FIGURE 6.

Helenalin acetate disrupts the cooperation of C/EBPβ and p300. A, QT6 cells transfected with expression vectors for C/EBPβ and p300, as indicated. Cells were treated with the indicated concentrations of helenalin acetate, harvested after 12 h, and analyzed by Northern blotting for expression of MRP126 and S17 mRNAs. The numbers below the blots indicate the relative expression levels of MRP126 mRNA. B, QT6 fibroblasts were transfected with the Gal4-dependent reporter gene pG5E4-38Luc, expression vectors for p300-VP16 and the indicated Gal4-C/EBPβ proteins. Cells were harvested after 12 h and analyzed for luciferase and β-galactosidase activities. The columns show the average luciferase activity normalized against the β-galactosidase activity. Error bars show the S.D. **, p < 0.01; ***, p < 0.001. C, in vitro pulldown experiment. Glutathione-Sepharose beads loaded with equivalent amounts of bacterially expressed GST (lane 1), GST-C/EBPβ (lane 2), GST-C/EBPβ(1–200) (lane 3), and GST-C/EBPβ(1–50) (lane 4) were incubated with identical amounts of a bacterially expressed GFP-Taz2 fusion protein. Bound GFP-Taz2 and aliquots of the input samples were analyzed by Western blotting with antibodies against GFP. The bottom panel shows a stained gel to confirm loading of the Sepharose beads with equivalent protein amounts. Numbers on the left side refer to molecular weight markers. D, QT6 cells were transfected with the C/EBP-dependent luciferase reporter gene p-240luc, the β-galactosidase expression vector pCMVβ, expression vectors for p300, and the indicated C/EBPβ constructs. Reporter gene activities were analyzed as in B. Error bars show the S.D.