Ls174t

The following cell lines (ATCC, except where noted) were grown in culture medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco), 100 units/mL penicillin, 100 μg/mL streptomycin (Gibco), and 1x GlutaMAX (Gibco) (except where noted), and maintained in a humidified incubator at 37°C in 5% CO₂; Rev-GFP U2OS [McCoy's 5A, 200 ug/ml G418 (Sigma)], MM.1S (RPMI), MV-4–11 (IMDM), THP-1 (RPMI), HCT-116 (McCoy's 5A), AML2 (DSMZ, RPMI), AML3 (DSMZ, RPMI), HT1080 (EMEM), HEL (DSMZ, RPMI), Kasumi-6 (RPMI, 2 mM L-glutamine, 1.5g/L sodium biocarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, 2 ng/mL GM-CSF, 20% FBS), SINE compound resistant HT1080 (EMEM, 600 nM KPT-185), A549 (RPMI), UCH1 (4:1 IMDM:RPMI), UCH2 (4:1 IMDM:RPMI), LS174T (EMEM), and ASPS-KY (gifted from A. Ogose, RPMI). The XPO1 SINE™ compounds KPT-330 (selinexor), KPT-335, KPT-350, KPT-8602, KPT-301, KPT-9159 (biotinylated LMB, b-LMB), KPT-9058 (biotinylated KPT-276), and KPT-9511 (biotinylated KPT-8602) were synthesized at Karyopharm Therapeutics, Inc. (Newton, MA). LMB was purchased from Cell Signaling.

The human CRC cell lines (HT29, HCT116- p53 wild type, and LS174T) were obtained from ATCC (Manassas, Virginia) and authenticated in February 2016 (Genetica DNA Laboratories, Cincinnati, OH). DLD1 PI3KCA mutant cells were obtained from ATCC, while DLD1 PI3KCA wild type cells were a gift from Dr. Jing Wang (The University at Buffalo-SUNY). Genetic mutations with potential implication for treatment resistance to one of the studied drug agents include a PI3K mutation in all four main cell lines tested (S2 and S3 Tables). HT29, HCT116, and LS174T cells were grown in McCoy’s 5A media (Sigma Aldrich, St. Louis, Missouri) containing 10% fetal bovine serum (FBS) and 1X antibiotic-antimycotic (Life Technologies, Carlsbad, California) and cultured at 37°C under an atmosphere containing 5% CO₂. DLD1 mutant and wild type cells were grown in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts) containing 10% FBS and 1X penicillin-streptomycin and cultured at 37°C and 5% CO₂. At the time of experimentation, cells were in a passage range of 15–20 and cells were seeded at 8x10⁵ cells per well.

COLO-205, SW48, SW480, LS180, LS174-T, HT-29, T84 and LoVo cells were purchased from the European Collection of Cell Cultures (ECACC). Cells in culture were maintained as a subconfluent monolayer in Dulbecco's Modified Eagle's medium supplied with non-essential amino acids (LS180 and LS174-T), Dulbecco's modified Eagle's medium-F12 nutrient mixture (T84), McCoy 5-A medium (HT-29), L15 Leivobitz medium (SW48 and SW480), Nutrient Mixture F12 HAM (LoVo) and RPMI 1640 (COLO-205) purchased from Sigma. Each cell line was grown under identical conditions, and cell culture medium supplements were provided according to the manufacturer's instructions. To ectopically overexpress c-Src kinase protein, subconfluent SW480 and T84 cells were transfected with 0.4 μg of pBABE-puro expression plasmid carrying cDNA from a c-Src gene. Stable clones of the T84 cell line were selected in F12/DMEM medium supplied with 0.5 μg of puromycin for 3 weeks. In the same way, stable clones of the SW480 cell line were selected in L15 Leivobitz medium supplied with 0.5 μg of puromycin. As a control, subconfluent SW480 and T84 cells were transfected with 0.4 μg of DNA containing an empty pBABE-puro expression plasmid. Positive clones were selected for protein expression measured by Western blotting. The entire set of transfected clones was used as a stable pool for transfection.

The human colorectal carcinoma cell lines HCT8, SW480, SW620, HCT116 and LS174T cells were obtained from the American Type Culture Collection (ATCC). SW480 and SW620 were cultured in L15 Medium (sigma, USA). HCT8 and HCT116 were grown in RPMI medium 1640 (sigma, USA). LS174T cells were cultured in Dulbecco's modified Eagle's medium (Sigma, USA). Fisher rat thyroid (FRT) cells and FRT cells transfected stably with human TMEM16A were obtained from Alan Verkman (University of California, San Francisco, CA, USA) [33] (link) and were cultured in Coon's modified F12 medium. All media was supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.

Three colorectal cancer cell lines were used in the experiments: HCA-2 (Duke C), LS 174T (Duke B), and SW1116 (Duke A), together with a normal cell line, CCD 841 CoN. All cell lines were supplied by ATCC (American Type Culture Collection ATCC^®, Old Town Manassas, VA, USA). To ensure optimal conditions for cell growth, appropriate culture media were used: Eagle’s minimum essential medium (EMEM) (ATCC 30-2003) for CCD 841CoN and LS 174T cell lines, and Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 Ham (DMEM), Sigma-Aldrich D8437). For SW1116 and HCA-2 cell lines, both media were supplemented with 10% foetal bovine serum ((FBS), ATCC 30-2020) and 1% penicillin–streptomycin–neomycin stabilised solution (Sigma-Aldrich P4083).