Figure 4. Inhibition of the activation of JAK₂/STAT3 or PI3K/AKT abrogated the impacts of AGEs or leptin on the divergent regulation of gene expression of RAGE and AGE-R1 in HSC.

Serum-starved HSC were pretreated with AG490 (20 μM), a specific JAK₂ inhibitor, or LY294002 (20 μM), a selective inhibitor of PI3K/AKT, for 1 h prior to the addition of leptin (100 ng/ml) or AGEs (100 μg/ml) for additional 24 h. Total RNA or whole cell extracts were prepared for real-time PCR or Western blotting analyses of RAGE or AGE-R1. A & B. Cells were treated with AG490 and analyzed by real-time PCR (A), or Western blotting analyses (B); C & D. Cells were treated with LY294002 (LY) and analyzed by real-time PCR (C), or Western blotting analyses (D). Values in A & C were expressed as mRNA fold changes (means ± SD) (n=3). *P<0.05 vs. the untreated control (the corresponding 1^st column); ‡P<0.05 vs. the cells treated with leptin only (the corresponding 2^nd column); §P<0.05 vs. the cells treated with AGES only (the corresponding 5^th column). β-actin was used as an invariant control in Western blotting analyses for equal loading. Representatives were presented from three independent experiments. [E. Luciferase activity assays of HSC co-transfected with the rage or age-r1 promoter luciferase reporter plasmid pRAGE-luc or pAGE-R1-luc and a cDNA expression plasmid pdn-STAT3 or pwt-STAT3, followed by the treatment with or without AGEs (100 μg/ml) for 24 h. The plasmids pdn-STAT3 and pwt-STAT3, respectively, encode dominant negative STAT3 and wild-type STAT3. Luciferase activities were expressed as relative units after normalization with β-galactosidase activities (means ± SD, n=6). *P<0.05 vs. the untreated control (the corresponding 1^st column); ‡P<0.05 vs. the cells only transfected with pRAGE-luc or pAGE-R1-luc, without pdn-STAT3 or pwt-STAT3 (the corresponding 2^nd column).]