Ncm460 cells

Human-derived intestinal epithelial NCM460 cells were obtained from INCELL (San Antonio, TX). [³H]-RF (specific activity: 21.2 Ci/mmol, radiochemical purity: > 98%) was purchased from Moravek Biochemicals (Brea, CA). Anti-hRFVT-1 polyclonal antibodies obtained from Abnova (Walnut, CA). Anti-Sp1, anti-hRFVT-2, anti-hRFVT-3 and anti-β-actin monoclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rabbit IRDye-800 and anti-mouse IRDye-680 antibodies were purchased from LI-COR Bioscience (Lincoln, NE). All cell culture supplies, transfection and molecular biology reagents were obtained from commercial vendors.

NCM460 cells were from INCELL (San Antonio, TX), and [³H]-TPP (specific activity 1.3 Ci/mmol; radiochemical purity 97%) was from Moravek Biochemicals (Brea, CA); qPCR primers were from Sigma Genosys (Woodlands, TX); Other chemicals/reagents were from commercial vendors and were of analytical/molecular biology grade. Human and mouse specific anti-SLC44A4 polyclonal antibodies were generated for us by Alpha Diagnostics International (San Antonio, TX) and by Thermofisher (Rockford, IL), respectively. Human anti-ELF3 (AV31639) antibody was from Sigma-Aldrich (Saint Louis, MO); anti-CREB-1 (#9197), anti-ERK1/2 (#9102S), anti-Phospho ERK1/2 (#9101S) and anti-Phospho NF-κB (#3033S) were from Cell Signaling Technology (Danvers, MA); anti-NF-κB (ab16502) antibody from Abcam (Cambridge, MA); anti-β-actin (sc-47778) and anti-Lamin B (sc-6216) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibodies, anti-rabbit IRDye-800 and anti-mouse IRDye-680, were purchased from LI-COR Bioscience (Lincoln, NE).

Caco2 cells were purchased from the American Tissue Culture Collection, USA, and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco, USA). NCM460 cells were purchased from INCELL Corporation LLC, USA, and cultured in M3: BaseF medium (INCELL, USA) supplemented with 10% fetal bovine serum (Gibco, USA). Cells were cultured in a temperature‐controlled incubator at 37 °C and 5% CO₂. Cells were treated with 500µM H₂O₂ for 12 or 24 h to induce oxidative stress.

HT29 cells were donated by Dr. J.C. Fernández-Checa (CSIC, Barcelona, Spain). NCM460 cells were obtained from INCELL Corporation (San Antonio, TX, USA). Dulbecco's Modified Eagle's Medium (DMEM), Penicillin, streptomycin, L-glutamine and fetal bovine serum were from Lonza (Basel, Switzerland). M3:10^TM medium was from INCELL Corporation (San Antonio, TX, USA). Detachin was from Gelantis (San Diego, CA, USA). Fura2/AM and Fura4F/AM were from Invitrogen (Carlsbad, CA, USA). AINEs and 2-APB are from Sigma-Aldrich (Madrid, Spain). 2-APThapsigargin is from Alomone Labs (Jerusalem, Israel). Anti β-actin was from ABcam (Cambridge, UK) anti MCU is from Santa Cruz Biotechnology (Dallas, TX, USA). SYBR green I was from Kappa Biosystems (Boston, MA, USA). Primers were obtained from Thermo Scientific (Ulm, Germany). mitGA plasmid was kindly donated by P. Brület (CNRS, France).

The NCM460 cells (normal human colon mucosal epithelial cell line) obtained from INCELL Corporation (San Antonio, TX, USA) were grown in M3 media supplemented with 10% FBS at 37 °C with 5% CO₂. The CRC HCT-116 and DLD-1 cells were purchased from the cell bank of the Taiwan Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS and 1% penicillin/streptomycin in a 37 °C, 5% CO₂ incubator.

NCM460 cells (INCELL Corporation, LLC, San Antonio, TX, United States) were grown in INCELL’s enriched M3Base medium supplemented with 1% (v/v) Penicillin/Streptomycin 100X (Corning, NY, United States), 1% (v/v) Non-Essential Amino Acids 100X solution (Euroclone, Pero, Italy), and 10% (v/v) heated inactivated Fetal Bovine Serum (FBS; Corning, NY, United States). Cells were grown in culture dishes at 37°C in a 5% CO₂ atmosphere, and seeded at 60–70% confluence (10⁵ cells/well in 96-well plate; 3 × 10⁶ cells/well in 6-well plate) for 24 h prior the co-incubation conditions.