Figure 3

H-rev107 increases PGD2 and cAMP levels and enhances the expression of SOX9 protein. NT2/D1 cells plated in triplicate in 6-well plates were transfected with 1 μg of indicated expression or control vector for 24 h. Cells were washed and then incubated with 1 μg/mL AA for 1 h (A). Alternatively, cells were transfected with indicated expression or control vector and then incubated for 18 h in the presence of 1 mM Br-cAMP or ethanol vehicle for 18 h or 500 ng/mL PGD2 for 30 min as described in the Methods. Cell lysates were prepared, and levels of PGD2 (A, B) or cAMP (C) were measured using an enzyme immunoassay. Representative results from triplicate samples were expressed as mean ± SD. Student’s t-test: **, P <0.01; ***, P < 0.001. Cells were transfected with H-rev107 expression or control vector for 24 h. Expression of SOX9 and phosphorylated SOX9 in total cellular extracts was determined by Western blot analysis (D).