of cotransfected β-Gal. (B and H) Time course of TN16 stimulation of BMP-2 promoter. The BMP-2-Luc cells were treated with TN16 at the indicated doses for 12, 24, or 48 h (B) or up to 3 weeks (H), and BMP-2 promoter reporter activity was measured as described above. (C and D) Effect of TN-16 on BMP-2 mRNA expression. 2T3 cells were treated with TN16 at the indicated doses for 24 h and total RNA purified from cell lysate and reverse transcribed to cDNA. Quantitative real-time PCR amplification was performed using Applied Biosystems’ primers (Mm01962381_s1 for mouse BMP-2 and 4319413E for 18S). (E) Immunofluorescence detection of Smads. 2T3 cells treated with TN16 at 1 μM for 24 h were fixed and incubated with an anti-Smad1/5/8 antibody (Cell Signaling) followed by a Cy3-conjugated second antibody. Fluorescence was visualized and captured with a confocal microscope system. (F) Effect of TN16 on BMP signaling. 2T3 cells were transfected with a 12×SBE-Luc reporter plasmid, containing multiple copies of Smad binding elements, and treated with TN16 at the indicated doses for 24 h. Luciferase activity was determined. (G) Lack of effect of TN16 on Wnt signaling. 2T3 cells were cotransfected with the TOP Flash construct (which has multiple binding sites for TCF) and with a β-catenin/TCF4 expression plasmid. The cells were then incubated without or with TN16 at the indicated doses for 24 h. The reporter activity was determined with β-Gal normalization. (H) Time course of TN16 stimulation of BMP-2 mRNA expression. 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. Total RNA was purified, and BMP-2 real-time PCR was performed with 18S as control. *, P < 0.05 versus results on day zero.