Enbrel

Bone loss was evaluated in vivo after a 7‐day pretreatment period. In brief, at 7, 5, 3, 1, and 0 days prior to RANKL injection, mice were administered a single intraperitoneal injection of anti‐FKN mAb (clone 5H8‐4, 400 μg/mouse), control IgG (400 μg/mouse), etanercept (Enbrel; 400 μg/mouse; Pfizer Inc., New York, NY), or PBS. Then, to induce bone loss, on the day after the last mAb treatment, mice in each group were intraperitoneally injected with recombinant human soluble RANKL (0.75 mg/kg) (Oriental Yeast Co. Ltd., Tokyo, Japan), followed by a second RANKL injection on the following day.⁽²⁴, ²⁵⁾ For bone histomorphometry and micro‐CT, hindlimbs were isolated 4 hours after the first RANKL injection and 24 hours after the second, respectively. For flow cytometry, peripheral blood and hindlimbs were collected before the first RANKL injection but after anti‐FKN mAb (5H‐8) or control IgG pretreatment described previously, and blood leukocytes and BMCs were stained with antibodies described in the Flow Cytometry section.

Human cell lines were obtained from American Type Culture Collection (ATCC;CEM, Manassas, VA, USA) or Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany) and cultured in DMEM medium or RPMI 1640 (Life Technologies, Inc., Eggenstein, Germany) or McCoy's medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany), 1 mM Sodium Pyruvate (Invitrogen, Karlsruhe, Germany) and 1% penicillin/streptomycin (Invitrogen, Karlsruhe, Germany). IFNα was obtained from Sigma-Aldrich (Taufkirchen, Germany), zVAD.fmk from Bachem (Heidelberg, Germany), Nec-1 from Merck (Darmstadt, Germany) and Enbrel from Pfizer (Berlin, Germany). The bivalent Smac mimetic BV6 was kindly provided by Genentech, Inc. (South San Francisco, CA, USA). All other chemicals were obtained from Sigma-Aldrich or Carl Roth (Karlsruhe, Germany) unless indicated otherwise.

Liver organoids were generated by digesting embryonic livers (E20) from vFlip^AlbCre+ and control mice and liver progenitor cells were cultured in a HepatiCult™ Organoid Growth Medium (Mouse) (STEMCELL technologies) for 7 days. Premature liver organoids were treated with selected factors Necrostatin-1 (30 µM, Sigma-Aldrich), Etanercept (25 µg/ml Enbrel®, Pfizer), Interferon β + γ (100 ng/ml each, PeproTech) in addition to A83-01 (50 nM, Sigma-Aldrich) for hepatocyte maturation.

MDA-MB-231 breast carcinoma and SK-N-AS neuroblastoma cells were grown in DMEM medium (Invitrogen, Karlsruhe, Germany), T24 bladder carcinoma cells in McCoy's medium (Invitrogen) and Kym1 rhabdomyosarcoma cells in RPMI medium (Invitrogen) supplemented with 1% penicillin/streptomycin, 1% sodium pyruvate (both from Invitrogen) and 10% fetal calf serum (Invitrogen). The bivalent Smac mimetic BV6 that antagonizes XIAP, cIAP1 and cIAP2^{6 (link)} was kindly provided by Genentech Inc. (South San Francisco, CA, USA) and Enbrel by Pfizer (Berlin, Germany). Recombinant human TNFα was purchased from Biochrom (Berlin, Germany). All chemicals were obtained from Sigma (Deisenhofen, Germany) unless indicated otherwise.

N-ε-carboxylmethyllysine (CML, CAS-No 941689-36-7, Cat#14580) with at least >97% purity (TLC) with water (<12.0%), dihydroethidium (DHE, 104821-25-2, Cat #37291), and acridine orange (AO, 65-61-2, Cat#A9231) were purchased from Sigma–Aldrich (St. Louis, MO, USA). CIGB-258 (Jusvinza^®), a recombinant peptide from HSP60 with 27 amino acids in a lyophilized powder formula (1.25 mg/vial, Lot# 1125J1/0), was obtained from the Center for Genetic Engineering and Biotechnology (CIGB, Havana, Cuba) under the agreement that it would be used for research purposes only. Remsima (Infliximab 100 mg powder, Celltrion, Incheon, South Korea Lot# 2B3C091), Enbrel (Etanercept 50 mg, Pfizer, Dublin, Ireland), and Actemra (Tocilizumab 400 mg/20 mL, Lot# 21K010E11778, JW Pharmaceutical, Seoul, Republic of Korea) were purchased from Shinsung Pharmaceutical (Seoul, Republic of Korea) under the agreement that it would be used for research purposes only.

The effects of etanercept (Enbrel®, Pfizer, Germany) on macrophage gene expression profiles and cytokine response were evaluated at a concentration of 2 µg/ml, which is comparable with concentrations found in plasma of arthritic patients^{45 (link),46 (link)}. MDM were challenged with etanercept for 6 hours at 37 °C in a 5% CO₂ atmosphere.