2.1 Data source and statistical analysis
Data on mRNA expressions and the clinical traits of human glioma samples and normal samples were obtained from the TCGA (The Cancer Genome Atlas Program (TCGA)), CGGA (Chinese Glioma Genome Atlas) and GTEx (Genotype-Tissue Expression) initiatives. The mRNA expression datasets underwent processing via the UCSC Toil RNAseq Recompute approach, ensuring a unified analysis across various datasets devoid of batch effects (https://xenabrowser.net/hub/). Statistical evaluations were performed utilizing the R software, version 4.2.0 (http://www.r-project.org/). To explore TCEAL5's clinical relevance in glioma, its expression levels were categorized into high and low groups, based on an optimal cutoff value determined by the surv_cutoff function of the survminer R package. The log-rank test was employed to compare survival curves across these groups.
2.2 Cell lines and cell culture
U251 cells and Ln229 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which performs routine cell line authentication testing with short tandem repeat analysis. Mycoplasma contamination was regularly examined and no contamination occurred during this study. Cells were cultured as previously described 14. TCEAL5 cDNA was cloned into the GV492 lentivirus vector (GeneChem, China) to enable ectopic expression in human glioma cells. Lentiviruses were produced by the transfection of 293T cells with plasmids using the packaging Mix (GeneChem, China). For TCEAL5 ectopic expression, glioma cells were infected with the lentiviruses and selected with puromycin.
2.3 RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)
Cells were subjected to total RNA extraction using Invitrogen's Trizol reagent (Cat. No. 15596026), adhering strictly to the provided guidelines. Post-extraction, the integrity and concentration of the RNA were assessed through gel electrophoresis and measured using the Thermoscientific Nanodrop2000 spectrophotometer. To eliminate genomic DNA contamination and synthesize cDNA, 2 µg of the RNA underwent reverse transcription utilizing Vazyme's HiScript III RT SuperMix for qPCR (+ g DNA wiper) (Cat. No. R123-01), in compliance with the supplier's protocol. The synthesized cDNA was then analyzed using quantitative real-time PCR (qPCR) on the ABI-7500 system, employing TAKARA's TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) (Cat. No. RR420A). β-actin was employed as the internal control for normalization.
2.3 Western blot and antibodies
The cells were processed for protein extraction by lysing them in RIPA (150 mM NaCl, 2 mM EDTA, 50 mM Tris pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail). Post-lysis, the lysate was centrifuged at 12,000 g and 4°C for 20 minutes. The protein content of the supernatant was then quantified using the Bradford protein assay. The whole cell extracts were subjected to SDS-PAGE, and were subsequently transferred onto a PVDF membrane. The membrane was then blocked using a 5% solution of nonfat dry milk in TBST to prevent non-specific binding. This was followed by incubation with primary antibodies overnight at 4°C, and then with HRP-conjugated secondary antibodies at room temperature for 2 hours. Finally, the chemiluminescence reaction was carried out according to the manufacturer's instructions. The primary antibodies used in Western blot are as follows: anti-FLAG (Sigma, F3165), anti-E-cadherin (Proteintech, 20874-1-AP), anti-N-cadherin (Proteintech, 22018-1-AP), anti-TWIST1 (Proteintech, 25465-1-AP), anti-MTA2 (Proteintech, 17554-1-AP), anti-HDAC1 (Proteintech, 10197-1-AP), anti-CHD4 (Proteintech, 14173-1-AP), anti-β-tubulin (Proteintech, 66240-1-Ig).
2.4 Immunofluorescence and antibodies
Cells, cultivated on glass coverslips placed in 24-well plates, underwent fixation using a 4% solution of formaldehyde and were then made permeable with a PBS solution infused with 0.2% Triton X-100. A blocking step was conducted using PBS mixed with 5% bovine serum albumin for one hour, followed by a room temperature incubation with the primary antibody for another hour. This was succeeded by a one-hour incubation with secondary antibodies conjugated to a fluorescent dye, and subsequent DAPI staining. Microscopic imaging of the cells was carried out using a Leica microscope setup. The following antibodies were used in immunofluorescence: anti-FLAG (Sigma, F3165), anti-E-cadherin (Proteintech, 20874-1-AP), anti-N-cadherin (Proteintech, 22018-1-AP), anti-TWIST1 (Proteintech, 25465-1-AP).
2.5 Wound healing assays and transwell cell invasion assays
In the wound-healing assay, cells were cultured in 6-well plates. An overnight serum starvation was initiated, post which a sterile 1ml pipette tip was used to create a scratch, simulating a wound in the cell monolayer. After removing the dislodged cells with a PBS rinse, the culture was continued in a medium enriched with 1% FBS. The healing process, particularly the closure of the gap, was monitored and documented at specified time points using a light microscope.
For the transwell in vitro cell invasion assays, cells, approximately 2×105, were placed in the Matrigel pre-coated top chamber of the transwell chambers (8.0 mm, Corning), while the lower wells received complete medium to act as a chemoattractant and encourage cell invasion. The setup was then incubated for a period ranging from 24 to 48 hours within a cell culture incubator. Following incubation, cells that remained on the upper side of the membrane were carefully wiped away with a cotton swab. The cells that successfully migrated and adhered to the bottom side of the membrane were then stained with 0.1% Crystal Violet. The quantification of these cells was carried out manually to assess the invasion capability.
2.6 Cell proliferation assay and clonogenic assay
Cell proliferation was assessed employing the Cell Counting Kit-8 (Beyotime, C0038), strictly in line with the instructions provided by the manufacturer. The procedure involved seeding cells at a density of 3×103 cells per well in 96-well plates. After the seeding, 100µl of serum-free culture medium, supplemented with 10µl of WST-8 reagent, was introduced to the cells at pre-determined time intervals. The plates were then placed in a cell culture incubator and allowed to incubate for 2 hours. Post-incubation, the optical absorbance of each well was measured at a wavelength of 450nm using a microplate reader to evaluate cell proliferation.
In parallel, clonogenic assays were executed to examine the colony-forming ability of the cells. This involved seeding cells in 6-well plates at a density of 1000 cells per well. The seeded cells were incubated in a cell culture incubator for a duration of 14 days, allowing colonies to form. At the end of the incubation period, cells were stained using crystal violet staining solution (Beyotime, C0121) and the colonies were counted manually to quantify the clonogenic potential of the cells.
2.7 Immunoprecipitation
To conduct co-immunoprecipitation experiments, cells were lyzed using lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, protease inhibitor cocktail) for 20 minutes at 4°C. Subsequent centrifugation at 14,000 g for 15 minutes at 4°C followed. The resulting protein supernatant was then incubated with 2 µg of specific antibodies for 12 hours at 4°C under constant rotation. Afterwards, 50 µl of a 50% solution of protein A or G agarose beads was added, extending the incubation for an additional 2 hours. The beads were washed five times with the original lysis buffer, with centrifugation at 500 g for 3 minutes at 4°C to collect the beads between washes. Proteins bound to the beads were eluted by resuspending in 2x SDS PAGE loading buffer and heated for 5 minutes. To identify the TCEAL5-interacting proteins, the eluates were then separated on a SDS-PAGE gel, silver stained, and analyzed through mass spectrometry.
2.8 Chromatin immunoprecipitation
Cells underwent crosslinking with 1% formaldehyde for 10 minutes at room temperature, which was then quenched by adding glycine to achieve a final concentration of 125 mM for 5 minutes. Subsequently, the cells were prepared in lysis buffer (1% SDS, 5 mM EDTA, and 50 mM Tris-HCl pH 8.0, protease inhibitors). This mixture was then exposed to cycles of sonication for 30 seconds on and off using a Bioruptor, resulting in chromatin fragments approximately 300 bp long. The lysates were then diluted in Dilution Buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.0, protease inhibitors). For immunoprecipitation (IP), this diluted chromatin was incubated either with normal IgG as a control or with specific antibodies for 12 hours at 4°C under continuous rotation. Following this, 50 µL of 50% (volume/volume) protein A/G Sepharose beads were added, and the incubation continued for an additional 2 hours. The beads were then washed using wash buffers sequentially: Wash buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris-HCl, pH 8.0); Wash buffer II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, and 20 mM Tris-HCl, pH 8.0); Wash buffer III (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.0); and TE (1 mM EDTA and 10 mM Tris-HCl, pH 8.0). Finally, the chromatin complexes and input were de-crosslinked at 55°C for 12 hours in elution buffer (1% SDS and 0.1 M NaHCO3), and the DNA was purified using the QIAquick PCR Purification Kit. The DNA was then analyzed using quantitative real-time PCR (qPCR) on the ABI-7500 system, employing TAKARA's TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) (Cat. No. RR420A).