Figure 2.

High glucose-dependent activation of the NFAT-luciferase reporter and NFAT5 expression are insensitive to the calcineurin-A inhibitor FK506. Fibers were transfected as in the protocol indicated in Figure 1a. One day after transfection, fibers were transferred to isotonic or high-glucose (50 mmol/L) media with DMSO (0.5% v/v) or with FK506 (0.5 μmol/L), a calcineurin-A inhibitor, during 24 h. (a) Luciferase activity driven by NFAT normalized to β-galactosidase activity driven by CMV relative to control fibers. Mean ± S.E. of four independent experiments (four mice per group) is shown. (b) Western blot analysis of whole cell homogenates prepared from FDB fibers cultured in control isotonic media or in high d-glucose (50 mmol/L) media with DMSO (0.5% v/v) or with FK506 (0.5 μmol/L) for 24 h by using NFAT5 antibody. The blot is representative of three independent experiments (three mice per group). The bar plot is the result of triplicate experiments, showing that the increase on NFAT5 expression induced by elevated glucose is insensitive to the calcineurin-A inhibitor FK506. *Indicates P< 0.05 compared with control. NFAT, nuclear factor of activated T-cell; DMSO, dimethyl sulfoxide; CMV, cytomegalovirus; FDB, flexor digitorum brevis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (A color version of this figure is available in the online journal)