Iec 6

HEK293T, SW480, HCT116, DLD-1, and IEC-6 cell lines were purchased from ATCC and RKO-pBAR/Renilla and SW480-pBAR/Renilla were a gift from Professor Xi He (Harvard Medical School). All cell lines were maintained in DMEM-F-12 (Gibco, Life Technologies Limited, Paisley, UK) enriched with 10% fetal bovine serum (Gibco). The chalcone lonchocarpin was purified by Nascimento and Mors, 1972 [50 (link)] and kindly donated for this study by professor Ricardo Kuster (Federal University of Espirito Santo). PNU-74654 was synthesized at the Laboratory of Evaluation and Synthesis of Bioactives substances (Biomedical Sciences Institute, UFRJ). Both compounds were diluted in DMSO (Sigma-Aldrich, Saint Louis, MO, USA) at the concentration of 10 mM. PNU-74654 has been previously described as an inhibitor of Wnt/β-catenin pathway by blocking the interaction between β-catenin and TCF [30 (link)]. L-cell conditioned medium (CM) and Wnt3a CM were obtained according to the ATCC protocol. L-cells and L-Wnt3a cells were plated into 75 cm² flasks at 50% confluence with 10 mL DMEM medium containing 10% FBS. After 4 days, the first batch of medium was obtained and kept at 4 °C. Three days later, the last batch of medium was obtained and combined with the first one. Finally, L-cell CM and Wnt3a CM were passed through a 0.22 μm filter, and kept at 20 °C.

All cell lines were maintained in DMEM F12 culture medium supplemented with 10% fetal bovine serum and 100U/mL and 100 µg/mL penicillin/streptomycin (Gibco, Life Technologies Carlsbad, CA, USA) at 37 °C and 5% CO₂. Dimethylsulfoxide (DMSO), anti-β-catenin and anti-α-tubulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Secondary antibodies were purchased from Life Technologies (CA, USA). Cell lines used were HEK293T, L-cell, L-Wnt3a, HCT116, DLD-1, SW480 and IEC-6 (ATCC). Wnt signalling reporters cell lines, RKO pBAR/Renilla and SW480-pBAR/Renilla were a kindly donated by Dr. Xi He (Boston Children´s Hospital). The alkaloid piperine used in this study was extracted from Piper nigrum, purified and identified at Research Institute for Natural Products in the Federal University of Rio de Janeiro (IPPN-UFRJ-Brazil).

The cell lines small intestinal epithelial cells (IEC-6) of SD female rats were purchased from ATCC cell bank. The IEC-6 cells were cultured according to the supplier’s recommendations. The IEC-6 cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 0.1 U/ml bovine insulin as described. SIRT1 was knocked down by siRNA in IEC-6 cells according to the manufacturer’s instructions. IEC-6 cells were seeded in 6-well plates, and then a specific SIRT1 siRNA and control negative (NC) siRNA were transfected with Lipofectamine RNAiMAX. After 48 h, total RNA of cells was extracted for qPCR detection.

The intestinal epithelial cell line, IEC-6, was obtained from ATCC (Rockville, MD, USA), which was cultured in DMEM mixed with 10% FBS. After LPS irritation and the corresponding vector transfection, the disposed IEC-6 cells were collected and inoculated into 96-well plates. After 24 h culture, cells were reacted with 20 μL MTT solution (5 mg/mL, Sigma, St. Louis, MO, USA) for an extra 4 h. When terminating the incubation, 150 μL DMSO (4%, Sigma) was used to accelerate the crystal dissolution. After accomplishing these procedures, a microplate reader (BioTek, Winooski, VT, USA) was applied to test the absorbance at 570 nm.