Patients, cell lines, and cultures
Human GBM tissue samples and normal brain contusion tissues were obtained from the First Affiliated Hospital of Soochow University, Suzhou, China. This study was approved by the Ethics Committee of Soochow University. Human SHG-140 cell lines were obtained from the Department of Neurosurgery & Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, Suzhou, China, after primary culture and identification by STR. T98G cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS).
Antibodies
Anti-USP7 (CST#4833; western blotting, 1:1000; immunoprecipitation, 1:50), anti-BAX (CST#89477; western blotting, 1:1000), anti-CLEAVED-CASPASE 3 (CST#9664; western blotting, 1:1000; immunofluorescence, 1:500), anti-β-tubulin (CST#2146; western blotting, 1:1000), and anti-Ub (CST#3933; western blotting, 1:1000) were purchased from CST, USA. Anti-Bcl-2 (Ab692; western blotting, 1:1000; immunofluorescence, 1:500), anti-ARF4 (Ab171746; western blotting, 1:1000; immunoprecipitation, 1:50), anti-K48 linkage-specific Ub (ab140601; western blotting, 1:1000), and anti-K63 linkage-specific Ub (ab179434; western blotting, 1:1000) were obtained from Abcam (UK). Horseradish peroxidase-labeled goat anti-mouse IgG and goat anti-rabbit IgG (immunofluorescence, 1:1000) were purchased from ZSGB-Bio (China). P5091 (s7132), CHX (s7418), and MG132 (s2619) were purchased from Selleck (USA).
Lentiviral transfection
GeneChem (China) designed two shRNAs against USP7, shUSP7-1: 5’ -UGUAUCUAUUGACUGCCCUTT- 3 ’ and shUSP7-2: 5’ -UGGAUUUGUGGUUACGUUACUC- 3’, and a non-silencing control. Lipofectamine 3000 was used for co-transfection for 8 h, and transfection efficiency was investigated after transfection. Lentiviral shRNA targeting the USP7 gene was generated using the GV112 vector (hU6-MCS-CMV-Puromycin; GeneChem, China). ARF4 overexpression lentivirus was prepared using the GV692 vector (Ubi-MCS- 3FLAG-CBh-gcGFP-IRES-Puromycin; GeneChem). The constructed lentiviral vector was transfected into cells followed by puromycin intervention, and the transfection efficiency was identified by western blotting analysis to screen for stably transfected cell lines.
Immunohistochemistry
Tissues were paraffin-embedded and sectioned for immunostaining. Slides were dewaxed in xylene, then rehydrated in graded ethanol, followed by quenching of endogenous peroxidase activity with 0.3% hydrogen peroxide (China), and strong antigen recovery solution was heated to 37 ◦C to recover the antigen. Non-specific proteins were blocked with 5% goat serum (Solarbio, China). Primary antibodies (1:100 dilution) were used to incubate the sections overnight at 4 ◦C, followed by addition of appropriate biotinylated secondary antibodies (1:100 dilution) (ZSGBBio, China) for 60 min at 37 ◦C. Next, slides were incubated with ABC peroxidase and diaminobenzidine (ZSGBBio) and then counterstained with Mayer's hematoxylin solution (Solarbio) for nuclear staining. For HE staining, slides were deparaffinized and rehydrated. Slides were re-stained by nuclear staining and subsequently using the HE kit (Solarbio). Images were acquired using an inverted microscope (Olympus, Japan).
Western blotting and immunofluorescence analyses
After induction, cells were collected in lysis buffer, incubated on ice for 30 min, and centrifuged at 12 000 × g at 4°C for 10 min. The supernatant was collected and the protein concentration determined. Equal amounts of protein samples were subjected to 8–12% SDS-PAGE, transferred to PVDF membranes, and incubated at room temperature or 1 h using 5% skim milk with primary antibody overnight. Membranes were washed with PBST buffer for 30 min and then incubated with a secondary antibody for exposure. For immunofluorescence, cells induced on slides were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton for 15 min. Slides were incubated at room temperature with 5% BSA for 1 h, BSA was discarded, and mouse anti-BCL2 antibody and rabbit anti-CLEAVED-CASPASE3 antibody were added for overnight incubation. The next day, the primary antibody was discarded and the secondary antibody was incubated with Alexa Fluor 594-labeled goat anti-rabbit (1:200) and Alexa Fluor 488-labeled goat anti-mouse (1:200) for 1 h at 37°C in a light-proof oven at a constant temperature. Fluorescence microscopy and image acquisition were performed under light-proof conditions.
Cell viability assay (CCK-8 assay)
P5091 were added to 96-well plates containing cell suspensions in a total volume of 100 µl. After induction for 24, 48, and 72 h, 10 µl of CCK8 reagent (Dojindo, Japan) was added and incubated at 37°C for 2 h in a constant temperature incubator. Cell viability was evaluated by measuring the difference in optical density values at 450 nm using an enzymatic standard.
Annexin V-FITC/PI flow cytometry for apoptosis detection
After apoptosis induction, cells were digested with 0.25% trypsin and centrifuged at 2 000 × g for 3 min at 4°C, resuspended in pre-chilled PBS, and centrifuged again at 2 000 × g for 3 min at 4°C. The supernatant was discarded and the cell precipitate was added to Annexin V-FITC, PI, and binding buffer (BD, USA). After incubation for 10 min at 4°C protected from light, cell fluorescence was detected by flow cytometry.
Tandem mass tags proteomics analysis
Tandem mass tag (TMT) proteomic analysis was performed as follows. Total protein was extracted for concentration determination by SDS-PAGE, trypsin digestion, and TMT peptide labeling. Equal amounts of labeled samples were mixed and separated by chromatography. Samples were loaded onto a pre-column Acclaim PepMap100 100 µm × 2 cm (RP-C18, Thermo Fisher) at a flow rate of 300 nl/min, and then separated on an analytical column (Acclaim PepMap RSLC, 75 µm × 15 cm; RP-C18, Thermo Fisher). Finally, samples were analyzed by LC-MS/MS with primary MS mass resolution set to 60 000, automatic gain control to 3e6, and maximum injection time to 50 ms. Mass spectrometry scan was set to full scan charge to mass ratio m/z range 350–1500, and the scan was performed for the 20 highest peaks. MS/MS spectra were acquired using data-dependent positive ion, the MS/MS resolution was set to 15000, the automatic gain control to 2e5, the maximum ion accumulation time to 40 ms, and the dynamic exclusion time to 30 s.
Immunoprecipitation (IP) and mass spectrometry
Cells were washed twice with pre-cooled PBS and lysed with cell lysis buffer supplemented with protease inhibitors. After removal of insoluble material by centrifugation at 12 000 × g, pre-lysates were incubated with protein A/G magnetic beads overnight at 4°C. Precipitates were washed three times with lysis buffer, boiled in 1 x SDS sample buffer for 5 min, and proteins resolved by SDS-PAGE on 8 %–12% gels. Immunoblot detection was performed using appropriate antibodies. For MS, immunoprecipitation was performed as described above, and immunoprecipitates were resolved on SDS-PAGE denaturing gels, stained with Coomassie Blue, and then analyzed by MS. MS was performed with a Q-Exactive mass spectrometer (Thermo Scientific). Parent ion scan range was 350–2000 m/z, mass spectrometry was scanned in data-dependent acquisition, and the most intense 20 fragmentation profiles were acquired after each full scan (MS2 scan) by high-energy collision dissociation (HCD), NCE energy at 28, and dynamic exclusion time at 25 s.MS1 resolution at M/Z 200 was 70 000, AGC target was set to 3e6, maximum injection time to 100 ms, MS2 resolution to 17 500, AGC target to 1e5, and maximum injection time to 50 ms.
Nude mouse intracranial xenograft model
Female BALB/c nude mice (4–5 weeks, 15–17 g) were purchased from the Animal Center of the Institute of Oncology, Chinese Academy of Medical Sciences (Beijing, China). A total of 5 × 104 SHG-140 cells with luciferase-encoding lentivirus (GeneChem, Shanghai, China) were stereotactically injected into mice (six per group). The lentiviral vectors were GV260 and Ubi-MCS-Luc-IRES-puromycin. P5091 was dissolved in 20% DMSO, 40% PEG-300, and 40% PBS. Seven days after implantation, mice were injected intraperitoneally with equal doses of 10 mg/kg/day, 5 mg/kg/day of P5091, or PBS 2 days per week during the survival period. Intracranial tumor size was assessed and radiance values recorded on days 7, 14, and 28 using the IVIS Spectral Real-Time Imaging System (Blandford, USA). Live mouse brains were removed, fixed in 4% paraformaldehyde, embedded in paraffin, and subjected to HE and IHC. Animal studies were performed according to internationally accepted norms and national regulations.
Statistical analysis
Statistical analyses were performed with SPSS 16.0 or GraphPad Prism 8.2.1 software. Bar statistical plots were expressed by the mean standard deviation of at least three times the number of experimental replicates. Differences between two groups were assessed by Student's t-test, and differences between multiple groups were tested by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Bars are expressed as mean ± s.d. or mean ± s.e.m. Statistical significance is shown at #P = NS, *P < 0.05,**P < 0.01,***P < 0.001, or ****P < .0001.