TeloTAGGG Telomerase polymerase chain reaction (PCR) ELISAPLUS kit was purchased from Roche Molecular Biochemicals (Basel, Switzerland). Chemiluminescent detection kit was from SuperArray Bioscience (Frederick, MD, USA). Bio-Rad protein assay kit and silver stain plus? kit were from Bio-Rad (Hercules, CA, USA). Isolation and culture of human SW1116 cells Human SW1116 inhibitor order us cells were maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco BRL, USA), 1 �� 105 U/L penicillin G and 100 mg/L streptomycin in an atmosphere containing 5% CO2 at 37��C. Adherent SW1116 cells were dissociated to single cell suspensions and seeded in serum-free medium (SFM). After spheres of SW1116 cells were formed, proliferation and differentiation potentials of SW1116 cells were observed.

SW1116 cells were isolated and maintained in a SFM (DMEM/F12 medium) containing 20 ��g/L human recombinant epidermal growth factor (EGF; Invitrogen, Carlsbad, CA, USA), 20 ��g/L human recombinant basic fibroblast growth factor (bFGF; Invitrogen, Carlsbad, CA, USA), 2 mmol/L L-glutamine, 4 U/L insulin, 1 �� 105 U/L penicillin G, and 100 mg/L streptomycin. Sphere formation assay Primary spheres of SW1116 cells were dissociated to single cell suspensions and inoculated in ultra-low attachment 96-well plates (Corning Life Sciences, Acton, MA) (100 cells per well) supplemented with 200 ��L SFM. Then, 25 ��L SFM per well was added every 2 d. The number of spheres of SW1116 cells in each well was evaluated 14 d after culture.

Differentiation assay of spheres Two days after primary culture, SW1116 cells were plated in 24-well culture plates with 10% FBS and cultured with FBS-supplemented medium every two days. Differentiation potentials of SW1116 cells were observed under a microscope. Cell proliferation assay SW1116 cells were seeded onto 35 mm Petri dishes at a density of 1 �� 104. Cultured SW1116 cells were stained with trypan blue and counted in triplicate under a microscope for 6 wk. Chemosensitivity test SW1116 cells were seeded onto 96-well plates at a density of 5 �� 103 per well. Twenty four hours after drugs were added at different concentrations, SW1116 cells were exposed to drugs for 2 d. Then MTT (5 mg/mL) was added and the plates were incubated at 37��C for 4 h before 100 ��L 100% dimethyl sulfoxide was added to each well.

The optical density of SW1116 cells in each well was detected with a microplate reader (Bio-Rad, Model 550) at a wavelength of 570 nm. The survival time of SW1116 cells was calculated as OD value of OM- exerted cells/OD value of mocked-cells �� 100%. Fluorescence-activated cell sorting analysis Stained SW1116 cells at a concentration of 1 �� 106 Carfilzomib cells per 100 ��L buffer contained PBS at pH 7.2, 0.5% BSA, and 2 mmol/L EDTA. Antibodies against CD133 (anti-CD133-PE, BD Pharmingen, Franklin Lakes, USA) and CD29 (anti-CD29-FITC, Chemicon, Billerica, MA, USA) were used. Antibody was incubated at 4��C for 30 min.