Reagents and primary antibodies

Primary antibodies against NPRL2, Gal-3 and GAPDH were obtained from Santa Cruz Biotechnology (CA, USA). TRIM16, CTR1, HSP70, ferredoxin 1(FDX1), dihydrolipoamide S-acetyltransferase(DLAT) were purchased from Thermo Fisher Scientific(HK, China). Total-ERK1/2, phospho-ERK1/2(p-ERK1/2), CD8, HA-tag, Flag-tag and His-tag were bought from Abcam(MA, USA). MG132 and IgG were purchased from Beyotime Biotechnology(Shanghai, China). Elesclomol, cycloheximide(CHX) and ERK1/2 activator BAY2965501(α-ERK) were purchased from MedChem Express(NJ, USA). The recombinant mouse His-tag Gal-3 protein was obtained from Abcam, CuCl2 was purchased from Sigma(Shanghai, China).

Cell culture

Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 (DMEM/F12), RPMI-1640 and fetal bovine serum (FBS) were purchased from Gibco(CA, USA). Two mouse glioma cell lines(GL261 and CT-2 A) were obtained from the Shanghai Life Academy of Sciences Cell Library(Shanghai, China) and cultured in DMEM/F12 plus 10% FBS. CD8+T cell isolation from mice was approved by the Ethics Committee of Chongqing Medical University. As described previously, the spleens of mice were collected after anesthesia via injection of 2% pentobarbital into the abdomen. After being minced and digested, the spleen fragments were added to Ficoll–Paque (Sigma, MO, USA) and centrifuged(500 ×g, 30 min, 20℃), the mononuclear cell layer was extracted. The CD8+T cells were purified via magnetic activated cell sorting (MACS) according to manufacturer’s protocol(Miltenyi Biotec, Germany), cultured in RPMI-1640 supplemented with 10% FBS, and activated by 2 µg/ml anti-CD3/anti-CD28 antibodies(BD Biosciences, NJ, USA) for 48 h [23]. All the cells were maintained in 5% CO2 at 37℃.

Cell transfection

The NPRL2 overexpression lentiviral vector(LV-NPRL2) and corresponding negative control lentivirus (NC), as well as the lentiviral TRIM16 shRNA vector(TRIM16-shRNA-LV) and matched empty lentivirus (NC1) were purchased from Genechem(Shanghai, China).The HA-tag UB plasmid was purchased from Miaoling Biology(Wuhan, China), and the Flag-tag TRIM16 plasmid was purchased from Dowobio Biotechnology(Shanghai, China). Cell transfection and transduction were performed as previously described according to the manufacturer’s protocol [5]. The medium from two glioma cell lines in control, NC and LV-NPRL2 groups cultured for 48 h was collected as conditioned medium(CM), such as CM-control, CM-NC and CM- LV, respectively.

Real-time PCR

Total RNA was extracted from two glioma cell lines using RNAiso Plus (Invitrogen, CA, USA). The concentrations of RNA samples were measured via a spectrophotometer, and the RNA was reverse-transcribed into cDNA via the Primescript RT reagent Kit (TaKaRa Biotechnology, Beijing, China). The primer sequence for Gal-3 was as follows: forward 5’-AACACGAAGCAGGACAATAA CTGG-3’ and reverse 5’-GCAGTAGGTGAGCATCGTTGAC-3’. For GAPDH was: 5’-CATCACTGC CACCCAGAAGACTG-3’ and reverse 5’-ATGCCAGTGAGCTTCCCGTTCAG-3’. The amplification conditions were as follows: 95℃ for 20 s, followed by 40 cycles at 95℃ for 15s, and 60℃ for 60s. Relative fold-changes in mRNA levels were determined via the 2-ΔΔCT method [5].

Western blot(WB)

WB analysis was performed as previously described [24]. Briefly, the cells were collected and lysed in RIPA lysis buffer containing 0.1% phosphatase inhibitor and 1%PMSF. The 30 µg protein samples were separated by SDS–PAGE and transferred onto PVDF membranes. The primary antibodies were used to incubate with PVDF membranes overnight at 4℃, including NPRL2(1:200), Gal-3(1:500), GAPDH(1:1000), TRIM16(1:200), HA(1:100), Flag(1:100), His(1:100), p-ERK1/2(1:200), total-ERK1/2(1:500), CTR1(1:200), HSP70(1:200), DLAT(1:300) and FDX1(1:400). PVDF membranes were incubated with the secondary antibodies(1:5000) for 1 h at 37℃, and the protein in each band was quantified using Quantity One 4.6 computer software.

Enzyme-linked immunosorbent assay (ELISA)

The medium was collected after culturing with glioma cells for 48 h. Then, Gal-3, interleukin-2(IL-2) and interferon gamma(IFN-γ) levels were calculated according to optical density values by an enzyme-labelled instrument. ELISA was performed following manufacturer’s instructions.

Coimmunoprecipitation (Co-IP)

The cells were lysed on ice using NP40 buffer containing a protease inhibitor. Total protein(60 µg) was divided equally. 30 µg of protein was used as input, and the other 30 µg was put into protein G-agarose beads(Thermo Fisher Scientific, MA, USA) which were pretreated with 2 µl of capturing antibodies, such as IgG, Gal-3, Flag or CTR1. After overnight incubation at 4℃, the beads were washed, and the final samples were used for WB analysis.

Ubiquitination assays

The ubiquitination of Gal-3 was required for transfection with HA-tag UB plasmid, and the cells were treated with 10µM MG132 for 6 h before collection. Then, the cells were lysed and subjected to Co-IP via anti-IgG or anti-Gal-3 agarose beads and subsequent SDS-PAGE. Ubiquitination of Gal-3 was detected using HA antibody(1:100).

Immunofluorescence(IF)

Glioma cells were seeded onto coverslips for attachment overnight. The CD8+T cell suspension was dropped onto slides coated with poly-lysine, and additional medium was added after 4 h for culturing these cells overnight. Then, the cells were fixed, blocked, and incubated with primary antibodies(against Gal-3 at 1:200, TRIM16 at 1:200, His at 1:100 and CTR1 at 1:100) overnight at 4℃. Next, the cells were incubated with FITC and TRITC-labelled secondary antibodies together with DAPI for IF.

Cell counting kit-8(CCK-8)

CD8+T cells were seeded into 96-well plates at a density of 4000 cells/well. After incubation with recombinant mouse Gal-3 protein, CuCl2, elesclomol or treatment with different CM for 24, 48–72 h, CCK-8 buffer(10 µl) was added to each well for 20 min. Absorbance values were calculated using an enzyme labelled instrument (450 nm).

Flow cytometry(FCM)

After incubation with the Gal-3 protein for 48 h, the CD8+T cells were harvested, resuspended and stained with Annexin V-FITC/PI apoptosis reagent kit(KeyGen Biotechnology, Nanjing, China). Early apoptosis was observed by flow cytometry.

Copper microplate assay

The copper assay kit(Abcam) was used to detect copper content according to user’ manual. The CD8+T cells(2 × 106) were harvested and lysed to analyze copper concentration via an enzyme labelled instrument (360 nm).

Intracranial tumor model

Following previous studies, C57BL/6 male mice (5–6 weeks) were used to establish brain tumor model [23]. After anesthetization and sterilization on a stereotaxic instrument, the mouse glioma cells were injected at 1.25 mm right lateral and cranium 0.7 mm anterior to the bregma as well as at a depth of 2.5 mm from the brain surface. The tumors were created and collected after 21d.

Patients and clinical specimens

The 93 paraffin-embedded high-grade glioma(grade III-IV) tissues, including 38 anaplastic astrocytoma and 55 glioblastoma multiforme samples, were obtained from the Second Affiliated Hospital of Chongqing Medical University between 2017 and 2023. All the patients’ clinical information is listed in Table 1.

Table 1 Clinical characteristics of 93 patientsImmunohistochemistry(IHC)

Paraffin sections were deparaffinized in xylene and rehydrated in ethanol at a descending concentration. Next, the sections were treated with 3% hydrogen peroxide, the antigens were retrieved in citrate buffer, the nonspecific binding was blocked with 5% goat serum for 35 min. Then, the tissues were incubated with NPRL2(1:100), TRIM16(1:100), Gal-3(1:200) and CD8(1:100) antibodies overnight at 4℃. After incubation with secondary antibodies at 37℃ for 40 min, the tissues were stained with diaminobenzidine and counterstained with hematoxylin. The isotype controls, IgG from the same species of primary antibodies, were processed along with the samples. No apparent immunoreactivity was observed in isotype controls. Finally, the slides were examined under a microscope(DM6000 B; Leica, Wetzlar, Germany). The NPRL2, TRIM16, Gal-3 and CD8 positive cellular percentages in each sample were estimated as follows: Five nonoverlapping fields of vision were selected randomly in one slice, and the numbers of positively expressed cells(brown stain) and total cells(blue stain) were calculated to obtain the ratio of positive cells to total cells in each field of vision. The average of five ratios was considered as percentage of positive cells in one patient.

Statistical analysis

Significant differences were identified using t-Test, ANOVA, Kruskal-Wallis multiplex analysis, and Pearson correlation coefficients. The analyses were performed by SPSS 25.0, and P < 0.05 was considered as the statistical significance.