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HNF4alpha and HNF1alpha Dysfunction as a Molecular Rational for Cyclosporine Induced Posttransplantation Diabetes Mellitus

Figure 1

Cyclosporine inhibits protein expression of HNF4α and binding to target gene promoters.

(A) HNF4α western blotting of 20 µg Caco-2 cell nuclear extracts [control or cyclosporine treatment, 25 µM (30 µg/ml) for 72 h]. (B) HNF1α western blotting of 30 µg Caco-2 cell nuclear extracts [control or cyclosporine treatment, 25 µM (30 µg/ml)for 72 h] (C) Actin western blotting of 15 µg Caco-2 cell nuclear extracts [control or cyclosporine treatment, 25 µM (30 µg/ml) for 72 h]. The lower panels represent the quantification of protein amounts for HNF4α (A) and HNF1α (B) relative to the actin expression. (D) Electrophoretic mobility shift assays with 2,5 µg Caco-2 cell nuclear extract [control or cyclosporine treatment, 25 µM (30 µg/ml) for 72 h] and 32P labeled oligonucleotides to probe for DNA binding to HNF4α binding-sites within promoters of HNF1α (HNF1α), apolipoprotein C2 (ApoC2), glycerol kinase (GK), pyruvate kinase (PKLR), aldolase B (ALDOB), and insulin2 (INS2). In EMSA supershift assays an antibody directed against HNF4α (+) was added. Shifted (HNF4α) and supershifted bands (HNF4α ss) were marked. (E) Dried EMSA gels were analyzed with a Molecular Imager (BioRad, Muenchen, Germany) using the Quantity One software (BioRad, Muenchen, Germany). HNF4α binding of control extracts to the respective binding sites was set to 100% and inhibition of binding to the respective binding sites after treatment with cyclosporine [25 µM (30 µg/ml) for 72 h] was quantified.

Figure 1

doi: https://doi.org/10.1371/journal.pone.0004662.g001