The LN-229 glioblastoma cell line was purchased from ATCC (Manassas, VA, USA). Cells were cultured in RPMI-1640 (Thermo Fisher Scientific, Inc., MA, USA) supplemented with 10% Gibco® fetal bovine serum (Thermo Fisher Scientific) and 100 μg/mL penicillin-streptomycin (Thermo Fisher Scientific) at 37℃ and 5% CO2. Cell transfection was performed using Lipofectamine™ 3000 Transfection Reagent (Invitrogen™, CA, USA) strictly according to the manufacturer’s instruction. After 48 h of transfection, the efficiency was determined by RT-qPCR and western blotting which are described below.

RT-qPCR was used for detection of EIF6 and glycolysis related genes GLUT1, HK1 and LDHA. Briefly, total RNA was extracted from LN-229 cells using TRIzol reagent (Tiangen Biotech, Beijing, China). The concentration of extracted RNA was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, DE, USA). Then reverse transcription was conducted and cDNA was obtained by a Prime-Script™ One Step RT-qPCR kit (Takara Biotechnology Co., Ltd., Dalian, China). A SYBR GREEN mastermix kit (Applied Biosystems, CA, USA) was used to perform the PCR reactions in Applied Biosystems 7500 Real Time PCR system (Applied Biosystems) with the following condition: 95 ℃ 15 min, 35 cycles, 4 ℃ denaturation for 15 sec, 55 ℃ annealing for 25 sec, 70 ℃ extension for 30 sec. The primer sequences were designed by Primer3 (https://bioinfo.ut.ee/primer3-0.4.0/) and were listed in table 1. The reverse transcription system and PCR reaction system were listed in table 2-3.

Table 1. Primers in this study.

Table 2. The reverse transcription system

Table 3. PCR reaction system.

1. Extraction of Total Protein: Total proteins were extracted from LN-229 cells using radio-immunoprecipitation assay (RIPA) buffer (Vazyme Biotec Co., LTD, Nanjing, China).
2. Protein Concentration Measurement: The protein amount was quantitated using the BCA protein assay reagent from Bio-Rad (Hercules, CA, USA). A BCA working reagent was prepared according to the manufacturer's instructions.
3. Electrophoresis Separation: Samples containing an equal amount of protein (20-40 μg) were loaded into each well of a 10% SDS-PAGE gel. Electrophoresis was performed at a constant voltage (100-200 V) until the dye front reached the bottom of the gel.
4. Transfer to PVDF Membrane: The membrane was equilibrated in transfer buffer (Tris-glycine buffer with 20% methanol) for 15-20 minutes. A transfer sandwich (gel-PVDF-membrane) was assembled, and transfer was carried out using a semi-dry or wet transfer system following the manufacturer's recommendations.
5. Blocking: The PVDF membrane was incubated in 5% non-fat milk at room temperature for 1 hour.
6. Primary Antibody Incubation: The primary antibody against EIF6 (anti-EIF6 antibody from Abcam, Cambridge, MA, USA) was diluted in 1% non-fat milk or 1% BSA in PBS following the manufacturer's recommended dilution. The membrane was incubated with the primary antibody solution overnight at 4℃ with gentle shaking.
7. Secondary Antibody Incubation: The secondary antibody (horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibody from Abcam, Cambridge, MA, USA) was diluted in 1% non-fat milk or 1% BSA in PBS. The membrane was incubated with the secondary antibody according to the manufacturer's instructions at room temperature for 45 minutes.
8. Chemiluminescent Detection: The chemiluminescent reagent (Super Signal West Pico Chemiluminescent Substrate kit from Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was prepared according to the manufacturer's instructions. Chemiluminescent signals were captured using an imaging system. Relative protein expression was quantified using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA).

The MTT assay was performed to assess cell viability following transfection at various time intervals. Initially, 5 × 10^3 cells were seeded into individual wells of 96-well plates and incubated for 48 hours at 37℃ in a 5% CO2 environment. Subsequently, 10 μl of MTT solution (5 mg/ml) was introduced to each well, and the cells were incubated for an additional 4 hours. Afterward, the MTT solution was carefully aspirated, and 100 μl of dimethyl sulfoxide (DMSO) was added to each well. Optical density (OD) values were measured at 490 nm to evaluate cell viability.

The clonogenic formation assay was carried out to assess cell proliferation following transfection, following established protocols. In summary, cells were initially seeded at a density of 5 × 10^3 cells per well in 6-well dishes. After a 14-day incubation period, cells were fixed using glutaraldehyde and subsequently stained with crystal violet. Positive colony formations were manually counted for each well, and all experiments were conducted in triplicate.

The Transwell invasion assay was conducted to assess the invasive capacity of cells through a Matrigel-coated membrane (24-well insert; pore size, 8 μm; BD Biosciences). A total of 1.25 × 10^5 cells were seeded in the upper chamber with serum-free media, while the lower chamber contained media with serum as a chemoattractant. Following a 24-hour incubation period at 37℃, non-invading cells on the upper surface of the membrane were gently removed, and the invaded cells on the lower surface were fixed with glutaraldehyde and stained using 0.1% crystal violet. Subsequently, five randomly selected fields of view within each sample were imaged and counted under a microscope. The mean number of invaded cells from triplicate experiments was determined, providing a quantitative measure of the cells' invasive potential.

Cell migration was assessed via the scratch wound assay, where cells were initially seeded into 6-well plates and cultured for 24 hours to achieve confluence. Subsequently, uniform wound gaps were generated within the cell monolayer using a 200 μL pipette tip. The cells were then cultured in DMEM supplemented with 10% FBS, and images were captured at both 0 and 48-hour time points to precisely document and quantify changes in wound width, providing insights into cell migration dynamics over the specified time frame.