Cell culture and transfection

The human astroglial normal cell line HEB (ScienCell, USA) and glioma cell lines LN229 (AW-CCH094, Abiowell, China), HS683 (AW-CCH053), U251MG (AW-CCH034), TJ905 (AW-CCH361), and SNB-19 (AW-CCH343) were cultured in DMEM medium containing 10% high-quality FBS and 1% penicillin–streptomycin.

Cells were grown in six-well plates at a density of 5 × 104/cm2. After 24 h, transfections were performed using Lipofectamine 2000 (11,668,500, Invitrogen, USA) with oe-BTRC, oe-AQP4, oe-nuclear factor of activated T cells 5 (NFAT5), and si-NFAT5 (HonorGene, China) into LN229 and U251MG cells, either individually or in combinations. The cells were then gathered for collection after 48 h post-transfection.

Reverse-transcription quantitative real-time PCR (RT-qPCR)

Total RNA from cells was extracted using TRIZOL reagent (15,596,026, ThermoFisher, USA), then it was reverse transcribed into cDNA with a HiFi-Script cDNA first strand synthesis kit (CW2569, CWBio, China). Fluorescent quantitative PCR was conducted using an UltraSYBR Mixture kit (CW2601, CWBio). Data was then analyzed through the 2−ΔΔCt method and normalized to β-actin (Table 1).

Table 1 Primer sequences for RT-qPCR amplificationWestern blot (WB)

Cell and tissue proteins were extracted with Lysis Buffer (P0013B, Beyotime, China). The concentration of protein was measured using a BCA protein concentration assay kit (PC0020, Solarbio, China). Proteins from the samples were separated on a 10% SDS–polyacrylamide gel and then transferred to a nitrocellulose filter membrane. The membrane was then sealed with 5% skim milk powder (AWB0004, Abiowell) and incubated with the corresponding primary antibody (Table 2) at 4 ℃ overnight. The membrane was incubated with HRP-labeled secondary antibodies (Table 2) at room temperature. The antibody binding band was detected using a SuperECL Plus hypersensitive luminescent solution (K-12045-D50, Advansta, USA).

Table 2 Antibody informationCell counting kit 8 (CCK8)

Cells were inoculated into a 24-well plate at a density of 5 × 103 cells/well, with each well containing 300 μL. After transfecting the cells with the appropriate plasmid, a 30 μL/well CCK8 solution (AWC0114a, Abiowell) was added to each well. After incubation for 4 h, the OD values at 450 nm were detected by a microplate reader (MB580, China) of Shenzhen Huisong Technology Development Co., Ltd.

Apoptosis detection

The apoptosis of cells was detected by a specified kit (KGA1107, KeyGen, China). Cells were digested with pancreatic enzymes, washed with PBS, and centrifuged for collection. They were then suspended in 500 μL of Binding buffer and mixed with 5 μL of Annexin V-APC and 5 μL of Propidium Iodide. Following 10 min at room temperature, flow cytometer (A00-1-1102, Beckman, USA) was employed to measure the reaction.

Scratch wound healing

In 6-well plates, cells were seeded at a density of 5 × 105 cells per well and incubated until they became confluent. A 250 μL pipette tip was used to make a scratch. Afterward, the medium was taken out, and the cells were rinsed with 500 μL of PBS. Then, a serum-free DMEM culture solution was introduced. Immediately after changing the medium, images were captured at 0 h using an inverted microscope (DSZ2000X, Cnmicro, China). After incubating at 37 ℃ and 5% CO2 for 24 and 48 h, images were retaken, and the distance of the scratch area was measured using the ImageJ software.

Transwell

Cells were digested and then adjusted to a density of 2 × 106 cells/mL using a serum-free medium. The Transwell (3428, Corning, USA) was precoated with Matrigel, then 100 μL of cells were seeded in the upper chamber, while 500 μL of complete medium (10% FBS) was added to the lower chamber. After incubation for 48 h, the upper chamber was removed and cleaned with PBS. The cells were then fixed with a 4% paraformaldehyde solution (N1012, NCM, China) for 20 min and subsequently stained in crystal violet solution (AWC0333, abiowell) for 5 min. Ultimately, the cells on the membrane's upper surface were examined using a microscope.

Co-immunoprecipitation (Co-IP)

Cells were lysed using an IP lysis buffer. The primary antibody anti-NFAT5 (ab3446, Abcam) was added to the cell lysate and co-incubated at 4 ℃ overnight. Anti-IgG (B900610, Proteintech) was used as a negative control. Cell lysate incubated with the antibody overnight was then added to pretreated Protein A/G agarose beads and gently shaken at 4 ℃ for 2 h to allow for conjugation with the Protein A/G agarose. The protein complex was then washed with IP buffer. Loading buffer was added to the obtained precipitated protein and boiled, and WB analysis was performed with BTRC (28,393-1-AP, Proteintech) and NFAT5 as target primary antibodies, respectively.

Cycloheximide (CHX) chase assay and ubiquitination assay

To investigate the regulatory relationship between BTRC and NFAT5, cells were transfected with oe-BTRC or oe-NC and treated with a 30 μM protein synthesis inhibitor CHX (T1225, TargetMol, China). Cells were collected at various time points (0, 2, 4, 6, 8 h) and were subjected to WB analysis using NFAT5 as the primary target antibody.

The ubiquitination modification of NFAT5 by BTRC was detected by transfecting cells with oe-BTRC or oe-NC and treating them with 10 μg/mL proteasome inhibitor, MG132 (Y16782, Biolab, China) for 4 h. The pretreated cell samples were then lysed with IP lysis buffer. Following this, cell lysates were immunoprecipitated using anti-NFAT5 (ab3446, Abcam) primary antibodies and Protein A/G agarose beads. Finally, WB was analyzed with Ubiquitin (10,201–2-AP, Proteintech) and NFAT5 as the primary target antibodies.

Chromatin immunoprecipitation (ChIP)

The transcriptional regulatory relationship between NFAT5 and AQP4 was confirmed by a ChIP assay using a ChIP kit (ab500, Abcam). Briefly, the cells were fixed with a final concentration of 1.1% formaldehyde solution for 10 min. The DNA of the cells was fragmented by sonication. The ultrasonicated supernatant was incubated with NFAT5 (ab3446, Abcam) primary antibody and agarose gel magnetic beads. Finally, the chromatin fragments were quantified by real-time PCR.

Subcutaneous tumor model

Male BALB/C nude mice (4 weeks old, SJA, China) were maintained under standard conditions of a 25 °C room temperature and a 12-h light/dark cycle. Food and water were freely provided. LN229 cells were co-transfected with lentivirus oe-NC + oe-NC (the NC group), oe-BTRC + oe-NC (the oe-BTRC group), oe-NC + oe-NFAT5 (the oe-NFAT5 group), or oe-BTRC + oe-NFAT5 (the oe-BTRC + oe-NFAT5 group), and stable cell lines were established. After adapting to the laboratory conditions for 1 week, 100 µL of 5 × 106 cells were subcutaneously injected into the right wing of each mouse. The tumor volume of the mice was recorded every 3 days. The formula for calculating the tumor volume is V = (length × width2) / 2. 21 days later, 150 mg per kilogram of pentobarbital solution was injected intraperitoneally into each nude mouse. After complete anesthesia, the mice with dislocated vertebrae were euthanized, and the tumors were extracted and weighed for subsequent analysis. All animal experiment protocols were approved by the medical ethics committee at our hospital.

Immunohistochemical staining (IHC)

After the paraffin sections of the tumor tissue were dewaxed and dehydrated, an appropriate dilution of the anti-Ki67 (AWA10320, 1:400, Abiowell) primary antibody solution was added and left at 4 °C overnight. The anti-rabbit-IgG antibody-HRP complex was incubated at 37 °C for 30 min. The DAB (AWA10320, AWB0175) working solution was added and incubated at room temperature for 1–5 min. Hematoxylin was used for counterstaining for 5–10 min. Neutral gum was used for mounting. Finally, the positive rate of Ki67 was observed under a microscope.

Data analysis

The statistical analysis was performed using GraphPad Prism 8.0. An unpaired t-test was used between the two groups. A one-way or two-way analysis of variance (ANOVA) was used for the multi-group comparison, followed by Tukey's post hoc test. A P-value of < 0.05 indicated statistical significance.