Fig. 9.

Multiple signaling pathways are involved in the regulation of TGF-α expression. (A) Astrocytes were treated with indicated concentrations of DX, followed by determination of TGF-α mRNA levels by qPCR. (B) Cells were treated with 10 μM of Mifepristone (Mif), a GR antagonist, and 100 nM of DX, followed by the measurement of TGF-α mRNA levels by qPCR. (C) Cells were transfected overnight with 0.5 μg of TGF-α promoter luciferase plasmid and pretreated with 50 μM of PD98059, 20 μM of LY294002 or 10 μM of H89 for 30 min., followed by treatment with 100 nM of DX for 24 h. The TGF-α promoter activity was measured with luciferase assay. (D and E) Cells were treated with kinase inhibitors and DX as described in (C) and RNA was isolated to run qPCR with GAPDH as an internal control. Equal amounts of PCR products were run on 1% agarose gel. The quantification of TGF-α mRNA levels is shown in (E). (***p<0.001; significantly different from the control; one-way or two-way ANOVA followed by Tukey’s post hoc test; N=3)