Proteomic analysis

Biospecimens were collected from newly diagnosed patients who underwent surgical resection (The First Affiliated Hospital of Wenzhou Medical University, Zhejiang, China). All sample collection procedures complied with routine clinical practice. Protein sample preparation and liquid chromatography-tandem mass spectrometry (LC–MS/MS) using the Tandem Mass Tag (TMT) were performed at PTM Biolab Co., Ltd. (Hangzhou, Zhejiang, China). Briefly, cells were harvested to fetch whole proteins, which were further proceeded to digestion. After the trypsin digestion procedure, peptides were labeled by TMT/iTRAQ according to the manufacturer’s protocol for TMT kit/iTRAQ kit. The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length). These peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. MS data were processed using Proteome Discoverer 1.3.

Plasmids, reagents, and antibodies

SelK knockdown plasmid and its control plasmid, as well as β-TrCP1 knockdown plasmid and its control plasmid, were purchased from Open Biosystems Company (Huntsville, USA). The PEGFP-CDK4 over-expression plasmids were constructed on-site as described in previous studies [46, 47]. HA-SKP2 over-expression plasmid, HA-β-TrCP1 over-expression plasmid, HA-β-TrCP1-C-term plasmid, HA-β-TrCP1-N-term plasmid, GFP-CDK4-N-term plasmid and GFP-CDK4-C-term plasmid were constructed on-site. MG132 and cycloheximide (CHX) were bought from Calbiochem (San Diego, CA, USA). Antibodies specific against SelK (PA5-52,529) were purchased from Invitrogen (Grand Island, NY, USA). Antibodies specific against CDK2 (Sc-6248), CDK4 (Sc-260), CDK6 (Sc-177), cyclinD1 (Sc-20044), cyclinE2 (Sc-481), GFP (Sc-9996), E6AP (Sc-166689), GRP78 (Sc-13539), and SMURF2 (Sc-518164) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific against β-TrCP1 (11984S), SKP2 (2652S), P-PERK (3179S), HA (3724S), Myc (2276S), ATF4 (11815S), and P-eIF2α (Ser51,3597S) were obtained from Cell Signaling Technology (Boston, MA, USA). Antibodies specific against IRE1 (DF7709) and P-IRE1 (Ser 724, AF7150) were obtained from Affinity. Antibody specific against GAPDH (10,494–1-AP) was obtained from Proteintech (Chicago, IL, USA).

Western blot analysis

Aliquots of cells (106) were lysed on ice for 30 s-1 min using cell lysis buffer Boiling Buffer (containing 10% SDS, 100 mM Na3VO4, 1 M Tris–HCl [pH7.4]), followed by sonication. After centrifugation for 10 min, the concentration of protein in the supernatant was measured using a NanoDrop One system (Thermo Fisher Scientific, USA). Each protein sample was homogenized, and equal amounts of protein were loaded into each well on 10% or 12% SDS-PAGE gels and the proteins were then resolved. Gel contents were then electrotransferred to a PVDF membrane. Each membrane was then incubated in a solution of TBS (Tris-buffered saline, pH 7.4) containing 5% skim milk powder for 60 min at room temperature. Dedicated membranes were generated for each protein of interest to negate any need for membrane stripping for re-analysis of another protein.

Each membrane was then placed in a solution of TBST (TBS-0.1% Tween-20) bearing a specific primary antibody (at manufacturer-recommended dilution) and incubated overnight at 4 °C with gentle shaking. The following day, each membrane was rinsed three times with TBST, then placed in a solution of 5% skim milk containing the specified secondary antibody (at manufacturer-recommended dilution) and incubated at 4 °C for 4 h. After a final rinsing with TBST, each membrane was treated with developing solution to visualize all bound antibody. In all cases, glyceral-dehyde-3phosphate dehydrogenase (GAPDH) expression was assessed to monitor for gel loading. Ultimately, all membranes were scanned using a Typhoon FLA 7000 phosphor imager system (GE Healthcare). All protein expression values were then normalized to GAPDH levels.

Clinical specimens, cell culture, and transfections

A total of 88 clinical GB samples were provided by the First Affiliated Hospital of Wenzhou Medical University (Zhejiang, China). Collection of all samples had been approved by the Clinical Research Ethics Committee of The First Affiliated Hospital of Wenzhou Medical University (permission: 2023-R262). Astrocytes NHA were purchased from ScienCell (San Diego); human GB cells U87, A172, LN229, and U251 cells were bought from ATCC (Manassas, VA). All lines were confirmed by STR typing without errors. U87 and U251 cells were cultured in minimum essential medium (MEM; Gibco, #11,095–080). NHA, A172, and LN229 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, #11,995–065). All media contained 10% fetal bovine serum (FBS, Gibco, #10,437–028). All cells were cultured in 37 °C incubators with 5% CO2. All stable cell lines were generated by lentiviral infection and infection for ectopic expression. Lentiviral infection was performed when the HEK293 cell density reached 70–80%, using transfection reagent 1.2 μg psPAX2 (Addgene, #12,260), 1.2 μg pMD2.G (Addgene, #12,259), and 2.0 μg target plasmid transfected into HEK293 cells. After 48 h, the medium containing viral particles was collected and centrifuged, and the supernatant was filtered and used to infect cells. Ectopic expression infection was a PolyJet™ DNA in vitro Transfection reagent (SignaGen Laboratory, SL100688) for transfecting plasmid into LN229 and U251 cells, and G418 (4000-5000ug/mL for LN229, 1000–2000 5000ug/mL for U251, Santa Cruz, sc-29065) was used to select cells stably expressing corresponding resistance constructs.

Soft agar colony formation assay (soft agar)

A lower gel (containing 0.5% soft agar in 10% FBS-basal medium Eagle [BME]) was prepared first in the bottom of 6-well culture plates; after it solidified, an upper gel (10% FBS-BME containing 0.33% agar) containing the quantitative cells (i.e., 104 cells) was overlayed. The plates were then incubated at 37 °C in 5% CO2 for 2–3 wk. Soft agar experiments were performed in three wells of a 6-well plate, with five independent areas photographed from each well using a microscope equipped with a camera; all clones containing > 32 cells were counted and analyzed. Representative images were selected based on the clone count results.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from 5 × 105 cells using TRIzol reagent (Invitrogen, #15596018). After quantification of the total RNA concentration using a BioDrop μLite spectrophotometer (BioDrop, Cambridge, UK), cDNA was generated by PCR using reverse transcription reagents (Takara, #RR037A). All procedures were performed according to manufacturer instructions. Then each sample was subjected to three wells in a 384-well plate and analyzed using the Q6 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The qPCR procedures were described in detail in a previous study [48], and the primers used are noted in Supplementary Table 3.

Nude mouse xenograft model

This study was approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University. BALB/c nude mice (female, 3–4 wk-of-age) were obtained from GemPharmatech (Nanjing, Jiangsu, China) and housed in an SPF-level experimental area maintained at 20–26 °C with a 40–70% relative humidity at the Animal Center of Wenzhou Medical University. All mice had ad libitum access to standard rodent chow and filtered tap water. Mice were housed for ~ 1 wk and then randomly allocated into three groups for treatments with Nonsense, shSelK#1 and shSelK#2, respectively (n = 6/group).

LN229 and U251 knockdown and control cells were stably transfected and identified for animal experiments. Into the right flank of each mouse, a total of 3 × 106 LN229 (Nonsense), LN229 (shSelK#1), LN229 (shSelK#2), U251 (Nonsense), U251 (shSelK#1) and U251 (shSelK#2) cells were subcutaneously injected into nude mice. After ~ 3 wk, when the tumor had grown to an appropriate size, all the mice were euthanized. At autopsy, the tumors were removed, measured in volume and weighed, and then photographed.

ATP assay

Cells were seeded into 96-well plates at a density of 1.5 × 103 cells per well, with each cell type represented in five wells. The cells were cultured in medium containing 0.1% FBS at 37 °C for 12 h. Subsequently, the medium was removed and replaced with medium containing 10% FBS for a varying number of days. After the defined incubation period, the medium in each well was removed, and equal amounts of ATP reagent (G7572, Promega) and PBS (phosphate-buffered saline, pH 7.4) were added to each well. The reagents were mixed, and after 10 min at room temperature, the mixture was measured for total fluorescence in a Centro LB 960 luminometer (Berthold Technologies, Berthold, Germany), and each assay was repeated 3 times independently.

Ubiquitination assay

Instantaneous transfection of ubiquitin and substrate granules into cells in a 3:1 ratio. After 24 h, ubiquitin was co-expressed with the substrate in cells; at that point, 10 μM MG132 was added and the cells were incubated a further 8 h. Co-immunoprecipitation was then performed as described above.

Intracellular Ca2+ concentration measurement

Cells were harvested from their culture dishes and prepared as suspensions in 1.5 ml tubes. Each tube then received 500 μL HBSS and 0.5 µl (5 mM) Calbryte™ 630 AM (AAT Bioquest, Sunnyvale, CA, USA) and was incubated for 1 h at room temperature, according to manufacturer instructions. The cells were then washed twice with HBSS to remove excess dye and were immediately analyzed using the CytoFLEX flow system three times to measure fluorescence intensity.

EdU assay

For these studies, cells were evaluated using an EdU Assay Kit (Ribobio, Guangzhou, China; #c10310-2). In brief, LN229 and U251 cells (104 cells/well) were plated into 96-well plates, with each cell type seeded in three wells. After 24 h of incubation, the medium in each well was removed and replaced with 100 μL EdU medium (50 μM) and incubated for a further 2 h. The cells were then fixed with 50 μL 4% paraformaldehyde/well, and then treated with 50 μL of a 2 mg glycine/ml solution. The cells were then treated with 100 μL kit-provided penetrant (PBS containing 0.5% TritonX-100), and incubated for 10 min at 37 °C. Thereafter, 100 μL kit-provided Apollo® staining reaction solution was added to each well. The cells were then examined using a fluorescence microscope and images captured for quantification of EdU staining (following manufacturer protocols and scoring recommendations).

Protein degradation experiment

Aliquots of cells (80–90%) were inoculated into 6-well plates and incubated in medium containing 0.1% FBS for 12 h, followed by 8 h of incubation first in a medium containing 10 μM MG132/10% FBS. The cells were then treated with a medium containing 50 μg cycloheximide (CHX)/ml and 10% FBS for 0, 6, 12, or 24 h or for 0, 3, 6, 12 h. Finally, the cells were collected and their proteins were isolated for Western blot analyses.

Flow cytometry

Aliquots of the test cells (106 cells in 2 ml medium) were inoculated into 6-well plates and incubated—sequentially—with medium containing 0.1% FBS for 12 h and then with medium bearing 10% FBS for 12 h. After that, the cells were removed from the plates using trypsin and transferred into Eppendorf tubes wherein they were then fixed overnight at 4 °C with 70% pre-cooled alcohol. Then, after pelleting the cells, to each tube, 40 μL of RNase A and 360 μL of PI were then added and the cells were incubated at room temperature for 30 min. The cells were then examined for cell cycle status in a CytoFLEX system (Beckman Coulter) for three times. The steps were described in detail in our previous study [49].

Immunohistochemistry

Immunohistochemical (IHC) staining was used to analyze levels of specific proteins in the 88 clinical GB samples and in mouse samples. A commercial kit (Boster Bio, #SA1022) that contained 3% H2O2, 5% BSA, rabbit secondary antibody, SABC (Strept Avidin–Biotin Complex), and DAB (3,3’-Diaminobenzidine) was used in conjunction with specific primary antibodies against SelK (Invitrogen, #PA5-52,529), Ki67 (Abcam, #ab16667), β-TrCP1 (Abcam, #ab233739), or CDK4 (Proteintech, #11,026–1-AP) for IHC staining according to manufacturer instructions. Each tissue section was analyzed with a single primary antibody to avoid mischaracterization of total staining intensity (due to use of the single type of secondary antibody). All stained samples were ultimately evaluated for staining intensity using a Nikon Eclipse Ni microsystem (DS-Ri2) and Image-Pro Plus (v.6.0, Media Cybernetics, Rockville, MD, USA) to capture images and to calculate the integrated optical density (IOD) of each stained area (IOD/area).

Co-immunoprecipitation

Target plasmids were transiently transfected into cells at a 1:1 ratio. After 36 h of transfection, at which the proteins were co-expressed in the cells. The cells were then lysed on ice using cell lysis buffer (Cell Signaling Technology, #9803) supplemented with a complete protein inhibitor mixture (Roche, #04693116001). After high-speed centrifugation (13,000 × g) for 10 min, each supernatant was collected, and protein concentration was determined using a BCA kit (Thermo Fisher Scientific, #23,225). For immunoprecipitation, equal amounts of labeled magnetic beads, i.e., anti-HA-tagged mAb magnetic beads (MBL, M180-11) and anti-GFP-tagged mAb magnetic beads (MBL, D153-8), were separately incubated with an equal amount of lysate protein for 4–5 h at 4 °C. Thereafter, the beads were washed several times with cell lysis buffer to remove unbound protein. The final precipitate was then rinsed with cell lysis buffer and re-suspended in cell lysis buffer and boiled for 5 min. The supernatant from this solution was then collected using an MBL magnetic rack system and the isolated/separated protein then analyzed by Western blotting.

Statistical analysis

Experimental data are presented as means ± SD. All data were processed and plotted with Prism software (v.6.0, GraphPad, San Diego, CA). Results from technical replicates are presented as means ± SD, with no statistical analysis performed. In contrast, results from biological replicates are also presented as means ± SD, and statistical analyses were conducted to compare differences; for each endpoint evaluated, differences between the two groups being compared at a given time were evaluated using a Student’s t-test. A p-value ≤ 0.05 was taken as statistically significant.