Cell viability/TNF cytotoxicity assay. YAMC, COX-1?/? MCE, and COX-2?/? MCE cells were plated in growth medium without interferon-�� and incubated for 2 days at 33��C. The cells were then starved in serum-free sellectchem RPMI at 37��C and treated with 100 ng/ml TNF for 48 or 72 h at 37��C. Cell number was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent from the CellTiter 96 Aqueous One Solution cell viability assay (Promega, Madison, WI) or the NucleoCounter (New Brunswick, Edison, NJ) automated cell counter according to the manufacturers’ instructions. Relative cytotoxicities are expressed as percentage of cell loss stimulated by TNF, calculated as follows: 100% ? A490(TNF)/A490(Mock) �� 100%, where A490 is absorbance at 490 nm.
Generation of stable cell lines. The generation of EGFR?/? MCE cells with empty pcDNA3.1/Zeo vector (Vec) or wild-type (WT) EGFR and TNFR1?/? MCE cells with empty bicistronic LZRS-green fluorescent protein retroviral vector (Vec) or hemagglutinin (HA)-tagged WT mouse TNFR1 is described elsewhere (16, 76). An HA-tagged mouse death domain deletion (��DD) TNFR1 mutant, in which the TNFR1 protein sequence ends at P348, and HA-tagged mouse WT TNFR2 were cloned from YAMC cells. The resulting amplified sequences were subcloned into the LZRS vector. TNFR1?/? and TNFR2?/? MCE cells were infected with the respective retroviral constructs selected for expression, as previously described (16). Small interfering RNA transfections.
YAMC cells were transfected with 200 nM siGenome SMARTpool number 2 nontargeting (NT) small interfering RNA (siRNA) or 200 nM mouse EGFR siGenome SMARTpool siRNA (Dharmacon, Lafayette, CO) using the Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA) transfection reagent according to the manufacturer’s instructions. Cell stimulation assays, preparation of cell lysates, and Western blot analysis. Cells were treated with 100 ng/ml TNF or 10 ng/ml EGF (Peprotech, Rocky Hill, NJ), unless otherwise indicated. For experiments using pharmacological inhibitors, cells were pretreated for 1 h with DMSO vehicle control or 1 ��M AG-1478 (Calbiochem, San Diego, CA), 2 ��M CGP-77675 (Pharma Research, Basel, Switzerland), 1 ��M PP2 (Calbiochem), 10 ��M SB-203580 (Calbiochem), or 10 ��M SB-202190 (Calbiochem).
Primary and secondary antibodies used in Western blot analysis include rabbit anti-EGFR (Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, Dacomitinib St. Louis, MO), rabbit anti-HA (Zymed), horseradish peroxidase-conjugated anti-mouse and anti-rabbit polyclonal antibodies (Cell Signaling Technology, Beverly, MA), and goat anti-COX-2 and horseradish peroxidase-conjugated anti-goat polyclonal antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA).