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2 protocols using tnf α

1

Regulation of Connexin 43 Phosphorylation

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TNF-α and IL-1β were obtained from Alomone (Jerusalem, Israel); Fasudil, Y-27632, ethidium (Etd+) bromide and lanthanum (La3+) chloride were obtained from Sigma-Aldrich (St. Louis, MO, USA). TBARS assay kit was obtained from Cayman Chemicals (Ann Arbor, MI, USA), and CellTiter96® Non-Radioactive Cell Proliferation Assay was obtained from Promega (Madison, WI, USA). The mimetic peptides gap19 (KQIEIKKFK, intracellular loop domain of Cx43) and TAT-L2 (YGRKKRRQRRRDGANVDMHLKQIEIKKFKYGIEEHGK, second intracellular loop domain of Cx43) were obtained from GenScript (Piscataway Township, NJ, USA) [25 (link)]. The monoclonal anti-α-tubulin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). The polyclonal anti-phosphorylated-MYPT1 (Thr696) antibody was obtained from Merck Millipore (Darmstadt, Germany). The monoclonal anti-MYPT1 antibody was purchased from BD Transduction Laboratories (San José, CA, USA). The monoclonal anti-unphosphorylated Cx43 antibody was obtained from Invitrogen (Carlsbad, CA, USA). Anti-mouse and anti-rabbit secondary antibodies conjugated to horseradish peroxidase were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
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2

Annexin V/PI Assay for CRC Sensitivity

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Sensitivity of CRC cell lines to each treatment was determined by Annexin V/propidium iodide high-content microscopy. Cells were seeded at appropriate densities and treated for 24, 48 or 72 h with TL32711 (Bir; Selleck Chemicals, Munich, Germany) alone, chemotherapy alone ['OX5FU': 10 μM 5FU (Medac, Chobham, Surrey, UK) plus 2 μM oxaliplatin (Accord, Barnstaple, Devon, UK)], or a combination of the two in the presence or absence of 10 ngÁmL -1 TNF-α (Alomone Labs, Jerusalem, Israel). Cells were stained with propidium iodide (Sigma, UK), FITC-labelled Annexin V (Thermo Fisher, Waltham, MA, USA) and Hoechst 33342 to visualise nuclei (Thermo Fisher). Cell death was quantified (AV+and/or PI+) on an Array Scan XTI high-content microscope (Thermo Scientific). To control for variabilities in basal cell death across the CRC panel, treatment-specific cell death was calculated as follows: {cell death in treatment group} -{cell death in control group}.
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