Tnfr1

Western blotting and immunoprecipitations were performed using the following primary homemade antibody: a monoclonal antibody against Morgana (P1PP0) was generated in our laboratory using GST-morgana fusion protein as an antigen^{20 (link)}. Other commercially antibodies were used: Vinculin (Sigma, SAB4200080, 1:5000), CHORDC1 (Sigma, HPA041040, 1:1000), MMP9 (Abcam, ab76003, 1:1000), MMP2 (Santa Cruz, 8835, 1:500), HSP90 (Santa Cruz, 13119, 1:1000), P-IkBα Ser32-36 (Santa Cruz, 8404, 1:500), P-IkBα Tyr42 (Abcam, ab24783, 1:1000), IkBα (Cell Signaling, 4814, 1:1000), IKKα (Cell Signaling, 11930, 1:1000), IKKα (Cell Signaling, 2682, 1:100 for co-immunoprecipitation experiments), IKKβ (Cell Signaling, 8943, 1:1000), IKKγ (Santa Cruz, 8032, 1:500), αTubulin (Sigma, T5168, 1:8000), MBP (Cell Signaling, 2396, 1:1000), TNF-R1 (Santa Cruz, 8436, 1:500), TAK-1 (Santa Cruz, 166562, 1:500), p-AKT (Cell Signaling, 9271, 1:1000), AKT (Cell Signaling, 4691, 1:1000), CDC37 (Santa Cruz, 5617, 1:500), P-IKKα/β (Cell Signaling, 2697, 1:1000).
TNFα (300-01A and 315-01A) was purchased from Peprotech, the IKKβ inhibitor PS1145 (P6624), ROCK inhibitor Y27632 (Y0503), the PTEN inhibitor VO-OHpic (V8639), and the AKT inhibitor GSK69093 (SML0428) were from Sigma. BEZ235 (S1009) was purchased from Selleckchem.

Adherent cells on passage 7 were resuspended in FACS (fluorescence-activated cell sorting) buffer, and the concentration adjusted to 10⁵cells/mL. For intracytoplasmic and nuclear markers, cells were permeabilized with 5 μl 0.1 % Triton X-100 for 30 min prior to incubation with primary antibodies (concentration of 1:100) specific for stem cells, inflammation, and cell cycle progression: Oct 3/4 (C-10, SC-5279), Nanog (n-17, SC-30331), CD45/OX1 (SC-53045), CD105 (2Q1707, SC-71042), CD90 (Thy-1, aTHy-1A1), CD34 (BI-3C5, SC: 19621), Caspase-3 (SC-7272), HSP-47 (SC-8352), P21 (SC-6246), Ki67 (Ab – 15580), Cyclin-D1 (AB-27618), P53 (Ab −26), TRA-1-81 (SC-21706), MCP-1 (SC- 32771), TNF-R1 (SC, 52746), all from Santa Cruz Biotechnology, as well as CD117 (c-Kit, SCF-Receptor Ab-6, RB-1518-R7, Thermo Scientific, Lab Vision Corporation, Fremont, CA, USA), VEGF-R1 (Clone VG1, M7273, DakoCytomation, CA, USA), COX-2 (Cayman Chemical Co, EUA), CD11b (MCA-551FT), Ly6a (Ab – 51317), CD1a (SC-18885), and CD133 (Mab4310, Merck Millipore), all for 45 min at room temperature. Then, cells were incubated for 2 h with a secondary antibody (Anti-Mouse FITC, DakoCytomation, Santa Cruz Biotechnology). The analysis was performed using a flow cytometer (FACSCalibur, BD). The expression of surface markers was determined by comparison with an isotype control analyzed by a histogram on the CELLQUEST program.

Samples were loaded on 10 % (v/v) sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membrane. After blocking with TBST (20 mM Tris, 150 mM NaCl, 0.1 % (v/v) Tween-20, pH 7.5) containing 5 % (w/v) of bovine serum albumin, blotting membrane was reacted with antibodies against FasL (Cell Signaling Technology, Inc., Danvers, MA, USA), Fas (Cell Signaling Technology), FADD (Cell Signaling Technology), tumor necrosis factor α (TNFα) (Cell Signaling Technology), TNFR1 (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), or TRADD (Santa Cruz Biotechnology) followed by a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or anti-goat IgG antibody. For β-actin detection, blotting membrane was reacted with an anti-β-actin antibody (SIGMA, Missouri, SL, USA) followed by an HRP-conjugated anti-mouse IgG antibody. Immunoreactivity was detected with an ECL kit (Invitrogen, Carlsbad, CA, USA) and visualized using a chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA). Protein concentrations for each sample were determined with a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA).

Maduramicin ammonium (molecular weight = 934.16, purity>97%, by HPLC) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution (5 mg/ml), aliquoted and stored at −80°C. Dulbecco's modified Eagle's medium (DMEM) and 0.05% trypsin-EDTA were obtained from Mediatech (Manassas, VA, USA). Fetal bovine serum (FBS) was from Atlanta Biologicals (Lawrenceville, GA, USA). One Solution Cell Proliferation Assay Kit was from Promega (Madison, WI). Cellular DNA Flow Cytometric Analysis Kit was purchased from Roche Diagnostics (Indianapolis, IN, USA). CF488A-Annexin V and Propidium Iodide (PI) Apoptosis Assay Kit was purchased from Biotium (Hayward, CA, USA). Enhanced chemiluminescence solution was from Perkin-Elmer Life Science (Boston, MA, USA). The following antibodies were used: cyclin A, cyclin B1, cyclin D1, cyclin E, CDK1, CDK2, CDK4, CDK6, CDC25A, CDC25B, CDC25C, p21^Cip1, p27^Kip1, Rb, p-Rb (S807/811), survivin, Mcl-1, Bcl-2, Bcl-xL, BAX, BAK, BAD, FasL, Fas/CD95, TNFα, TNFR1, TRAIL, DR4, DR5, FLIP S/L, FADD, TRADD, RIP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase 3, cleaved PARP (Cell Signaling, Beverly, MA, USA), β-tubulin (Sigma, St Louis, MO), goat anti-mouse IgG-horseradish peroxidase, and goat anti-rabbit IgG-horseradish peroxidase (Pierce, Rockford, IL, USA).