2.8 Cell culture and treatment
The human epithelial colorectal adenocarcinoma HT-29 cell line was obtained from the Procell Life Science and Technology Company, and it was maintained in DMEM with 10% FBS, 1% nonessential amino acids, and gentamicin (50 μg/mL) at 37° C and 5% CO2, and the medium was replaced every two days. For parameter measurements, the culture medium was seeded onto 6-well plates at a density of 5 ×105 cells/well (Corning Inc., Corning, NY, USA). The cells were incubated with 1 mM 3-MA or 10 μM rapamycin for 1 hour. To induce inflammatory damage, the cells were exposed to LPS (1 μg/ml) at 37 °C for 6 hours. HT-29 cells were transfected with 10 pmol of siRNA for TLR4 using Lipofectamine RNAiMAX according to the manufacturer’s instructions. The cellular proteins were obtained for western blot analysis as previously described (M. Wang et al., 2017).