Bay 11 7082

Differentiated 3T3-L1 cells were incubated overnight using the contact method in serum-free DMEM with 2% FFA-free BSA. The cells were then co-cultured with RAW264.7 cells in the same medium for 24 h with butyrate (0, 0.5 mmol/L) and PGE2 (0, 5 ng/mL), after which the culture medium was harvested. The levels of FFAs in the co-culture medium were measured using an acyl-coenzyme A oxidase–based colorimetric assay method, which enables detection of C8 (octane) and longer-chain fatty acids. The assay was conducted in accordance with the manufacturer’s instructions [37 (link)]. Levels of free glycerol in the medium were measured using an Adipolysis Assay kit (Cayman Chemical). To examine the effects of BAY 11-7082 and SC 58635, 3T3-L1 cells were pretreated by culturing at 37 °C for 60 min in serum-free DMEM with 0.5% (v/v) DMSO (vehicle), 5.0 ng/mL PGE2 (Cayman Chemical) dissolved in DMSO, 10 μmol/L BAY 11-7082 (Santa Cruz Biotechnology, Santa Cruz, CA) dissolved in DMSO, or 1 μmol/L SC 58635 (R&D Systems) dissolved in DMSO. 3T3-L1 cells were then stimulated with TNF-α (with or without butyrate) in 2% FFA-free BSA medium and cultured for 24 h, after which levels of FFAs and free glycerol released into the medium were measured.

Anti-p-p65 antibody, Gö6976, heparin, PMA, Röttlerin, and PKC-δ siRNA were purchased from Sigma-Aldrich (St.Louis, MO). Bay 11-7082, Bay 61-3606, piceatannol, and caffeic acid phenethyl ester (CAPE) were purchased from Cayman Chemicals (Ann Arbor, MI). Lipofectamine 3000 reagent was acquired from Invitrogen Life Technologies (Gaithersburg, MD). Antibody specific for Thy-1, and p-PKC-δ was purchased from Cell Signaling Technology (Danvers, MA). Anti-Syk, p65, PKC-δ, and G3PDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody specific for p-Syk was purchased from EMD Millipore (Temecula, CA). Dual-Luciferase assay kit was purchased from Promega (Madison, WI). Penicillin and streptomycin were purchased from HyClone (South Logan, Utah). Endothelium cell growth factor (ECGS) was purchased from Biomedical Technologies (Stoughton, MA). Fetal calf serum (FCS), Medium 199 (M199), and GlutaMAX Supplement were obtained from GIBCO (Grand Island, NY). Endothelial Cell Growth Medium MV was purchased from PromoCell (Heidelberg, Germany).

Antibodies against p-RelA-S536 (#3033; 1:1000), RelA (#8242; 1:1000), RelB (#4922, 1:2000; IHC: #10544), c-Rel (#4727; 1:1000), NF-κB2 (#3017; 1:1000), LKB1 (#3050; 1:1000), and NUAK1 (#4458; 1:1000) were purchased from Cell Signalling Technology (Danvers, MA). Antibodies against lamin A/C (MAB3211; 1:500) and actin (A2066; 1:20000) was purchased from Millipore (Temecula, CA, USA). Antibody against tubulin (T5168; 1:20000) was purchased from Sigma. HRP-conjugated antibodies against mouse IgG (NA931; 1:10000) and rabbit IgG (NA934; 1:10000) were purchased from Cytiva. All antibodies were diluted in tris-buffered saline-Tween 20 containing 5% bovine serum albumin, with the exception of NUAK1 (tris-buffered saline-Tween 20 containing 5% nonfat milk). BAY 11-7082 was purchased from Cayman Chemical (cat. 10010266; Ann Arbor, MI, USA). ROS-Glo H₂O₂ Assay was purchased from Promega (cat. G8820).

Reagents were purchased from the following companies: nicotine, LPS and ovalbumin (OVA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant murine GM-CSF, IL-4 and TGF-beta were obtained from R&D (Minneapolis, MN, USA). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, USA). H-2K^b CTL peptide of OVA (SIINFEKL) was synthesized by Sangong, (Shanghai, China). BrdU cell proliferation kit and IFN-gamma Elispot kit were obtained from Roche (Basel, Switzerland) and U-CyTech Biosciences (Utecht, Netherlands) respectively; Recombinant murine IL-6, TNF-alpha, Brefeldin A solution and Fluorescein-conjugated antibodies to CD4, CD25, Foxp3, CD80, CD86, CD40, OX40L, 4-1BBL, MHC class I, MHC class II, CD11c, TGF-beta, TNF-alpha, and IL-6 were obtained from eBioscience (San Diego, CA, USA). Recombinant Mouse IFN-beta, IFN-beta neutralizing antibody (MIB-5E9.1), B7H1 neutralizing antibody (10F.9G2), fluorescein-conjugated antibodies to IFN-beta, SIINFEKL-H2K^b, B7H1, GITRL, GITR were purchased from Biolegend (San Diego, CA, USA). The NF-kappaB inhibitor PDTC and Bay 11-7082 were purchased from Cayman Chemical (Ann Arbor, MI, USA). TRI-zol was purchased from Invitrogen life technologies (Carlsbad, CA, USA). SYBR Premix Ex Taq and PrimeScript Reverse Transcriptase were purchased from Tarkara (Dalian, Liaoning, China).

To investigate the regulation of serpinA3N gene expression specifically in neurons, the embryonic immortalised hypothalamic cell line, N42, was used (Cellution Biosystems Inc., Ontario, Canada. Please see https://www.cedarlanelabs.com/Products/Detail/CLU122 for a comprehensive list of all genes expressed by these cells including the leptin receptor). These cells have been previously shown to provide a good model for hypothalamic neuron gene expression studies [44 (link)].
Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies) maintained at 37 °C under 5% CO₂. Neurons were grown in 60-mm plates (n = 6) to 90% confluence then challenged with either 50 nM leptin (R&D Systems, Abingdon, UK), 200 μM palmitic acid or 200 μM oleic acid conjugated to fatty acid-free bovine serum albumin (BSA) (Sigma-Aldrich, UK), or the appropriate vehicle as a control. For leptin, this was a mixture of 12 μM HCl and 6 μM NaOH and for fatty acids 50 μM fatty acid-free BSA. To inhibit the NFkB pathway, N42 neurons were treated with 50 μM BSA or 200 μM palmitate in the presence and absence of 25 μM of the NFkB inhibitor BAY 11-7082 (Cayman Chemical, UK). All solutions were filter sterilised prior to use. For all treatments, medium was removed after 6 h.