Cell culture
The human GC cell lines PAMC-82 and SNU16 were obtained from the Chinese Academy of Sciences. The PAMC-82 cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics. The SNU16 cell line was cultured in RPMI-1640 medium with the same additives. All the cells were incubated in a moist atmosphere with 5% CO2 at 37°C.
Sphere-forming culture and self-renewal assay
In order to obtain sphere cultures, cells were cultured in an ultra-low adhesion flask (T75, Corning, NY, USA) at a density of 3 × 105 cells/flask in 15 mL serum free medium (SFM) DMEM/F12 containing 20 ng/mL EGF (Invitrogen), 10 ng/mL LIF (Invitrogen), B27 (1:50; Invitrogen), 20 ng/mL bFGF (Invitrogen), 1% glutamine, and antibiotics. After 7–10 days of culture, the GC sphere cells were collected for follow-up experiments.
We used these sphere formation experiments to explore the self-renewal capacity of these cells. The cells were seeded in 24-well ultra-low attachment plates (Corning) at a density of 500 cells/well and cultured in SFM that was supplemented with 0.8% methylcellulose (Sigma), 20 ng/mL EGF, B27 (1:50), 10 ng/mL LIF, and 20 ng/mL bFGF. The cells were cultured at 37°C in 5% CO2 for 7–13 days, and then the quantity of spheres was counted using a microscope.
Acquisition of stable transfection cell lines
We used lentivirus for ENO1 overexpression and shENO1 to infect PAMC-82 and SNU16 cells in the presence of polybrene (Sigma, St. Louis, MO, USA). Then, the infected cells were selected using puromycin (Sigma). Finally, ENO1-overexpression and ENO1-knockdown stable cell lines were successfully obtained.
Antibodies for western blot and immunohistochemistry
The ENO1 antibody (ab227978), β-Tubulin antibody (ab52901), anti-CD44 antibody (ab157107), SOX2 antibody (ab97959), anti-Nanog antibody (ab80892), and anti-Oct4 antibody (ab18976) were from Abcam.
Western blot
Cells were lysed in cold radio-immuno-precipitation assay buffer for 20 minutes. The lysate was removed after centrifugation and the appropriate buffer was supplemented. Equivalent amounts of protein were transferred to PVDF membranes after SDS-PAGE electrophoresis. Proteins of interest were detected using a LI-COR Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA) after incubation with appropriate secondary antibodies.
Immunohistochemistry
Tissues were fixed in 4% neutral buffered formalin, embedded in paraffin, sectioned and stained for routine histological examination. Biotinylated secondary antibody and the Elitevectastain kit (Vector Laboratories) were used for signal detection and DAB was used as a chromogen substrate. The anti-ENO1 primary antibody was used at 1:1000.
Transwell™ invasion assay
To evaluate invasive activity, a total of 2 × 104 serum-starved cells were resuspended in 200 μL SFM and plated in the top of a Transwell™ chamber (24-well insert; pore size, 8 μm; Corning) that was coated with diluted Matrigel (BD Biosciences). The inserts were placed in wells that contained media with 10% FBS. After cells was incubated at 37°C in 5% CO2 for 24 h, the cells that had not invaded the pores were removed with cotton swabs, while the invasive cells on the lower membrane surface were fixed in 1%–4% formaldehyde, and stained with 0.25% crystal violet. The number of infiltrating cells was counted using a light microscope, and the invasion of cells was analyzed quantitatively.
Chemosensitivity assay
Cells were seeded in 96-well plates (4000 cells/well) and cultured with medium supplemented with 10% FBS for 24 h. Then the cells were treated with different concentrations of cisplatin for 48 h. A Cell Counting Kit-8 (CCK8) was used to evaluate the number of viable cells, and the absorbance at 450 nm was measured using a microplate reader (Bio-rad, USA).
Tumorigenicity in BALB/c nude mice
BALB/c nude mice (4–5 weeks old) were obtained from HFK Bioscience Company (Beijing, China). All mice were raised in specific pathogen-free conditions and relevant experiments were approved by the Animal Care and Use Committee of the Cancer Hospital, Chinese Academy of Medical Science and the Peking Union Medical College (Beijing, China). For tumorigenesis assays, 2.4 × 106 cells were subcutaneously injected into the back of nude mice (five mice/group). The tumor size was recorded every week. All mice were then sacrificed on day 30 after inoculation and the tumor weight of each mouse was measured.
Glucose consumption
We seeded cells in six-well plates for 24 h and then replaced the medium with 3 mL of fresh medium. After a fixed time, we collected the supernatant and measured glucose consumption using a Glucose and Sucrose Assay Kit (Sigma-Aldrich, MAK013). The number of cells was counted three times. The glucose consumption was normalized to μmol/106 cells.
Lactic acid measurement
Cells were collected after culturing for the same length of time as indicated above, and then lactic acid of cells were measured by colorimetry according the instructions of a Lacate Colormetric Assay Kit (Biovision, K627-100). The cells left were counted and the lactic acid production was normalized to μmol/106 cells.
Glycolysis level analysis
The Seahorse XFe/XF Analyzer was preheated on the previous day before each assay. Cells were plated in a Seahorse XF96 plate at a density of 15,000 cells per well, were placed at room temperature for 1 hour, and then incubated at 37°C and 5% CO2 for 24 hours. In a 37°C non-CO2 incubator, the sensor cartridge was hydrated overnight in Seahorse XF Calibrant. On the day of assay, the compounds that included glucose, oligomycin, and 2-Deoxy-D-glucose (2-DG) were loaded into appropriate ports of a hydrated sensor cartridge. Then the growth medium of cell culture was replaced with warmed assay medium using a multichannel pipette and placed in a non-CO2 incubator at 37°C for 1 h before measured. Finally, the cells’ glycolysis stress was tested using the Seahorse XFe/XF Analyzer.
Statistical analysis
All data are shown as the mean ± standard deviation (SD) and results were considered significant if P < 0.05. SPSS 13.0 and GraphPad Prism 5.0 were used to perform all analyses.