The cancer genome atlas (TCGA) dataset analysis
Glioma and normal brain tissue data were obtained from the TCGA database in UALCAN (http://ualcan.path.uab.edu/index.html).
Cell culture
The glioma cell lines U87 and U251 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37°C with 5% CO2.
Transfection
U87 cells were transfected with CBX2-shRNA to knock down the expression of CBX2, and the negative control group was transfected with Scramble-shRNA. The sequences are shown in Table S1. The overexpression plasmid, pcDNA3.1-C-(k)DKY encoding CBX2 protein.
Quantitative real-time PCR(qRT-PCR)
Total RNA isolation was followed by reverse transcription, and SYBR Green master mix (Promega, USA) was used for qRT-PCR. The CBX2 primer sequences were as follows: forward: 5’-GCCCAGCACTGGACAGAAC-3’ and reverse: 5’-CACTGTGACGGTGATGAGGTT-3’. GAPDH was used as the reference gene, and the primer sequences for GAPDH were as follows: forward: 5’-TGTGGGCATCAATGGATTTGG-3’ and reverse: 5’-ACACCATGTATTCCGGGTCAAT-3’. The 2-△△Cq method was used to calculate the relative expression.
Western blotting
Total protein was extracted and mixed with 5× SDS loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, USA). The membranes were incubated with primary antibodies overnight and then incubated with HRP-conjugated secondary antibodies. Protein expression was visualized using an enhanced chemiluminescence kit (Millipore). The bands were analyzed with ImageJ software.
Transwell invasion assay
Matrigel and DMEM were diluted at a ratio of 1:2 and served as the extracellular matrix, and the Matrigel solidified after being incubated for 30 min at 37°C. After transfection for 48 h, the cells were cultured in the wells with Matrigel under general conditions for an additional 48 h. The invaded cells were detected by staining the cells with crystal violet, and the cells were counted and imaged with a microscope at ×200 magnification.
Cell counting kit-8 assay
The transfected cells were cultured in 96-well plates at 2000 cells per well. Twenty microliters of CCK-8 solution (Solarbio, Beijing, China) was added to each well and incubated at 37°C for 4 h after 24, 48, 72 and 96 h of culture. The results were measured via spectrophotometry at 450 nm (BioTek Instruments, Inc., Winooski, VT, USA).
Colony formation assay
After transection for 48 h, the cells were seeded in 6-well plates at a density of 1000 cells/well and cultured for 14 days. The colonies were fixed with paraformaldehyde (4% w/v) and stained with crystal violet (0.5% w/v). The number of colonies was counted with a microscope (Olympus Corporation).
Extreme limiting dilution analysis
Glioma cells were seeded into 96-well plates and cultured with 100 μl of serum-free DMEM/F12 medium containing B27, insulin, FGF and EGF. After 14 days, GSCs were detected by extreme limiting dilution analysis.
Animal experiments
To verify the effect of CBX2 knockdown, transfected U87 cells were injected into the brains of BALB/c-A nude mice by intracranial inoculation. The mice were imaged for Fluc activity using bioluminescence imaging. We recorded the weights of the mice and their survival.
Immunohistochemical staining
The paraffin-embedded sections were used for immunohistochemical staining detected with the avidin-biotin complex method. The sections were incubated with antibodies specific for CBX2 and Ki67 overnight at 4°C, followed by secondary antibody incubation at 37°C for 1 h. The expression of CBX2 and Ki67 was determined by coloration with DAB.
Statistical analysis
The significance of Kaplan-Meier analysis was determined by the log-rank test. Multivariate analysis was performed with a multivariate Cox regression model. The data in the study are presented as the mean ± SD. A value of P<0.05 was determined to be statistically significant. Significance was set at *P<0.05, **P<0.01, *** P<0.001. SPSS 17.0 (SPSS, Chicago, IL) was used in the present study.