Cli 095

The murine MS1 (Mile Sven 1) pancreatic islet endothelial cell line, obtained from American Type Culture Collection (ATCC®, CRL-2279^TM), is a commonly used model of microvascular endothelium. MS1 cells were cultured in high glucose (4.5 g/l) Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% decomplemented (heat inactivated) fetal bovine serum (FBS), 120 U/mL streptomycin/penicillin and 4 mM L-Glutamine, in 5% CO₂ at 37 °C. MOVAS (ATCC®, CRL-2797^TM) is an established VSMC line from C57BL/6 mice. Cells were grown in DMEM supplemented with 10% FBS, 2 mM L-Glutamine and 0.2 mg/ml G-418 (all reagents obtained from Lonza). For experiments, cells at 80% confluence were growth-arrested by serum starvation for 24 h. Endotoxin-free CsA (Calbiochem, Merck Chemicals) and tacrolimus (USBiological) stock solutions (10 mg/ml) were dissolved in ethanol. CLI-095 (InvivoGen) was used according to manufacturer’s time and dose recommendations. The NF-κB inhibitor parthenolide, the TRIF inhibitor resveratrol and the antioxidants 4′-hydroxy-3′methoxyacetophenone (apocynin) and diphenyleneiodonium chloride (DPI) were from Sigma-Aldrich. These reagents were used at concentration derived from prior dose-response studies in our laboratory or from bibliographic data.

The reactive oxygen species (ROS) scavenger MCI-186 (Biomol Research Laboratories) was dissolved in DMSO and diluted in a range from 1mM to 50μM. The MoDCs were pre-treated with MCI-186 for 1hr. The TLR4 inhibitor CLI-095 (Invivogen, San Diego, CA) was used according to the manufacturer’s protocol. CLI-095 is a selective TLR4 inhibitor shown not to inhibit TLR2, 3, and 9 signaling [32 (link)]. Lipid IVa (Pepta Nova, Sandhausen, Germany), a lipid A antagonist which binds MD-2, was dissolved in DMSO and used at a final concentration of 1μg/mL. The caspase-1 inhibitor peptide (ICE Inhibitor V, Z-Asp-[(2,6-dichlorobenzoyl)oxy]methane, Z-D-CH2-DCB (Calbiochem,Billerica, MA) was used at 100μM final concentration. Control DC samples contained equimolar amounts of BSA, EtOH, or DMSO as used in experimental groups.

For flow cytometry, the following fluorochrome-conjugated monoclonal antibodies were used. BD Biosciences: HLA-DR-APC (Clone: G46-6), CD86-FITC (Clone: FUN-1), CD80-PE (Clone: L307.4), CD54-APC (Clone: HA58), CD25-FITC (Clone: M-A251), CD127-BV421 (clone HIL-7R-M21), IFN-γ-FITC (Clone: 4S.B3), IL-4-PE (Clone: MP4-25D2); eBioscience: FoxP3-APC (Clone: 236A/E7), IL-17A-PE (Clone: Ebio64cap17); Beckman Coulter: CD40-PE (Clone: MAB89); Biolegend: CD4-PerCP (Clone: SK3). Cell viability was detected using the fixable viability dye eFluor 506 (eBioscience).
Antigen affinity-purified polyclonal anti-human TLR4 goat IgG was purchased from R&D systems. Cytokines (recombinant human granulocyte-macrophage colony-stimulating factor and IL-4), MicroBeads (CD14 and CD4) and cell purification units were obtained from Miltenyi Biotec. Protein-A agarose beads were from Cell Signalling Technology, and E. coli 055:B5 LPS and Polymyxin B-conjugated agarose beads were from Sigma-Aldrich. TLR4 signaling inhibitor CLI-095 and CpG ODN 2006 were procured from InvivoGen.

After 12 h of pre-incubation with or without palmitate (50 µM), cells were treated with Pg-LPS (100 ng/ml) for another 12 h. Cells were also pretreated by TLR4 signaling inhibitor Cli-095 (InvivoGen, San Diego, CA, USA) for 6 h and stimulated by palmiatate (50 µM) for indicated time. Primary antibodies against Polyclonal anti-poly (ADP-ribose) polymerase (PARP), P-p65, P-p38, P-JNK and P-ERK were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Monoclonal anti-β-actin was purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA) Western blotting was performed as previously described [18] . Cell pellets (1.5×10⁵ cells) were resuspended in ice-cold lysis buffer. Proteins were separated by SDS-PAGE and electro-blotted onto a nitrocellulose membrane. After blocking of the membrane with 3% milk for 30 min, membranes were incubated with primary antibodies (1∶1000) and anti-rabbit secondary antibody. β-actin (1∶10,000) was used as internal control. The results were visualized by Amersham ECL western blotting detection system (GE Healthcare, Japan).

Syk inhibitor (ER 27319 maleate), TLR2 inhibitor (human TLR2 mAb, clone 383936), isotype control for TLR2 mAb (mouse IgG2B, clone 20116), pan-caspase inhibitor (Z-VAD-FMK), caspase-8 inhibitor (Z-IETD-FMK), NADPH-oxidase inhibitor (apocynin) and phagocytosis inhibitor (cytochalasin D) were purchased from R&D Systems. TLR4 inhibitor (CLI-095) was from Invivogen, IRAK-1/4 inhibitor from EMD Millipore, glycine from Sigma-Aldrich. All inhibitors were used at concentrations indicated in the figure legends. All reagents were reconstituted in sterile dimethyl sulfoxide except for the antibodies that were dissolved in PBS and glycine that was dissolved in distilled water.