Ccl 228

Five different cell lines were used in this experiment. U373MG cells, currently reclassified as U251 (HTB-17, ATCC, Manassas, VA, USA) but here referred to with their original name for continuity with earlier publications from the group, were maintained in DMEM (Lonza, Basel, Switzerland) with 5% heat inactivated fetal bovine serum (FBS), 1% GlutaMAX (Gibco, Carlsbad, CA, USA), and gentamycin. HCE cells (kindly provided by Arto Urtti from the University of Helsinki and the University of Eastern Finland, Kuopio, Finland) and SW480 cells (CCL-228, ATCC, Manassas, VA, USA) were maintained in the same medium as the U373MG cell line. Vero cells (CCL-81, ATCC, Manassas, VA, USA) were maintained in M199 medium (Gibco), 5% heat inactivated FBS, and gentamycin. Raji cells (CCL-86, ATCC, Manassas, VA, USA) were maintained in RPMI 1640 (Lonza) with 10% heat inactivated FBS, 1% GlutaMAX, and gentamycin.

The cell line SW480 (human colon carcinoma, ATCC® CCL-228, RRID: CVCL_0546) and primary murine embryonic fibroblasts NIH3T3 (ATCC® CRL-1658, RRID: CVCL_0594) were obtained from American Type Culture Collection (ATCC®). Cells were cultured in DMEM/F-12 or DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Invitrogen; Thermo Fisher Scientific, Inc.) at 37 °C in a humidified 5% CO₂ atmosphere.

Human normal colonic epithelial cell line FHC (ATCC® CRL-1831), as well as human CRC cell lines HCT116 (ATCC® CCL-247™, Rockville, MD) and SW480 (ATCC® CCL-228™), was procured from ATCC (Manassas, VA). HCT116 cells were cultured in McCoy’s 5a medium containing 10% FBS (ATCC, No. 30-2007), while SW480 cells were maintained in Leibovitz’s L-15 medium (ATCC, No. 30-2008) containing 10% FBS. Meanwhile, FHC cells were cultured in DMEM/F12 medium. All the aforementioned cells were cultured in a humid incubator at 37 °C with 5% CO₂ in air.
Subsequently, the cells were seeded into a six-well plate (3 × 10⁵ cells/well). Upon reaching 50% cell confluence, transfection was carried out using Lipofectamine 2000 reagent (11668-019, Invitrogen, Carlsbad, CA). siRNAs targeting SP1 (si-SP1-1, si-SP1-2), siRNAs targeting TUG1 (si-TUG1-1, si-TUG1-2), siRNA targeting KDM2A (si-KDM2A) and its negative control (si-NC), miR-421 mimic, mimic NC, TUG1 over-expression plasmid (oe-TUG1) and oe-NC were all synthesized and provided by GenePharma (Shanghai, China). The siRNA sequences are shown in Supplementary Table 1.

The HCT116 (p53wt) was purchased from ATCC and HCT116 p53 double knockout (HCT116 p53^−/−) human colon adenocarcinoma cell lines was kindly provided by Prof. Bert Vogelstein (Johns Hopkins University, Baltimore, USA) and maintained in McCoy’s 5A medium (Sigma-Aldrich). The SW480 human adenocarcinoma colorectal cancer cell line (ATCC#CCL-228™; ATCC, USA) which carries two-point mutations (R273H/P309S) was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (Sigma-Aldrich). LS174T (p53wt, ATCC#CL-188™; ATCC, USA) [63 (link)] human adenocarcinoma colorectal cancer cells were cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich) and 1 mM sodium pyruvate (Sigma-Aldrich) at 37 °C in a 5% CO₂ atmosphere. After seeding at the desired density, cells were incubated overnight prior to the experiments. Cells were routinely checked for mycoplasma contamination.

CCD841 (ATCC^® CRL-1790™) healthy human mucosa cell lines and CaCo-2 (ATCC^® HTB 37™) human colorectal cancer cells were grown in an EMEM medium supplemented with heat inactivated 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1% non-essential amino acids, 100 U ml^–1penicillin, and 100 μg ml^–1 streptomycin. E705 (kindly provided by Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy) and SW480 (ATCC^® CCL-228™) human colorectal cancer cells were grown in an RPMI 1640 medium supplemented with heat-inactivated 10% FBS, 2 mM L- glutamine, 100 U ml^–1penicillin, and 100 μg ml^–1streptomycin. All the cell lines were maintained at 37°C in a humidified 5% CO₂ incubator. ATCC cell lines were validated by short-tandem repeat profiles that are generated by simultaneous amplification of multiple short-tandem repeat loci and amelogenin (for gender identification). All the reagents for cell cultures were supplied by Lonza (Lonza Group, Basel, Switzerland).

The human embryonic kidney cell line HEK393T (ATCC® CRL-11268™), human colon cancer cell line SW480 (ATCC® CCL-228™), and human cervix cancer cell line Hela (ATCC® CCL-2™) were purchased from ATCC. These cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco). Clofibrate was purchased from Toronto Research Chemical Inc. Taxol was purchansed from Ruibio. Cisplatin was purchased from Tokoyo Chemical industry. Etoposide and camptothecin were purchased from Hefei Bomei Biotechnology of China. Puromycin was purchased from Life Technologies.