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).