Ors and 0.5 mg/mL BSA (Sigma, St. Louis, MO) as described previously [25]. Cytomix treatment consisted of supplementing the maintenance medium with 0.2 ng/mL each of TNF + IL-1 + IFN (R D Systems, Minneapolis, MN) for 24 h. Fresh medium (without cytomix) was then applied and cells were exposed to DEP at 25 g/cm2 for 2 h (for fluorescent end points) or 24 h (for cytotoxicity) (Figure 1A). For DEP exposures, freshly prepared particle suspensions in saline (sonicated 3-times on ice; 10 sec each) were spiked into medium. In select experiments, to block iNOS expression, cells were pre-treated with the selective iNOS buy MG-132 inhibitor, 1400W dihydrochloride (100 M; Sigma, St. Louis, MO) for 24 h prior to applying cytomix. To decrease O2- levels, superoxide dismutase (SOD; 200 U/mL; Sigma, St. Louis, MO) was added to the medium 1 h prior to DEP exposure. To catalyze decomposition of peroxynitrite, cells were treated with a synthetic porphyrin complexed to iron (FeTMPyP; 10 M; Cayman Chemical, Ann Arbor, MI) during DEP exposure. Unless otherwise indicated, n = 4 wells per treatment group per time point assessed.In vitro assessments Nitric oxideMaterials and methodsDEP samplesFor the in vitro studies, particles generated in 1999 by a diesel powered automobile were used (a gift from Dr. Daniel Costa, US EPA). As previously reported, these particles closely resembled urban PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28499442 tunnel traffic emissions in that they were comprised of 35 OC, 35 EC, and low levels of soluble metal [25]. Due to limited quantities of this sample, the murine inhalation studies were performed using a DEP sample of comparable OC content ( 35 ) that had been generated in bulk and characterized by the Inhalation ToxicologyChanges in iNOS gene expression, protein, and NO production were assessed in LA-4 cells 24 h after addition of cytomix. Total RNA was isolated using an RNeasy kit (Qiagen, Valencia, CA). cDNA synthesis and realtime PCR using gene-specific primers and probes for iNOS, xanthine dehydrogenase (XdH), and -actin (Applied Biosystems, Foster City, CA) were performed using SuperScript III Platinum One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA). Differential expression was determined using the 2-CT method [80]. Cell lysate iNOS protein levels were assessed by separating equal amounts of protein on E-PAGE 8 gels (Invitrogen, Carlsbad, CA). Protein was transferred, blocked for 1 h, probed overnight at 4 with antibodies to iNOS (1:500; BD Transduction Laboratories, San Jose, CA) or -actin (1:5000; Sigma, St. Louis, MO), washed again, and incubated for 1 h with corresponding secondary antibodies. Signals were detected using chemiluminescence (LumiGlo, Cell Signaling Technology, Danvers, MA) with images acquired using an Alpha Innotech 8900 imaging station (San Leandro, CA). NO production was assessed after 30 min incubation with theManzo et al. Particle and Fibre Toxicology 2012, 9:43 http://www.particleandfibretoxicology.com/content/9/1/Page 12 offluorescent probe, DAF-FM (10 M; Invitrogen). Fluorescence was quantified on a plate reader (Packard FluoroCount BF10000).CytotoxicityUsing a commercially available kit for lactate dehydrogenase (LDH) (Thermo Fisher Diagnostics, Middletown, VA), LDH release was used to assess LA-4 cytotoxicity. Cell lysate protein was determined using a kit (Thermo Scientific, Rockford, IL). Assays were modified and adapted for use on the KONELAB Arena 30 clinical chemistry analyzer (Thermo Clinical Labsystems, Espoo, Finland).Intrac.
