The upper a part of panel C shows representative Western blots using anti-pAkt, anti-p-ERK1/2 or anti-Tubilin antibodies and each antibody was used in different membranes. phosphorylation whereas DHA preserved insulin action. Palmitic acid up-regulated TLR4 as well as pro-inflammatory cytokines IL6 and TNF contrasting with DHA effect. Similarly to palmitic acid, resistin treatment induced the up-regulation of IL6 and TNF as well as NFB activation. Importantly, palmitic acid potentiated the resistin-dependent NFkB activation whereas DHA abolished it. The recruitment of TLR4 to membrane lipid rafts was increased by palmitic acid treatment; this is concomitant with the augmentation of resistin-induced TLR4/MYD88/TIRAP complex formation required for TLR4 signaling. In conclusion, palmitic acid increased TLR4 expression promoting resistin signaling through TLR4 up-regulation and its recruitment to membrane lipid rafts. 106 cells per well in a six-well plate and transfected with the different Silencer Select siRNA to a final concentration of 5?nM using the Lipofectamine 2000 (Invitrogen, Illkirch, France) transfection reagent according to the manufacturers instructions. 48?h after transfection, serum starved cells were treated for 4?h with palmitic acid (200?M) or FFA-BSA as a control then challenged with resistin (200?ng/mL) for 15?min. Cells were then solubilized and protein lysates were subjected to Western blot as explained before. Crosslinking experiments Cross-Linking experiments were performed as previously explained12. Briefly, serum starved SH-SH5Y cells were washed with ice-cold PBS then treated with resistin (200?ng/mL) for 1?h at BCL2A1 4?C. BS3 crosslinking agent (Thermo Scientific, Illkirch, France) was then added directly to the incubation treatment for a final Mitoquinone mesylate concentration of 2.5?mM and followed by an additional incubation for 1?h at 4?C. Crosslinking reaction was stopped by the addition of a quenching answer (20?mM TrisCHCL pH 7.5). Mitoquinone mesylate Cells were solubilized and protein lysates were then subjected to immunoprecipitation using antibodies raised against TLR4. Western blots were performed as explained before and membranes were immunoblotted with antibodies raised against TLR4 or Mitoquinone mesylate resistin. Stable transfection and reporter gene luciferase assay NF-B luciferase reporter gene cells were generated by stably transfecting SH-SY5Y cells with the?pGL4.32[and a mammalian selectable marker for hygromycin resistance. After hygromycin selection (200?g/mL), surviving cells were transfected with a control vector containing -galactosidase with a cytomegalovirus promoter. 48?h after transfection, serum starved cells were treated 4?h with resistin (200?ng/ml), palmitic acid (200?M) or DHA (20?M) then luciferase and -galactosidase activity were measured using the Mithras LB 940 apparatus (Berthold technologies, Bad Wildbad, Germany) and the Dual-Light Chemiluminescent Reporter Gene Assay System (Applied Biosystems, llkirch, France) according to the manufacturers instructions. Luciferase activity was normalized by -galactosidase activity. All transfections were performed using Lipofectamine 2000 (Invitrogen, llkirch, France) according to the manufacturers instructions. Immunocytochemistry and microscopy analysis SH-SY5Y cells were seeded onto Lab-Tek chamber Slides (Millipore, Darmstadt, Germany). Serum starved cells were treated 4?h with palmitic acid, DHA or FFA-BSA as a control, then washed with ice-cold PBS and incubated or not with 1?g/mL of isothiocyanate-conjugated cholera toxin B subunit (FITC-CTxB) for 15?min at 4?C. Cells were washed with PBS then fixed with 4% paraformaldehyde for 20?min at 4?C followed by incubation with 50?mM NH4Cl for 20?min and were blocked/permeabilized with a solution containing 0.1% Triton X-100, 0.2% Fish Gelatin, 2% normal donkey serum for 1?h at room temperature. Fixed cells were then incubated with 1/300 dilution of rabbit anti-TLR4 (Santa Cruz Biotechnology, Dallas, TX, USA) in Blocking/permeabilization answer overnight at 4?C. Cells were washed with ice-cold PBS and incubated with 1/500 dilution in PBS of Alexa Fluor 488 conjugated donkey anti-rabbit IgG (Life Technologies, Illkirch, France) or DyLight 649 conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 1?h at room temperature. Cells were then washed with PBS and incubated for 3?min with 4,6-diamidino-2-phenylindole (DAPI) for DNA staining. After washing with PBS coverslips were mounted in Vectashield antifade mounting medium (Vectorlabs, Burlingame, Ca, USA) and slides were sealed with nail polish. Immunofluorescence was examined by confocal microscopy (Zeiss MRC1024ES; Zeiss microscopy, Jena, Germany) for TLR4 recruitment into lipid rafts. Hormones and chemicals Human resistin was purchased Mitoquinone mesylate from Shenondoah Biotechnology (Warwick, PA, USA). Human insulin Actrapid was provided by Novo Nordisk (Bagsvaerd, Denmark). Sodium palmitic acid, DHA and FFA-BSA were purchased from Sigma-Aldrich (St. Quentin Fallavier, France) and all cell culture reagents were purchased from Euromedex (Souffelweyersheim, France). All other chemicals were purchased from Sigma-Aldrich (St. Quentin Fallavier, France). Data analysis and statistics Repeated-measures two-way ANOVA followed by Bonferroni test were used to test the changes in signaling and gene expression experiments and value 0.05 was considered as statistically significant. The results are expressed as means S.E.M. Results Palmitic acid reduced insulin-dependent Akt/ERK phosphorylation and increased TLR4 Mitoquinone mesylate expression in SH-SY5Y neuroblastoma cells To investigate the effect of palmitic acid on neuronal insulin responsiveness, SH-SY5Y cells were treated with palmitic acid, DHA or placebo. SH-SY5Y cells treated with palmitic acid exhibited lower insulin-dependent Akt and Erk1/2 phosphorylation as compared to untreated cells.