with retinol had an increase in RAGE immunocontent and that co-incubation with ZD-1839 antioxidants reversed this effect (Gelain et al 2008a) Furthermore the concentrations of retinol that caused this effect were the same concentrations that increased the production of ROS in Sertoli cells indicating that retinol affected RAGE expression by a mechanism dependent on free radical production Here we investigated if the effect of retinol on RAGE upregulation was mediated by the activation of signal pathways that are known to be modulated by free radicals Our results indicate that pro-oxidant concentrations of retinol induce the activation of redox-sensitive pathways which result in the up-regulation of RAGE in cultured Sertoli cells .

Pregnant Wistar rats were housed individually in Plexiglas cages Litters were restricted to Afatinib eight pups each Animals were maintained on a 12-h light/dark cycle at a constant temperature of 23  C with free access to commercial food and water Male immature rats (15 days old) were killed by cervical dislocation All procedures were approved by the Local Ethics Committee Board of UFRGS All-trans retinol alcohol Trolox 27-dichloro hydrofluo rescein diacetate (DCFH-DA) 3-(45-dimethyl)-25- diphenyl tetrazolium bromide (MTT) Tween-20 and mercaptoethanol were from Sigma Chemical Co (St Louis MO USA) Retinol was dissolved in ethanol Concentrated stocks were prepared immediately before experiments by diluting retinol into ethanol and determining final stock concentration by UV supplier Cilostazol absorption;

solution was kept protected from light and temperature during all procedures Appropriate solvent controls were performed for each condition Treatments were initiated by adding concentrated solutions to reach final concentrations in the well The final ethanol Bay 43-9006 475207-59-1 concentration did not exceed 02% in any experiment Tissue culture reagents were from Gibco (Invitrogen Corporation Carlsbad CA USA) and were of tissue culture grade Rabbit polyclonal anti-RAGE was from Santa Cruz Biotechnology (Santa Cruz CA USA) Mouse monoclonal anti actin was from Sigma Rabbit polyclonal anti-p38 (phosphorylated form) and anti-Akt (phosphorylated form) were from Santa Cruz Sodium dodecyl sulphate (SDS)Cpolyacrylamide gel electrophoresis (PAGE) reagents were from Bio-Rad Laboratories (Hercules CA USA) nitrocellulose membrane (Hybond ECL) enhanced chemiluminescence kit (ECL plus) and anti-rabbit immunoglobulin (horseradish peroxidase-linked whole antibody from donkey) were from Amersham Pharmacia Biotech .

(Amersham UK) UO126 was from Promega Corporation (Madison WI USA) G?6983 and SB203580 were from Merck Biosciences (Darmstadt Germany) and H89 was from Biomol Research Laboratories (Plymouth Meeting PA USA) Other kinase inhibitors were a kind gift from Professor Peter Dunkley (University of Newcastle NSW Australia) 22 Isolation and  bruises culture of Sertoli cells and assays Sertoli cells were isolated as previously described (Pasquali et al 2008) Briefly testes of 15-day-old rats were remote.