Figure 4

TNF-α induces Msx2 expression via NF-κB activation. (A) Inhibition of NF-κB activation by BAY-11-7082 (BAY, 10 µM) blocked TNF-α-induced Msx2 expression. C2C12 cells were treated with the indicated reagents for 10 min (upper panel) or for 24 h (middle and lower panels), and then immunoblotting, RT-PCR and ALP staining were performed. The levels of phospho-IκBα and total IκBα were examined using whole cell lysates whereas the Msx2 protein level was examined using nuclear extracts. (B) BAY-11-7082 did not exert any cytotoxic effects at a concentration of 10 µM. The cell cytotoxicity/proliferation assay was performed using the CCK-8 reagent as described in Methods. Data represent the mean + SD of quadruplicates. ^*, P < 0.01 (Student's t-test) (C) Over-expression of dnIκBα suppressed TNF-α-induced Msx2 expression. C2C12 cells were transiently transfected with pcDNA or the dnIκBα expression vector and incubated in the presence of TNF-α for 24 h. Next, RT-PCR and immunoblot analyses were performed.