Fig. 8: IMD 0354 does not suppress osteoblast-related gene expressions.

a Primary calvarial osteoblasts were pretreated with or without 1 μM IMD 0354 for 2 h and then treated for the indicated periods. Cell lysates were analyzed using western blotting. The expression of phosphorylated IKKα/β, IκBα, and P65 and total IKKα, IKKβ, IκBα, and P65 was assessed (n = 3). b The gray levels of phosphorylated IKKα/β, IkBα, and P65 were quantified and normalized to total IKKα, IKKβ, IκBα, and P65 using Image J (n = 3). c Primary calvarial osteoblasts were pretreated with or without 1 μM IMD 0354 for 2 h and then treated for the indicated periods. Cell lysates were analyzed using western blotting. The expression of phosphorylated ERK, P38, and JNK and total ERK, JNK, and P38 was examined (n = 3). d The gray levels of phosphorylated ERK, JNK, and P38 were quantified and normalized to total ERK, JNK, and P38 using Image J (n = 3). e qRT-PCR analysis of Runx2, ALP, COL1a, OCN, and OSX mRNA expression in calvarial osteoblasts treated with the indicated IMD 0354 concentrations for 4 days (n = 3). f, g Western blot analysis of Runx2 and ALP expression in calvarial osteoblasts treated with the indicated IMD 0354 concentrations for 7 days (n = 3). Values were normalized to α-tubulin and negative control expression values. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005, compared with the controls