Ht 29 cell line

The HT29 cell line (cells: colon, disease: colorectal adenocarcinoma) from ATCC (Manassas, VA, USA) was cultured in McCoy’s 5A Medium (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% FBS (Thermo Fisher Scientific) with antibiotics, streptomycin, penicillin (Sigma-Aldrich, St. Louis, MO, USA), and primocin (InvivoGen, San Diego, CA, USA), in a 90–95% humidified atmosphere with 5% CO₂ . HT29-Snail clones generated as described previously^{4 (link)} were cultured with 200 μg/mL G418/Geneticin (Gibco/Thermo Fisher Scientific). Cells were tested monthly for mycoplasma (PlasmoTest, InvivoGen). The HT29 cell line and HT29-pcDNA control clone were authenticated by ATCC using the Short Tandem Repeat (STR) analysis.

PC3 cell line derived fron human prostate adenocarcinoma was obtained from ATCC (CRL-1435, Rockville, MD, USA) and maintained in RPMI medium containing 10% foetal bovine serum and 1% penicillin/streptomycin. Cells were used between passages 10 and 20 and seeded at a density of 20,000 cells/cm². Sixteen hours post-seeding, medium was changed to serum free medium and treatments were performed 24 hours later. HT-29 cell line derived from a human colon adenocarcinoma (ATCC, Rockville, MD), was used between passages 15 and 25 and cultured in DMEM glucose concentration 4.5 g/L supplemented with 20% fetal bovine serum, penicillin (100 U/mL) and streptomycin sulfate (100 μg/mL) in a humidified 5% CO₂ atmosphere at 37 °C. According to the solubility, the compounds assayed were dissolved in a maximum DMSO concentration of 0.5%.

In the presented study, HT29 cell line (human colon adenocarcinoma) from the ATCC was used as a model of human intestine. The cells were maintained in McCoy's medium supplemented with antibiotics (100 U/mL streptomycin and 100 g/L penicillin) and foetal bovine serum (100 mL/L)^{13 (link)}. The HT29 cell line was maintained at 37 °C under 5% CO₂ atmosphere in a cell incubator (Heal Force)^{13 (link)}. The cell line was employed between passages 6 and 11. Cultured cells were tested for mycoplasma contamination using Universal Mycoplasma Detection Kit from ATCC (USA).

To assess the cytotoxicity of the new compounds, the A549 cell line (lung carcinoma from human) (European Collection of Cell Culture) was selected. Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (PAN-Biotech), which includes 10% Fetal Bovine Serum (Sigma Aldrich), 2 mM Glutamine (Sigma Aldrich), and 100 units/mL penicillin and 100 mg/ml streptomycin (Sigma Aldrich).
HT29 cell line (colorectal adenocarcinoma from human) (American Type Culture Collection) was chosen to evaluate the cytotoxicity of novel compounds. Cells were grown in McCoy’s Medium (Biological Industries) which includes 10% Fetal Bovine Serum (Sigma Aldrich), 2 mM Glutamine (Sigma Aldrich), and 100 units/mL penicillin and 100 mg/ml streptomycin (Sigma Aldrich).
To compare the cytotoxicity of the new compounds, the EAhy926 cell line (the human umbilical vein, somatic cell hybrid) (American Type Culture Collection) was selected. Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (PAN-Biotech) which includes 10% Fetal Bovine Serum (Sigma Aldrich), 2 mM Glutamine (Sigma Aldrich), and 100 units/mL penicillin and 100 mg/ml streptomycin (Biological Industries). Before the initiation of the assay, cells were plated and grown in an incubator at 37 °C with 5% CO₂ to 80% confluence.

The HT-29 cell line (American Type Culture Collection, Manassas, VA) was maintained in the McCoy’s 5A medium (Life Technologies, Carlsbad, CA) containing 10% fetal bovine serum. The HepG2 cell line (American Type Culture Collection) was maintained in the Dulbecco’s Modified Eagle Medium (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum. For the experiments, the HT-29 cells were plated in 12-well plates at 3×10⁵ cells, the HepG2 cells were plated in 12-well plates at 5×10⁵ cells. The cells were cultured in serum-free media for 24 h, and incubated with a compound and oleic acid-albumin (Sigma-Aldrich) solubilized in dimethyl sulfoxide (final 0.1%) for 30 min. The triglyceride synthesis was initiated by the addition of ¹⁴C oleic acid (0.2 µCi). The cells were washed in PBS and harvested into the organic phase using hexane:2-propanol (3∶2) 30 min after initiation of the triglyceride synthesis. The solvent was evaporated, the extracts were solubilized in chloroform, and lipids were separated via TLC. Incorporation of the radiolabel into the triglyceride fraction was analyzed using BAS2000 in triplicate. The IC₅₀ of each compound was calculated using GraphPad Prism.