Wednesday, September 27, 2023

BCL2L13 promotes mitophagy through DNM1L-mediated mitochondrial fission in glioblastoma – Cell Death & Disease

Ethics statement

All experimental protocols were approved by the Ethics Committee of the Qilu Hospital (Jinan, China) and performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained from all patients. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shandong University (Jinan, China).

Database searches

The Cancer Genome Atlas (TCGA,, Rembrandt (, Oncomine (, the Human Protein Atlas (, and the Cancer Cell Line Encyclopedia (CCLE, were mined for relevant molecular data.

Cell lines and cultures

Human glioma cell lines U251, A172, and LN229 were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China; TCHu58, TCHu138). NHA, GBM#P3, GBM#BG5, luciferase-stable GBM#P3 were kindly provided by Prof. Rolf Bjerkvig (University of Bergen). U251, A172, LN229, and NHA cells were cultured in Dulbecco modified Eagle medium (DMEM; Thermo Fisher Scientific, SH30022.01B) supplemented with 10% fetal bovine serum (FBS, GE Healthcare Life Sciences, 10082147). GBM#P3 and GBM#BG5 cells were cultured in Neurobasal Medium (Thermo Fisher Scientific; Waltham, MA, USA) containing B27 supplement (20 μL/mL), FGF (20 ng/mL) and EGF (20 ng/mL) for cell culture and passage. Culturing medium of GBM#P3 and GBM#BG5 cells were changed into DMEM supplemented with 10% FBS for cell plating and further functional experiments. Cells were maintained at 37 °C in a humidified chamber containing 5% CO2.

BCL2L13 silencing and overexpression

Lentiviral vectors expressing human shRNA targeting BCL2L13 (sh-BCL2L13#1: 5’‐GGA AGA GAG CCU UGU GGA ATT‐3’; sh-BCL2L13#2: 5’‐GGA CAA AGA AAU UUC UGA ATT‐3’; sh-BCL2L13#3: 5’‐CUG CAG AAG AUA GCA AUG ATT‐3’, GenePharma Shanghai) or scrambled‐control (sh-NC) were used to generate stable cell clones expressing sh-BCL2L13 or a nonspecific shRNA as the control. Cells were plated and infected with lentiviruses expressing sh-NC/BCL2L13 for 24 h, according to the manufacturer’s protocol. Cells were transfected with pENTER-BCL2L13 to induce the overexpression of BCL2L13 and with an empty pENTER vector as a control. Transfection was performed with Lipofectamine 3000 (Life Technologies, Carlsbad, CA). Transfected clones were selected using 1 mg/mL of puromycin (Selleckchem). qRT-PCR and western blotting analysis were used to evaluate shRNA knockdown or overexpression efficiency.

Quantitative real-time PCR (qRT-PCR)

Total RNA was prepared from treated cells using Trizol (Thermo Fisher Scientific; MA, USA). Briefly, after centrifugation, the aqueous layer was transferred to a new Eppendorf tube, and isopropanol was added to precipitate total RNA. cDNA was generated from total RNA using the ReverTra Ace qPCR RT Kit (TOYOBO; Osaka, Japan). qRT-PCR was performed with SYBR Green Master (Roche; Basel, Switzerland) on the 480II Real Time PCR Detection System (Roche; Basel, Switzerland). GAPDH mRNA was used to normalize mRNA expression. The results are representative of at least three independent experiments. The sequences of the PCR primers used are the following: GAPDH-F 5’-GCACCGTCAAGGCTGAGAAC-3’, R 5’- TGGTGAAGACGCCAGTGGA-3’; BCL2L13-F 5’-AGGACTATTCGGCAGAGTACAT-3’, R 5’-TGATTCCAGGGTATTCCTCCTC-3’.

Western blot analysis

Cell lysates (20 µg protein) were subjected to western blot analysis, according to previously described protocols. Membranes were incubated with the following antibodies from Cell Signaling Technology: DNM1L (8570), phospho616-DNM1L (4494), phospho637-DNM1L (6319), MFN1 (14739), BECN1 (3495), MAP1LC3B (12741), GAPDH (5174). Additional antibodies were BCL2L13 (Proteintech, 16612-1-AP) and FIS1 (Abcam, ab156865).

Wound-healing assay

Cells in the logarithmic phase were put on a six‐well plate and cultured for 24 h until 90% confluence. After making a vertical wound on the board’s surface with the tip of a P200 pipette, cells were washed several times with PBS to remove cell debris. Then cells were cultured in a serum-free medium. Cell positions were recorded, and migrating rates were measured at 0 and 24 h.

Transwell assay

Matrigel (BD Biosciences, NJ) was obtained to cover the bottom membrane of transwell chambers (24 holes, Corning Inc., NY) and measure cells’ invasive ability. Each transwell membrane enclosed a mixture of Matrigel and medium at the proportion of 1:2 at 50 µl. The upper chamber was inoculated with 2.5 × 104 cells, while the serum, growth factors, and chemokines were placed into the lower chamber and cultured in 5% CO2 at 37 °C for 24 h. Then, chambers were stabilized with paraformaldehyde for 10 min, and 500 µl 0.1% crystal violet was added for 10 min before being washed out. After air-drying, the stained cells were photographed and counted under the light microscope (×200) in four randomly selected fields.

3D Invasion

Cell spheres that were cultured in low-attachment U-bottom 96-well plates were embedded in Matrigel (Trevigen; Gaithersburg, MD, USA) and cultured for 36 h (GBM#P3) or 120 h (GBM#BG5) at 37 °C for the 3D tumor spheroid invasion experiment. The sphere’s diameter was considered the starting point for quantification.


Cells were fixed with 4% glutaraldehyde and post-fixed with 1% OsO4 in 0.1 M cacodylate buffer for 2 h. The samples were then stained with 1% Millipore-filtered uranyl acetate, dehydrated in increasing ethanol concentrations, and infiltrated and embedded in epoxy resin (ZXBR, Spon 812). Electron photomicrographs were taken from GBM cells’ ultrastructures using a transmission electron microscope (JEM-1200EX II, JEOL; Tokyo, Japan).

GFP-LC3B transfection and evaluation

GFP-LC3B (pBABEpuro, 22405)-expressing vectors were obtained from Addgene and deposited by EndoFree Plasmid Maxi Kit (QIAGEN, 12362). Lentiviral supernatants were prepared according to the manufacturer’s instructions and provided by GenePharma. Lentiviral infections were performed accordingly. GFP-LC3B stable GBM cells were fluorescently labeled with 25 nM MitoTracker Red (Invitrogen, Molecular Probes) for mitochondrial morphology. Cells were viewed using an SP8 confocal microscope (Leica, Germany). All images were analyzed by ImageJ software (MD, USA). Autophagy/mitophagy was quantified by calculating the average number of GFP-LC3B puncta/GFP-LC3B, and RFP-mito merged puncta per cell in five high-power fields.

Detection of in vitro mitophagy using mt-Keima fluorescent reporter

Imaging of mt-Keima GBM#P3, GBM#BG5 and A172 cells was performed according to the instruction, using different settings for GFP and red fluorescent protein (RFP). mt-Keima is a ratiometric pH-sensitive fluorescent protein that exhibits green fluorescence (excitation 440 nm) in basic or neutral conditions and red fluorescence (excitation 550 nm) in acidic conditions. For our experiments, the settings used were green channel (excitation 440 nm, emission 570–695 nm) to visualize normal mitochondria, and red channel (excitation 550 nm, emission 570–695 nm) to visualize mitochondria undergoing mitophagy. The mt-Keima GBM#P3, GBM#BG5 and A172 cells were treated as previously mentioned, followed by confocal imaging. Data were quantified, using ImageJ software, as the total number of red pixels divided by the total number of green pixels.

Cell counting kit (CCK)-8 assay

Cell viability was assessed with the cell counting kit-8 (CCK-8; Dojindo, CK04-500). Cells (1.0 × 104 cells/well) were seeded into 96-well plates and incubated at 37 °C overnight. 3-MA (Sigma-Aldrich, M9281) or Baf (Sigma-Aldrich, B1793) were dissolved in DMSO (Sigma-Aldrich, D2650) and diluted to working concentrations in a culture medium. After the desired treatment, cells were incubated for an additional 4 h at 37 °C with 10 μL of CCK-8 in 100 μL of serum-free DMEM. The absorbance at 450 nm was measured using a microplate reader (Bio-Rad, model 680; Hercules, CA, USA).

Intracranial xenograft model

Athymic mice (male; 4 weeks old; ~22 g) were provided by Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). The mice were anesthetized with chloral hydrate and secured on a stereotactic frame. A longitudinal incision was made in the scalp, and a 1 mm-diameter hole was drilled 2.5 mm lateral to the bregma. Luciferase-stable GBM#P3 glioma cells (2 × 105) in 20 μL of serum-free DMEM were implanted 2.5 mm into the right striatum using a Hamilton syringe. Mice were monitored by bioluminescence imaging every week. Briefly, mice were injected with 100 mg luciferin (Caliper, 122796), simultaneously anesthetized with isoflurane, and subsequently imaged with a cooled charge-coupled device camera (IVIS-200, Xenogen; Alameda, CA, USA). Bioluminescence values of tumors were quantified using the Living Image 2.5 software package (Xenogen). Mice were euthanized after 28 days and perfused with 4% paraformaldehyde in PBS. Brains were coronally sectioned for immunohistochemistry assays.


Paraffin-embedded samples were sectioned (4 µm) and mounted on microscopic slides. Heat-induced epitope retrieval was performed in 10 mmol/L citric acid buffer at pH 7.2 in a microwave. Sections were incubated with the primary antibody at 4 °C overnight (LC3B, 1:200, Cell Signaling Technology, 12741 S; Ki67, 1:200, Cell Signaling Technology, 9027; BCL2L13, 1:20, Proteintech 16612-1-AP), rinsed with PBS, and incubated with horseradish peroxidase-linked goat anti-rabbit secondary antibody (ZSGB-BIO, PV-9000). Visualization was achieved using diaminobenzidine (ZSGB-BIO, ZLI-9033) as the substrate, and slides were counterstained with Mayer hematoxylin (Beyotime Biotechnology, C0107).

Statistical analysis

Three independent experiments were performed, and results were expressed as the mean ± the standard deviation (SD). Data were compared using paired Student t tests for two-group comparison and one-way analysis of variance (ANOVA) for multi-group comparisons in GraphPad Prism 8 software (San Diego, CA, USA). Kaplan–Meier survival curves were generated and compared using the log-rank test. P values determined from different comparisons <0.05 were considered statistically significant and are indicated as follows: *P < 0.05; ** P < 0.01; ***P < 0.001.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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