Cell culture and treatment
Tu212 cells were purchased from the Cell Research Institute of the Chinese Academy of Sciences (Shanghai, China). Tu212 cells were cultured in Roswell Park Memorial Institute-1640 medium (Gibco-BRL, Gaithersburg, MD), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT), 100 U/mL penicillin, and 100 g/mL streptomycin at 37°C in an atmosphere containing 5% CO2.
Sorting and identification of Tu212 laryngeal carcinoma cells
Magnetic sorting. Briefly, 1 × 107 Tu212 cells were resuspended in phosphate-buffered saline (PBS). The resuspended cells were added to 100 µL FcR Blocking Reagent and 100 µL CD133 MicroBeads, mixed, and incubated at 4°C for 30 min. Next, 2 mL PBS was added, and the cells were centrifuged at 300 × g for 10 min; the supernatant was discarded. Subsequently, the cell pellet was resuspended in 500 µL PBS. The magnetic separation (MS) column was clipped to the magnetic separator and 500 µL PBS was added to moisten the column. Next, the cell suspension was added to the MS sorting column. The MS separation column was washed three times with 500 µL PBS to remove unbound cells. The column was removed from the magnetic separator, 1 mL PBS was added, and the cells were expelled from the column using a push rod. After centrifugation at 300 × g for 10 min, the supernatant was discarded, and the cells were resuspended in 1 mL PBS and enumerated.
Determination of the purity of Tu212 CD133+ cells. Cells (2 × 105) were removed before and after separation, centrifuged, and the supernatant was discarded. Next, 80 µL PBS and 10 µL anti-human CD133-PE were added. The sample was gently mixed using a micropipette and incubated at 4°C for 10 min. The cells were centrifuged at 1000 rpm for 5 min and the supernatant was discarded. Precooled PBS (1 mL) was added, and unbound excess antibody components were removed by two centrifugation and washing steps. After adding 4% paraformaldehyde and incubation at 4°C for 20 min, the supernatant was centrifuged. The cells were transferred to a flow tube and stored at 4°C protected from light. Flow cytometry was performed using the standard procedure (Beckman, Fullerton, CA).
Experimental groups
Relationship between the proliferation and migration of Tu212 CD133+ cells and the levels of GLUT-1 and autophagy under hypoxia and low-glucose conditions. Eight groups were used in this experiment: CD133+ (20% O2, 25 mM Glu); CD133– (20% O2, 25 mM Glu); CD133++hypoxia (1% O2, 25 mM Glu); CD133–+hypoxia (1% O2, 25 mM Glu); CD133++low Glu (20% O2, 2.5 mM Glu); CD133–+low Glu (20% O2, 2.5 mM Glu); CD133++hypoxia+low Glu (1% O2, 2.5 mM Glu); and CD133–+hypoxia+low Glu (1% O2, 2.5 mM Glu).
Cell growth, migration, and levels of GLUT-1 and autophagy-related proteins. Fourteen groups were used in this experiment: CD133++hypoxia+low Glu (1% O2, 2.5 mM Glu); CD133–+hypoxia+low Glu (1% O2, 2.5 mM Glu); CD133++hypoxia+low Glu (1% O2, 2.5 mM Glu)+NC shRNA; CD133–+hypoxia+low Glu (1% O2, 2.5 mM Glu+NC shRNA); CD133++hypoxia+low Glu (1% O2, 2.5 mM Glu+GLUT-1 shRNA); CD133–+ hypoxia+ low Glu (1% O2, 2.5 mM Glu)+GLUT-1 shRNA); CD133++hypoxia+low Glu (1% O2, 2.5 mM Glu+beclin-1 shRNA); CD133–+hypoxia+low Glu (1% O2, 2.5 mM Glu+beclin-1 shRNA); CD133++hypoxia+low Glu (1% O2, 2.5 mM Glu+3-MA); CD133–+hypoxia+low Glu (1% O2, 2.5 mM Glu+3-MA); CD133++hypoxia+low Glu (1% O2, 2.5 mM Glu+CQ); CD133–+hypoxia+low Glu (1% O2, 2.5 mM Glu+CQ); CD133++hypoxia+low Glu (1% O2, 2.5 mM Glu)+rapamycin); and CD133–+hypoxia+low Glu (1% O2, 2.5 mM Glu)+rapamycin).
Clonogenic assay
The cell suspension was dispersed; the percentage of individual cells was greater than 95%. Next, cells were counted, and the cell density was adjusted to 250/mL by adding culture medium. The cell suspension was added to the wells of a six-well plate (2 mL per well), and the plate was gently shaken. The plate was placed in an incubator for 2 to 3 weeks, and the medium was replaced every 3 days. The culture was terminated when clones became visible. The medium was discarded, and the cells were gently washed twice with PBS, stained with 1% crystal violet at room temperature for 1 h, and photographed.
Cell-Counting Kit-8 assay
Cells were incubated at 37°C in an atmosphere containing 5% CO2 for 48 h. Next, 20 µL Cell Counting Kit-8 (CCK-8) solution was added, and the cells were incubated in the dark for 1 h. The absorption at 450 nm of the suspension was measured using a Spectra Plus Microplate Reader (Molecular Devices, Sunnyvale, CA).
Flow cytometry
Briefly, 10× Binding Buffer was diluted 1:10 with deionized water. Cells were collected by centrifugation for 5 min, exposed to reagents, digested, and resuspended in 500 µL Annexin V binding buffer. Next, 5 µL fluorescein isothiocyanate and 10 µL propidium iodide (Sigma Aldrich Co., St. Louis, MO) were added for 10 min in darkness at RT. Finally, the proportions of non-apoptotic and apoptotic cells were determined in triplicate by flow cytometry with ModFit LT software (Becton Dickinson, Mountain View, CA)
Transwell assay
Cells were digested with trypsin and the culture medium was discarded. Next, the cells were washed once or twice with PBS and resuspended in serum-free medium (containing 0.2% bovine serum albumin) to a density of 1 × 106/mL. Cell suspension (200 μL) was added to the upper Transwell chamber and 600 µL FBS was added to the lower chamber. The cells were incubated in a 5% CO2 atmosphere at 37°C for 24 h. The Transwell chamber was removed, and the culture medium was discarded. Then the chamber was washed twice with calcium-free PBS, fixed in formaldehyde for 30 min, air-dried, and the cells were stained with 0.1% crystal violet for 20 min. Finally, the upper layer of unmigrated cells was gently removed using cotton swabs and washed three times with PBS.
Quantitative real-time polymerase chain reaction
The cells were collected, washed three times with precooled PBS, centrifuged at 1500 rpm for 3 min, and lysed on ice in the presence of TRIzol. Total RNA was extracted from the cells according to the manufacturer’s instructions. Briefly, 1 µg RNA was reverse-transcribed using a First-Strand cDNA Synthesis Kit (K1622; Fermentas, Burlington, ON, Canada) and amplified by PCR using a SYBR Green qPCR Kit (Merck, Darmstadt, Germany). The PCR program was 37°C for 60 min, 85°C for 5 min, and 4°C for 5 min. The amplification products were stored at –20°C. The primers for GLUT-1, Beclin-1, Atg7, Atg5, and LC3 were designed and synthesized by Sangon Biotech (Table 1). The 2ΔΔCt method was used to calculate relative gene expression levels.
Western blotting
Total proteins were extracted from cells and tumor tissues in radioimmunoprecipitation assay buffer. The cells were collected, washed three times with precooled PBS, and centrifuged at 1500 rpm for 3 min. An appropriate volume of cell lysate was added, and the cells were left on ice for 30 min. The supernatant was centrifuged at 1200 rpm at 4°C for 30 min and stored at –80°C. After assaying the protein concentration, samples were added to 4´ sodium dodecyl sulfate loading buffer, boiled for 5 to 10 min, and centrifuged at 12,000 ´ g for 1 min. Proteins (30 μg) were subjected to SDS-polyacrylamide gel electrophoresis (SDS‑PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore). Primary antibodies against GLUT-1 (Abcam), beclin-1, LC3 (Proteintech), Atg7, Atg5, and β-actin (Abcam) were added and incubated at 4°C overnight; β-actin served as the internal control. After washing three times with Tris-buffered saline/Tween 20, the secondary antibodies were added for 1 h at room temperature. The signal was developed using an enhanced chemiluminescence assay kit (Beyotime Biotech) and analyzed semi-quantitatively using the ChemiDoc XRS+ System (Bio-Rad).
Transmission electron microscopy
Cells were collected, washed in PBS, fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, and dehydrated in ethanol and acetone. After embedding in epoxy resin, sections were cut and stained with uranyl acetate and lead citrate. Autophagy was visualized by transmission electron microscopy (TEM; Thermo Fisher Scientific, Waltham, MA).