Fig. 8. Shh inhibits the expression of the adipocyte markers aP2, leptin, C/EBP and PPAR2. (A) C3H10T1/2 were stimulated with Shh (10 µg/ml)) and total RNA was extracted after 4-day stimulation. Control cells were cultured without any stimulation and total RNA was extracted at the same time as stimulated cells. (B) C3H10T1/2 cells were stimulated for 1 hour with Shh (10 µg/ml); cells were washed and cultured for 4-days and total RNA was extracted. Control cells were similarly treated but did not receive any stimulation. The mRNA expression level of aP2, leptin (Lep), C/EBP and PPAR2 in both stimulated and unstimulated cells was determined by real-time TaqMan PCR. Alkaline phosphatase (ALP) mRNA was quantified and shown as a positive control of Shh stimulation. The expression level of tested markers was normalized on the basis of GAPDH expression. Data are presented as the relative expression level of adipocyte markers in stimulated versus unstimulated cells. Results (mean±s.d.) are representative of two independent experiments, both performed in triplicate.