TGF-β inhibition restores OB differentiation and potentiates Smad1 phosphorylation by BMP-2.

<p><b>A.</b> MC3T3-E1 cells were cultured for 14 days in osteogenic media in the absence or presence of conditioned media from RPMI8226 and U266 cells at 20%. SB431542 and Ki26894 were added at 3 and 10 µM to the indicated wells, respectively. <b>B.</b> MC3T3-E1 cells were cultured for 14 days in osteogenic media in the absence or presence of bone marrow plasma from patients with MM at 5%. SB431542 was added at 3 µM to the indicated wells. <b>C.</b> A chimeric firefly luciferase reporter plasmid (Topflash) and a renilla luciferase reporter plasmid (pRL-TK) were cotransfected into MC3T3-E1 cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009870#s4" target="_blank">Materials and methods</a>. The MC3T3-E1 cells were cultured in osteogenic media with rhBMP-2 at 50 ng/ml in the absence or presence of conditioned medium from RPMI8226 cells at 10%. SB431542 was added at 3 µM to the indicated wells. After 12 hours of incubation luiferase reporter activity was measured as renilla luiferase activity. Data were expressed as means +/− SD for six independent experiments. <b>D.</b> MC3T3-E1 cells were cultured in α-MEM with 1% FBS for 24 hours in 10-cm dishes at 60% confluence. SB431542 (3 µM), rhTGF-β (10 ng/mL), and the two in combination were added to the indicated dishes and the cells were cultured for another 48 hours, after which rhBMP-2 was added at 200 ng/ml (upper). SB431542 (3 µM), rhTGF-β (10 ng/mL), and the two in combination were added to the indicated dishes (lower). Following incubation for 30 minutes, cell lysates were collected. The protein levels of phosphorylated Smad1, Smad1, phosphorylated Smad2 and Smad2 were estimated by Western blotting. β-actin was used as a protein loading control.