Anti cox 2

Polyclonal or monoclonal antibodies were used to detect the expression of cell-proliferation related proteins, including anti-E2F1 (sc-193; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-Ki-67 (SP6; Abcam, Cambridge, MA, USA). The other antibodies included anti-Cox-2 (c-9897; Cayman Chemicals, Ann Arbor, MI, USA) and anti-cytokeratin-19 (Ab53119; Abcam) as cancer-related makers, and anti-connexin 26 (138100; Invitrogen, Carlsbad, CA, USA), anti-connexin 32 (358900; Invitrogen), and anti-connexin 43 (138300; Invitrogen). An antibody against calnexin (BD 610523) used as a control was purchased from Transduction Laboratories (BD Biosciences, San Jose, CA, USA) and used at a 1:1,000 dilution. Anti-mouse, anti-rabbit, and anti-goat IgG antisera conjugated with horseradish peroxidase (HRP) were purchased from DAKO (Glostrup, Denmark).

Immunohistochemistry (IHC) was performed on 4 μm paraffin sections of 10 NOE, 21 OE, 30 BE, 64 ADE, and 25 ESCC samples. For PLIN2 and COX-2 antigen retrieval, sections were incubated in water bath while submerged in a target buffer solution (DAKO), pH 6.0 and pH 9.0, respectively, for 40 min at 98 °C. Sections were then incubated with the primary antibodies anti-PLIN2 (AP125; Research Diagnosis Inc., working dilution 1:100), and anti-COX-2 (Cayman Chemicals, working dilution 1:200) during 12 h at 4 °C. The detection system used was the NovoLink Max Polymer Detection System (Leica Biosystems), following the protocol described by the manufacturer. Sections were counterstained with Harris’ hematoxylin. The staining positiveness evaluation was performed by three independent pathologists.

Anti-p65-NF-kB, anti-phospho-p65-NF-kB, anti-MMP-9, anti-PARP and anti-activated-Caspase-3 were from R&D Systems (Heidelberg, Germany). Anti-β1-integrin, anti-β-actin, BMS-345541, 4′,6-diamidino-2-phenylindole (DAPI), MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)), dithiothreitol (DTT), curcumin and alginate were from Sigma-Aldrich (Taufkirchen, Germany). Anti-Sox9 was from Acris Antibodies GmbH (Hiddenhausen, Germany) and anti-Cox-2 was from Cayman Chemical (Ann Arbor, MI, USA). Rhodamine-linked secondary antibodies for immunofluorescence were from Dianova (Hamburg, Germany). Sheep anti-mouse/sheep anti-rabbit alkaline phosphatase-associated secondary antibodies for Western blot, anti-CSPG, and anti-collagen type II were from EMD Millipore (Schwalbach, Germany). We prepared curcumin in DMSO (dimethyl sulfoxide) at 5000 µM strain concentration and kept it at −80 °C. For the studies, the final dilutions were carried out in a cell growth medium, and the final DMSO level did not increase above 0.1% during the experiments. Cell growth medium used, was composed of Dulbecco’s modified Eagle’s medium/Ham’s F-12 (1:1) with 10% fetal bovine serum (FBS), 1% glutamine, 1% penicillin/streptomycin dosing (10,000 IU/10,000 IU), 75 μg/mL ascorbic acid, 1% essential amino acids, and 0.5% amphotericin B solution from Seromed (Munich, Germany).