Increasing glucose (D-Glu) concentrations decreases TNF-mediated A20 protein up-regulation without affecting its transcriptional activation in SMC.

<p>(A) Analysis of A20 expression by WB. SMC were cultured in 5, or 30 mM D-Glu, or the osmotic control (25 mM L-Glu +5 mM D-Glu), and treated with TNF for 6 h. βactin was checked as a loading control and used to quantify relative A20 expression by densitometry, as reported beneath the WB. Densitometry of the bands of interest and was determined as the mean intensity of the areas delineated by Image J, then corrected by the main intensity of the corresponding housekeeping gene band. Fold induction was determined using the non-treated 5 mM D-glucose condition sample as one (1). Data are representative of 3 independent experiments. The cuts between samples reflect the fact that these samples, while on the same gel and same experiment, were not contiguous. (B) Analysis of A20 mRNA levels by real-time PCR. SMC were cultured in 5 or 30 mM D-Glu or mannitol (25 mM Mannitol +5 mM D-GLu), as an osmotic control, and treated with TNF for 1 or 3 h. 18S ribosomal RNA was used to normalize the data. Natural log transformed data (ln) are presented as mean±SEM of 3 independent experiments performed in duplicate. No significant differences (P>0.05) were noted between all groups and at all time-points.