LP9/TERT-1 (LP9), an hTERT-immortalized cell line that functionally resembles normal human peritoneal mesothelial cells  and human primary peritoneal mesothelial cells (HM3) were obtained from Dr. James Rheinwald (Brigham and Women’s Hospital, Harvard University, Boston, MA). Human pleural mesothelial cells (NYU474) were isolated surgically from cancer-free patients by Dr. Harvey I. Pass (New York University, New York, NY), as was the H2373 sarcomatoid MM cell line . The H2373 MM line gives rise to biphasic MM after injection into SCID mice as verified by a board-certified pathologist (Dr. Kelly Butnor at UVM) . LP9 and H2373 cells were cultured as described previously . NYU474 cells were grown to near confluence in DMEM containing 10% FBS and supplemented with penicillin (50 units/mL) and streptomycin (100 μg/mL). HM3 cells were grown in 50:50 M199:MCDB106 medium (Invitrogen, Carlsbad, CA) supplemented with 15% FBS, 10 ng/mL EGF, 0.4 μg/mL hydrocortisone, 50 units/mL penicillin and 100 μg/mL streptomycin. All cells were incubated at 37°C in 5% CO2 and grown to 80-90% confluence before addition of agents.
Sources of crocidolite asbestos, erionite and glass beads
The physical and chemical characterization of the NIEHS reference sample of crocidolite asbestos has been reported previously . Erionite fibers from Pine Valley, NV, were provided and characterized using similar methods by Dr. Ian Steele (University of Chicago, Chicago, IL). The SA of asbestos and erionite fibers and GB (Polysciences Inc., Warrington, PA) was measured using nitrogen gas sorption analysis. Asbestos, erionite fibers and glass beads were exposed to UV light overnight to inactivate any pathogens growing on them before adding to cell culture.
Scanning electron microscopy (SEM)
Cells grown on Thermonox coverslips (Nalge Nunc International, Naperville, IL) were fixed for imaging as previously described , and critical point-dried using liquid CO2 as the transition fluid in a Samdri PVT-3B critical point dryer (Tousimis Research Corporation, Rockville, MD). Cells on coverslips and fibers on carbon tape were then mounted on aluminum specimen stubs and dried before being sputter-coated with gold and palladium in a Polaron sputter coater (Model 5100; Quorum Technologies, Guelph, ON, Canada) and examined using a JSM 6060 scanning electron microscope (JEOL USA, Inc., Peabody, MA).
Following sterilization under ultraviolet light overnight to destroy lipopolysaccharide or microbial contaminants, minerals were suspended in 1 X Hanks’ Balanced Salt Solution (HBSS) at 1 mg/mL, sonicated for 15 min in a water bath sonicator, and triturated 5 X through a 22-gauge needle. This suspension was added to cells in medium to achieve the desired SA-based concentrations. After 24 h intervals, cells were collected by trypsinization, and counted using a hemocytometer .
Quantitative Real-Time PCR (qRT-PCR)
Total RNA (1 μg) was reverse-transcribed with random primers using the Promega AMV Reverse Transcriptase kit (Promega, Madison, WI) according to the recommendations of the manufacturer as described previously . Transcription was evaluated using the ∆∆Ct method. Duplicate or triplicate assays were performed with RNA samples isolated from at least two independent experiments. The values obtained from cDNAs and hypoxanthine phosphoribosyl transferase (hprt) controls provided relative gene expression levels for the gene locus investigated. The primers and probes used were purchased from Applied Biosystems (Foster City, CA).
Western blot analysis for NLRP3, activated Caspase-1 p20 and HMGB1 in whole cell lysates and supernatants (medium) of asbestos-exposed mesothelial cells
Medium was collected, cells were lysed as previously described , and protein content in cell lysates was determined using the RC DC protein assay (Bio-Rad, Hercules, CA). Medium (500 μL) was concentrated using Amcion® ultracentrifugal filters with a 10 K membrane (Millipore, Billerica, MA) by spinning at 14,000 X g for 30 min. Columns were then reversed into new collection tubes and spun for 2 min at 1,000 X g. Sample buffer was added to the concentrated supernatant, and samples were boiled for 15 min. Western blots were performed as previously described  on both cell lysates (NLRP3, HMGB1) and concentrated supernatants (HMGB1, Capase1-p20). Rabbit polyclonal antibodies for HMGB1 (Abcam, Cambridge, MA), NLRP3 (Novus Biologicals, Littleton, CO) and Caspase-1-p20 (Cell Signaling, Danvers, MA) were used at 1:500 dilutions.
Caspase-1 activity assay
Caspase-1 activity was measured using the Caspase-1 Colorimetric Assay (R&D Systems, Minneapolis, MN), according to the manufacturer’s directions. Caspase-1 activity was determined as OD405 (after subtraction of a blank control) relative to total protein. Protein concentrations were determined by the Bio-Rad protein assay  using the remaining lysate.
ELISA assay for IL-1β and IL-18
The Quantikine Human IL-1β/IL-1f2 Immunoassay (R&D Systems, Minneapolis, MN, measures predominantly mature IL-1β) was used on concentrated cell medium, prepared as described earlier, and the assay performed according to the manufacturer’s instructions. A total of 500 μL of cell supernatant was concentrated. Two hundred μL samples with assay diluents were loaded into 96 well plates pre-coated with IL-1β antibody. For IL-1β release studies in erionite-exposed cells, asbestos exposed HM3 cells and TNFα priming of siCon and siNLRP3 LP9 cells, an ELISA MAX™ Human IL-1β (Biolegend, San Diego, CA, measures both pro and mature form) was used. Wells were pre-coated with an IL-1β capture antibody overnight at 4°C. Fifty μL of concentrated samples or standards then were prepared in assay diluent and allowed to attach to plates overnight at 4°C. IL-18 release was measured using the Human IL-18 ELISA kit (MBL International, Woburn, MA, measures predominantly active IL-18, 0.7% pro IL-18). Medium was collected (500 uL), concentrated and processed according to the ELISA protocol provided. Values were expressed as pg/mL of IL-1β or IL-18 from the original supernatant (non-concentrated).
Transfection of cells with NLRP3 siRNA
On-Target plus non-targeting siRNA #1 (scrambled control), and On-Target plus SMART pool human NLRP3 siRNA (100 nM; Dharmacon, Lafayette, CO) were transfected into LP9 cells at near confluence using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. The efficiency of NLRP3 knockdown was determined by qRT-PCR.
SCID mouse xenograft model, IP injection of human MM, and retrieval of PLF
H2373 cells (5 × 106 cells in 50 μL 0.9% NaCl, pH 7.4) were injected into the peritoneal cavity of 6 wk-old male Fox Chase SCID mice (n = 3 mice/group/time point) as described previously . Mice were euthanized using sodium pentobarbital before the peritoneal cavity of each mouse was instilled with 5 mL of cold sterile PBS using an 18-gauge needle. The abdomen was gently massaged, and PLF was aspirated back into the syringe and placed on ice. PLF was then centrifuged at 1,000 rpm for 5 min at 4°C, and the supernatant removed and stored at -80°C for Bio-Plex cytokine analysis. Cytospins were prepared from cell pellets to determine total and differential cell counts .
Bio-Plex assays on cell medium in vitro and in PLF
To quantify cytokine and chemokine levels in cell supernatants and PLF, a multiplex suspension protein array was performed using a Human Cytokine 27-plex panel (Bio-Rad) as described previously . Concentrations of each cytokine and chemokine were determined using Bio-Plex Manager Version 3.0 software. Data were expressed as pg cytokine/mL medium.
In vitro and in vivo studies using the IL-1-ra, Ana
For in vitro experiments, cells were pre-treated with 100 ng/mL of IL-1ra Ana (Insight Genomics, Falls Church, VA) for 1 h before administration of asbestos . For in vivo experiments, SCID mice either injected with 500 μL sterile 0.9% NaCl (pH 7.4) or human MM cells (H2373) in saline were then injected with 100 mg/kg Ana (Kineret®) (Amgen, Thousand Oaks, CA) in 500 μL sterile 0.9% NaCl (pH 7.4)  or 500 μL sterile 0.9% NaCl (pH 7.4) alone. Mice were injected IP daily (with saline or Ana) for 1 wk observations or 3 X IP weekly for 4 wks before euthanization and collection of PLF for Bio-Plex cytokine analyses and cytospin preparations as described above.
Data were evaluated either by analysis of variance (ANOVA) using the Student Neuman-Keul’s procedure for adjustment of pair-wise comparisons between groups or a Student’s t-test. Differences in gene expression values determined by qRT-PCR were evaluated using a Student’s t-test. Differences with p values ≤ 0.05 were considered statistically significant. All experiments were repeated in duplicate or triplicate. Figures represent individual experiments, unless otherwise noted, with Mean ± SEM presented.