Antibodies and reagents
The antibodies included the following: anti-SQSTM1/p62 (M162-3, Medical Biological Laboratories, Japan), anti-SQSTM1/p62 (ab109012, Abcam, USA), anti-p53 (sc-126, Santa Cruze Biotechnology, USA), anti-p53 (10,442–1-AP, Proteintech, China), anti-mutant p53 (ab32049, Abcam), anti-NRF2 (M200-3, Medical Biological Laboratories), anti-NRF2 (16,396–1-AP, Proteintech), anti-ubiquitin (10,201–2-AP, Proteintech), anti-GAPDH (#5174, Cell Signaling Technology), anti-β-actin (#4970, Cell Signaling Technology), anti-HA (M180-3 and M561, Medical Biological Laboratories), anti-His (D291-3, Medical Biological Laboratories), anti-Flag (M185, Medical Biological Laboratories), anti-Flag (20,543–1-AP, Proteintech), anti-SLC7A11 (NB300-318, Novus, USA), anti-SLC7A11 (26,864–1-AP, Proteintech) anti-HO1 (ab68477, Abcam), anti-HO1 (10,701–1-AP, Proteintech), anti-NQO1 (ab80588, Abcam), anti-Keap1 (10,503–2-AP, Proteintech).
The reagents included the following: Erastin (S7242, Selleck, USA), APR-246 (HY-19980, MCE, USA), Pifithrin-α (HY-15484, MCE), Nutlin-3 (S1061, Selleck).
Cell culture and treatment
Human glioblastoma cell lines (U251, U118, U87 and A172) and HEK 293 T cells were obtained from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in high-glucose DMEM (Gibco, Thermo Fisher Scientific) containing 10% foetal bovine serum (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Erastin was used at 10 μM. APR-246 was used at 10 μM. Pifithrin-α was used at 10 μM. Nutlin-3 was used at 10 μM.
Human tissue samples
Human glioma specimens were collected from the Department of Neurosurgery, Renmin Hospital of Wuhan University, Wuhan, China. Patients were asked to submit written informed consent and were approved by the Institutional Ethics Committee of the Faculty of Medicine at Renmin Hospital of Wuhan University (approval number: 2012LKSZ (010) H). The collected specimen were histologically determined by pathologists at Renmin Hospital of Wuhan University.
DNA construction and transfection
The HA-p62 plasmids were constructed based on the pcDNA3.1. The HA-p62 mutant fragments (Δ1–385), (Δ124–440), (Δ230–440) and (Δ1–227, 245–442) were generated from HA-p62 wild type. “Δ1–385” means that residues 1–385 are retained and other region residues are removed. HA-p53 (wild type) plasmids, flag-p53 (wild type) plasmids, flag-R273H-p53 (mutant) plasmids and HA-NRF2 plasmids were constructed based on the pcDNA3.1. His-ubiquitin and its indicated mutants (His-K48R-Ub, His-K63R-Ub) were constructed based on the pcDNA3.1. Transfections were performed using Lipofectamine 3000 transfection reagent (L3000015, Thermo Fisher Scientific) according to the manufacturer's instructions.
CRISPR/CAS9-mediated SQSTM1/p62 depletion was applied according to Zhang Feng Lab’s protocol (http://www.addgene.org/crispr/zhang/). Two sgRNA targeting SQSTM1/p62 was designed using CRISPRdirect (http://crispr.dbcls.jp/) and the oligo-sequences for used sgRNAs:
oligo 1 (5′-caccgcgttcgctacaaaagccgcg-3′ and 3′-cgcaagcgatgttttcggcgccaaa-5′).
oligo 2 (5′-caccgcgccagctcgccgctcgcta-3′ and 3′-cgcggtcgagcggcgagcgatcaaa-5′).
The designed sgRNA were cloned into lentiCRISPRv2 vector (#52961, Addgene, USA).The constructed vectors were validated by sequencing. LentiCRISPRv2-p62 or lentiCRISPRv2 was co-transfected with the packaging vectors pSPAX2 (#12260, Addgene) and pMD2.G (#12259, Addgene) into 293 T cells using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) to generate lentiviruses. The supernatants containing the lentiviruses were collected and used to infect U251 or U87 cells. Cells were selected with 8 mg/mL puromycin for at least 48 h. Western blot was performed to verify the knockout efficacy.
Total RNA were isolated by using the trizol reagent (Invitrogen, USA), and cDNA was synthesized using a PrimeScript RT Reagent Kit with gDNA Eraser (RR047A, Takara, Japan) according to manufacturer’s guidelines. For quantitative RT-PCR, SYBR® Premix Ex Taq™ II (RR820A, Takara) and CFX96 Real-Time PCR System (Biorad, USA) were used according to the standard protocols. Expression levels were normalized to GAPDH. The primers are listed as follows:
GAPDH: 5′-GGAGCGAGATCCCTCCAAAAT-3′ (Forward),
SQSTM1/p62: 5′-GCACCCCAATGTGATCTGC-3′ (Forward),
SLC7A11: 5′-TCTCCAAAGGAGGTTACCTGC-3′ (Forward),
Cells were grown on coverslips and fixed in 4% paraformaldehyde for 15 min, permeabilized with 1% Triton X-100 in 1 × PBS for 10 min, blocked in 1% bovine serum albumin for 1 h at room temperature. Then cells were incubated with indicated primary antibodies overnight, followed by Alexa fluor-labelled secondary antibody (Antgene, Wuhan, China). Images were captured using Olympus microscope.
Lipid peroxidation analysis
Analysis of lipid peroxidation was performed by Lipid Peroxidation (MDA) Assay Kit (S0131S, Beyotime, China) following manufacturer’s instructions. MDA concentration was normalized to protein concentrations, which were detected by the BCA method with the kit (#AR0197, Boster Biological &Technology, China).
Transmission electron microscopy (TEM)
Cells were fixed with a 2.5% glutaraldehyde solution. The cells were then post-fixed in 1% osmic acid. A graded series of ethanol was used to dehydrate the specimens. They were then placed in capsules contained embedding medium and heated at 70 °C for approximately 9 h. The specimen sections were stained by uranyl acetate and alkaline lead citrate. Images were acquired using a TEM (Hitachi HT7700, Japan).
Tissue samples embedded in paraffin were sectioned, deparaffinized, and subjected to antigen retrieval performed in citrate buffer (pH 6.0). Peroxidase was blocked using the 3% H2O2 for 10 min. After being blocked with 1% BSA for 1 h, the sections were incubated in primary antibodies overnight, followed by HRP-labelled secondary antibody (Servicebio, China). Signals were detected using DAB staining (Servicebio), and the samples were counterstained with haematoxylin. Images were obtained using an Olympus BX51 microscope (Olympus).
Measurement of GSH
The relative glutathione (GSH) concentration in cell lysates was determined by the GSH and GSSG Assay Kit (S0053, Beyotime, China) according to the manufacturer’s instructions.
Total proteins from the cultured cells was extracted using RIPA Lysis Buffer (P0013B, Beyotime, China). BCA assay kit (PC0020, Solarbio, China) was used to determine the protein concentration. Proteins were subjected to SDS-PAGE and transferred onto PVDF membranes. After antigen blocking with 5% non-fat milk, the blots were incubated with primary antibodies overnight at 4 °C, followed by Alex Fluor 680/790-labelled secondary antibodies (LI-COR Bioscience, USA) or HRP-labelled secondary antibodies at room temperature for 1 h. Subsequently, the bands were visualized with LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences) or ChemiDoc™ Touch Imaging System (BIO RAD).
Proteins were lysed in IP buffer (2 mM Tris–Cl, pH 8.0, 0.5 mM EDTA, 150 mM NaCl, 1% NP-40, 50 mM NaF, 2 mM Na3VO4, 4 mM Na pyrophosphate, and 25 μL protein inhibitor/mL). The proteins were incubated with primary antibodies or IgG (Beyotime) for 4 h at 4 °C. Subsequently, 30 μL of Protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology) was added to the mixture and incubated at 4 °C overnight. The immunoprecipitates were washed five times in IP buffer, resuspended in 40 μL of 1.5 × loading buffer, and analyzed by western blot analysis.
Intracranial xenograft model
Cells were stereotactically implanted into the brain of nude mice under anesthesia as previously described . Mice were monitored periodically and sacrificed when they showed severe neurological symptoms and/or obvious weight loss (> 20% of their body weight). For histological analyses, the whole brains of the mice were removed, fixed in formalin and embedded in paraffin. Animal experiments were performed in accordance with animal care ethics approval and guidelines.
Luciferase assay was performed to detect the activity of NRF2 signaling pathway. ARE luciferase reporter (wild-type NRF2 binding sites) plasmids were constructed by Miaoling Biology (Wuhan, China). The pGMLR-TK luciferase reporter plasmid was purchased from Yeasen Technology (Shanghai, China) and used for renilla luciferase reporter as a control. The dual luciferase assay was performed using Dual Luciferase Reporter Gene Assay Kit (Yeasen Technology, Shanghai, China) according to the manufacturer’s instructions. Luciferase activity was calculated by normalizing the luciferase with the corresponding renilla value and represented as relative luciferase activity.
Single-sample gene set enrichment analysis (ssGSEA)
In TCGA-glioma dataset, 587 tumor samples were divided into four subtypes, p53-mutant LGG, p53-Wild-type LGG, p53-mutant GBM and p53-Wild-type GBM, according to the status of p53 mutation. In each subtype, samples were assigned to low or high expression group by the median value of p62 or NRF2. Furthermore, the gene set “WP_Ferroptosis” representing the activity of ferroptosis pathways were obtained from Molecular Signatures Database (http://www.broad.mit.edu/gsea/msigdb/), then the gene set was quantified for their enrichment degrees (enrichment score, ES) within respective in each glioma samples using single-sample gene-set enrichment analysis (ssGSEA) by R package “GSVA”. Difference analysis was performed among low and high expression group in each subtype by R package “ggpubr”, p < 0.05 was considered significant.
According to p53 mutation status and the median of p62 or NRF2 respectively, patients with glioma were divided into p53 mutant + p62/NRF2 low expression, p53 wild-type + p62/NRF2 low expression, p53 mutant + p62/NRF2 high expression and p53 wild-type + p62/NRF2 high expression group. Kaplan-Maier curve were performed by R package “survival” among four groups in p62 or NRF2 respectively to evaluate the association of overall survival (OS) and four groups.
Cell death assays
Cells after indicated treatment were trypsinized, and stained with Trypan blue. Viable cells were then counted using a hemocytometer under a microscope. The blue-stained cells were considered dead.
Statistical significance was determined by Student’s t-test. Survival analysis was performed by the Kaplan–Meier method and the log-rank test. Data are expressed as means ± SEM. P values were calculating by Graphpad prism 5.0, and P < 0.05 was considered significant.