Fig. 2

A HSP90 inhibitor onalespib upregulates TGF-β-induced HSP27 expression in MC3T3-E1 cells. The cultured osteoblasts were pretreated with 10, 20 or 30 nM of onalespib for 60 min, and subsequently incubated by 10 ng/ml of TGF-β or vehicle for 12 h. Cell extracts were analyzed by SDS-PAGE and Western blotting using antibodies of HSP27 or GAPDH. The histogram shows the quantitative representations of the levels of HSP27 normalized with each GAPDH gained from laser densitometric analysis. The levels were expressed as the fold increase to the basal levels presented as lane 1. Triplicate determinations of Western blot analysis were performed corresponding to three independent cell preparations. Each value represents the mean ± S.E.M. of triplicate determinations from three independent cell preparations. *P < 0.05, compared to the value of the control cells without TGF-β-stimulation. **P < 0.05, compared to the value of TGF-β alone. B TGF-β inhibitor SB431542 reduces the enhancement by onalespib of the TGF-β-induced HSP27 expression in MC3T3-E1 cells. The cultured osteoblasts were pre-incubated with 3 μM of SB431542 or vehicle for 60 min. The cells were subsequently pretreated with 30 nM of onalespib or vehicle for 60 min, and then stimulated by 10 ng/ml of TGF-β or vehicle for 12 h. Cell extracts were analyzed by SDS-PAGE and Western blotting using antibodies of HSP27 or GAPDH. The histogram shows the quantitative representations of the levels of HSP27 normalized with each GAPDH gained from laser densitometric analysis. The levels were expressed as the fold increase to the basal levels presented as lane 1