Ls174t cells

Authenticated human embryonic kidney (HEK) 293 cells and human colon cancer cell lines (LoVo and LS174T cells) were purchased from ATCC, immediately prepared frozen cell stocks and stored in liquid nitrogen freezer. Cells were passaged for fewer than 6 months after resuscitation. Cell line authentication was performed by ATCC using short tandem repeat DNA profiles. All cell lines were routinely maintained in ATCC-recommended conditions. 293-CD133 cells overexpressing CD133 were established by transfection with CD133-expressing plasmid, pcDNA3.1-CD133 (CD133 cDNA cloned into pcDNA3.1, Invitrogen), and were maintained with G418 (600 μg/ml, Invitrogen). Cells were incubated in a 37°C and 5% CO2 environment under humidified conditions. Human cDNA CD133 expression plasmid (EX-Z0396-M02) and empty vector plasmid (EX-NEG-M02) were obtained from GeneCopoeia.

Three colon cancer cell lines (LOVO, HCT8, LS174T) were used in this study. LOVO cells were grown in Harm’s F12K medium (Welegene, Daegu, Korea), LS174T cells (ATCC, Manassas, VA, USA) were grown in MEM (Welegene, Daegu, Korea), and HCT8 cells (ATCC, Manassas, VA, USA) were grown in RPMI 1640 medium (Welegene, Daegu, Korea); all media contained 10% fetal bovine serum (FBS; Hyclone, South Logan, UT, USA), and all cells were grown at 37 °C in a humidified environment with 5% CO₂ and 95% O₂. For this study, the CRC cells were infected with live F. nucleatum at a MOI (multiplicity of infection) of 1:500 for 4 h at 37 °C in 5% CO₂. Then, the cells were washed with phosphate-buffered saline (PBS, Welegene, Daegu, Korea) and the media was replaced with fresh media containing 2.5% 50,000 Da DSS (MP bio, Santa Ana, CA, USA) until the cells were harvested. Controls were subjected to similar media changes and wash conditions but without bacterial inoculation.

The IEC-6 cells (original from rat small intestinal crypts) and LS174T cells (original from human colonic gland) were purchased from ATCC. IEC-6 cells were cultured with DMEM-high glucose medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 5 μg/mL insulin, while LS174T cells were cultured with RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. All cells were incubated at 37°C, provided with 5% CO₂ and 95% air. Before treatment, cells were passaged and planted into 6-well plates or a 100-mm dish for 12 h following treatment with drug-containing medium for another 24 h. Series concentrations of GA, GRA, GAMG, GKW, APG, HGKW, LUT, LUTG, TLS, YHC, and YHP were tested for ZO-1 and MUC-2 expressions. The doses used in cell experiments were decided according to MTT cytotoxicity assay, and doses that did not inhibit cell growth were selected. For the proteomics experiment, GA and GKW were both used at concentrations of 50 μM. LPS and SB were used as positive control, respectively. Three biological repetitions were made for the Western blot experiment and two for the proteomics experiment. After rinsing with cold phosphate buffer saline, cells were treated with lysis buffer for Western blot assay or iTRAQ-labeled proteomics assay.