2.1 Cell culture and treatment

Two glioma cell lines, including U251 (Cat.No.SCSP-559) and LN229 (Cat.No.TCHu244), were obtained from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China). U251 and LN229 cells were respectively isolated from two GBM patients. The cells were cultured in an incubator at 37 ℃ with 100% humidity. The cell medium was Dulbecco's Modified Eagle's Medium (DMEM) (Cat.No. SH30022.01, HyClone) containing 10% fetal bovine serum (FBS, Cat.No. SH30070.03, HyClone) and 1% Penicillin &streptomycin (Cat.No. SV30010, HyClone). Bay 11–7082 (an NF-κB inhibitor, Cat.No. B5556), U0126 (an ERK inhibitor, Cat.No. 662009) and SP600125 (a JNK inhibitor, Cat.No. S5567) and TMZ (Cat.No. T2577) were purchased from Sigma‒Aldrich (St. Louis, MO). Glioma cells were treated with varying doses of TMZ (0–2000 μM) and Bay 11–7082 (10 μM) [14]. The U0126 (5 µM) [15] and SP600125 (5 μM) [16] were respectively added into the medium for inhibiting ERK and JNK, respectively. The glioma cells were treated with increasing doses of TMZ or Bay 11–7082 for 24 h. Then CCK8 assay, BrdU staining and Calcein/PI staining were conducted to evaluate cell viability or proliferation. LAMB1 overexpression plasmids, a set of two shRNAs targeting LAMB1, and corresponding negative controls were transfected into glioma cells using Lipofectamine® 3000 (Invitrogen). The above overexpression plasmids or shRNAs were supplied by GenePharma (Shanghai, China). sh-NC plus vehicle were used as the control group, vehicle was also used in the sh-LAMB1 group, and sh-NC was added in the Bay 11–7082 group.

2.2 TMZ-resistant cell modeling

The establishment of TMZ-resistant cell was referred to a previous study [17]. Briefly, the U251 and LN229 cells were treated with increasing doses of TMZ (1 μM–500 μM). After achieving stable cell growth, the TMZ dose was enhanced in multiples. Each dose was maintained for 15 days until the completion of the fifth month.

2.3 Cell counting kit-8 (CCK-8) assay

U251 and LN229 cells were grown in 96-well plates at 1 × 104 cells/well and cultured for 24h, 48h, 72h, and 96h, respectively. 10 μL of CCK-8 solution (Beyotime, Shanghai, China) was added to each well, and the incubation was terminated 2 h later. The optical density (OD) of each well at 450 nm was measured with a microplate reader (Bio-Rad Laboratories, Inc.). To determine the EC50 value of glioma cells to TMZ, the cells were treated with different doses of TMZ for 24 h. Following that, the CCK-8 assay was used for detecting cell viability and EC50 value was calculated by non-linear regression analysis.

2.4 5-bromo-2'-deoxyuridine (BrdU) staining and Calcein/PI staining

U251 and LN229 cells were cultured in 24-well plates at 5 × 104 cells/well and then incubated for 24 h. Then, the proliferation of the cells was measured using the BrdU staining method. Briefly, the cells were incubated with 10 μM BrdU (Cat.No.40204ES60, Yeasen, Shanghai, China) at 37 ℃ for 45 min. Later, the cells were washed with PBS twice and fixed by 4% PFA (15 min, room temperature). The cells were incubated with 2M HCl for 30 min at room temperature, washed with PBS twice, and incubated with 0.1M Na2B4O7 for 15 min at room temperature. After being blocked with 5% bovine serum albumin, the cells were incubated with BrdU Mouse mAb (Cat.No.31515ES50, Yeasen, Shanghai, China) overnight at 4 ℃. The next day, the cells were incubated with YSFluor™488 Rabbit Anti-Mouse lgG(H + L) (Cat.No.33906ES60, Yeasen, Shanghai, China) for 1 h at room temperature. The DAPI Stain Solution (Cat.No.40728ES03, Yeasen, Shanghai, China) was used for labeling the nucleus. Finally, the observation and photography of the cells were conducted under a fluorescence microscope (IX73, Olympus, Japan). The proliferation of the tumor cells was counted as BrdU+ + DAPI+/DAPI+*100%.

For Calcein/PI staining, the two cell lines were inoculated at 5 × 104 cells/well in 24-well plates and incubated for 24 h. TMZ was used to treat the cells for 24 h. Phosphate-buffered saline (PBS) was used to wash the cells three times, after which the cells were subjected to calcein/PI staining for 30 min using a calcein/PI cell viability/cytotoxicity assay kit (C2015S, Beyotime, Shanghai, China). The fluorescence signals were observed and taken under a fluorescence microscope (IX73, Olympus, Japan).

2.5 Transwell assay

The treated glioma cells were harvested, and the cell concentration was adjusted to 1 × 105 cells/mL (serum-free medium). The upper chamber of the Transwell (pore size 8 μm, Corning, Beijing, China) was precoated with Matrigel (BD, San Jose, USA). Then, the transwell assay was conducted to measure cell invasion. 24 h after cell seeding, the cells were fixed by 4% PFA and stained by 0.1% crystal violet (Cat.No.C0121, Beyotime, Shanghai, China). The invaded cells were observed and counted on a light microscope (IX53, Olympus, Japan).

2.6 Western blot

The cells were lysed by RIPA Lysis Buffer (Cat.No.P0013B, Beyotime, China) then the total protein of glioma cells was extracted on ice. The BCA Protein Assay Kit (Cat.No. P0011, Beyotime, China) was used for protein quantitation. 20 μg of total protein was separated by 10% SDS‒PAGE, and transferred to PVDF membranes (Millipore, Billerica, MA). The membranes were subsequently incubated overnight at 4 °C with the following primary antibodies: anti-LDHA (ab52488, Abcam, 1:1000), anti-PKM1/2 (ab137791, Abcam, 1:1000), anti-HK1 (AF1726, Beyotime, 1:200), anti-HK2 (AG2142, Beyotime, 1:200), anti-LAMB1 (ab109293, Abcam, 1:2000), p-NF-κB p65 (ab76302, Abcam, 1:1000), NF-κB p65 (ab16502, Abcam, 1:1000), and anti-GAPDH (ab8245, Abcam, 1:1000). Afterward, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (ab6721, Abcam, 1:2000) for 60 min at 37 °C. The binding was detected using western ECL substrate (Cat.No.P0018S, Beyotime, Shanghai, China), and the signals were exposed to X-films. The original images of the protein bands are shown in the supplementary figure.

2.7 Detection of glucose uptake and lactic acid production

U251 and LN229 cells were seeded in 24-well plates for 24 h. The contents of glucose and lactate in the cell supernatant were estimated by a colorimetric assay. Glucose uptake was tested with Glucose Uptake Assay Kit (ab65333, Abcam) and lactate production was assessed with the Lactate Assay Kit (ab65330, Abcam). The procedures were performed strictly under the instructions of the two kits.

2.8 Seahorse extracellular acidification rate (ECAR)

Glioma cells were inoculated at 5 × 104 cells/well in Seahorse XFe96 cell culture microplates (Agilent, 101,085-004). 10 mM glucose (glycolysis induction), 1 μM oligomycin (maximal glycolysis induction) and 50 mM 2-DG (glycolysis inhibition) were added sequentially according to the protocol of the Seahorse XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, USA). The data were analyzed using Seahorse XFe96 Wave software, and the ECAR was expressed as mpH/min.

2.9 Animal experiments

BALB/c nude mice (16–18 g, 6 weeks old) were obtained from the Animal Experimentation Center of Hebei Province. U251 cells (1 × 106) were inoculated subcutaneously into the axilla of the right forelimb of the mice. Tumor size was checked daily, and the mice were randomly grouped when the tumor grew to approximately 100 mm3. Mice were treated with Bay 11–7082 (i.p., 5 mg/kg, once every two days) as described previously [18], during which the tumor size was gauged every seven days. Tumor volume (mm3) = (length × width2)/2. The volume of tumors in mice should not exceed 2000 mm3 and the diameters are less than 15 mm in any dimension. All mice were euthanized using CO2 asphyxiation and then they were immediately anaesthetized by cervical dislocation. This study strictly followed the 3R principles and all the animal experiments were approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University (Approval No.2022-041).

2.10 Immunohistochemical (IHC) and IF

The tumor tissues formed in the nude mice were embedded in paraffin and then sectioned (4 µm thick) for IHC and IF. The sections were dewaxed, hydrated with ethanol, and incubated with 3% H2O2 to block endogenous peroxidase activity. The sections were blocked in 5% goat serum (Thermo Fisher Scientific) for 30 min at room temperature and then incubated with primary antibodies, including Ki67 (ab15580, Abcam, 1:100), anti-HK1 (AF1726, Beyotime, 1:50), anti-HK2 (AG2142, Beyotime, 1:50), or anti-p-NF-κB p65 (ab76302, Abcam, 1:500), at 4 °C overnight. HRP-labeled sheep anti-rabbit IgG secondary antibody (ab6721, Abcam, 1:500) or Alexa Fluor 488-labeled goat anti-rabbit IgG (H + L) was then added and incubated with the sections for 60 min at RT. For IHC, the sections were washed three times with PBS, subjected to DAB visualization, restained with hematoxylin, and sealed with neutral gum. For tissue IF, the sections were incubated with DAPI for 5 min at RT and washed with PBS twice.

2.11 Statistics analysis

The data were processed with GraphPad Prism 8.0 for plotting and statistical analysis. The results are presented as the mean ± standard deviation (SD). A t test was utilized to analyze the statistical significance between the groups, with a difference of P < 0.05 indicating statistical significance.