GTX protects osteoblast differentiation against inhibition from TNF-α in C2C12 cells; evidence from ALP activity, as well as Col I, Ocn and Bsp gene expression.(A) Chemical structure of GTX. (B) MTT cell viability assay suggested that, in the groups treated with 4 or 5 μg/ml GTX for 24 or 72 h, cell viability rate was significantly decreased, compared with that of the DMSO-treated control group. (C) Caspase-3 activity assay indicated that following 4 or 5 μg/ml GTX treatment for 24 h or 72 h, caspase-3 activity was markedly enhanced, compared with that of the DMSO control group. (D and E) ALP activity assays performed following 72 h of culture indicated the protective effect of GTX for osteoblast differentiation. (D) TNF-α inhibition of BMP-2-induced ALP activity was blocked by treatment with 1 μg/ml GTX. (E) GTX protected ALP activity in a dose-dependent manner. (F–H) The protective effects of GTX on osteoblast differentiation were determined via analysis of gene expression of Col I, Ocn and Bsp. *P<0.05, **P<0.01, n>3. GTX, gliotoxin; TNF-α, tumor necrosis factor-α; DMSO, dimethyl sulfoxide; ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein-2.