Figure 6.

TGF-β signal activation is required for the TMZ-induced CTGF upregulation. U87 and U251 cells were treated with different TMZ concentrations for 24 h and the culture supernatants were collected for TGF-β1 expression by ELISA assay, *P<0.05 compared to non-treated cells (a). qPCR analysis of TGF-β1 expression in U87 and U251 cells treated with or without different TMZ concentrations for 24 h, *P<0.05 compared to non-treated cells (b). Immunofluorescence analysis of CTGF and TGF-β1 expression with or without TMZ treatment in U87 and U251 cells, scale bar=100 μm (c). U87 and U251 cells were, respectively, treated with TGF-β receptor inhibitor SB431542 or TGF-β1/2/3 neutralizing antibody for 24 h, and analyzed by qPCR (d) and western blot analysis (e). *P<0.05 compared to non-treated cells, ^&P<0.05 compared to cells treated with TMZ alone. U87 and U251 cells were treated with recombinant TGF-β1 and/or TMZ for 24 h and analyzed for TGF-β1 and CTGF expression by western blot (f). The GB shCTGF and shCtrl cells were treated with TMZ for 24 h, western blot (g) and ELISA (h) analysis were performed for TGF-β1 and CTGF expression, β-actin served as the loading control, *P<0.05 compared to the shCtrl cells. Results above are expressed as the mean±S.D. of three independent experiments