Figure 7.

Blockade of TGF-β1 secretion, its receptor (RI and RII) induction and Smad 2/3 activation by chrysin in renal mesangial cells treated with AGE. Human renal mesangial cells were challenged for 3 days with 100 μg/mL AGE-BSA in the absence and presence of 1–20 μM chrysin or 10 μM TGF-β RI kinase inhibitor. Cell extracts and media were subject to Western blot analysis with a primary antibody against TGF-β RI, TGF-β RII, α-SMA and FSP-1 (A,C). β-Actin protein was used as an internal control. The bar graphs in the right panels represent densitometric results obtained from Image analysis. TGF-β1 in culture media was measured by using an ELISA kit (B). Cy3 staining was conducted for the nuclear Smad 2/3 detection with nuclear counter-staining of DAPI (D,E). Each photograph is representative of at least 4 different experiments and fluorescent images were taken with a fluorescence microscope. Scale bar = 100 µm. Fluorescent Cy3 staining intensity of Smad 2/3 was measured using an optical Axiomager microscope system (D). Values in bar graphs (mean ± SEM, n = 3 independent experiments) not sharing a same lower case indicate significant different at p < 0.05.