A microarray dataset (accession number GSE33290) was collected from the National Institutes of Health (NIH)-National Center for Biotechnology Information (NCBI)-GEO Dataset databases . Expression data from imatinib treatment cytogenetic responders (R) and non-responders (NR) with CML chronic phase were compared to identify significantly differentially expressed genes. Using the “cluster profiler” package of “R×64 3.4.1” software, differentially expressed genes were subjected to gene ontology (GO)-based enrichment analysis (biological process, cellular component, and molecular function) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses.
Cell lines and cultures
Human KBM5 cells, which are imatinib-sensitive cells, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). KBM5 cells with T315I mutation (KBM5-STI cells), which were provided by Prof. Michael Andreeff (Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA), were used as imatinib-resistant cells. The cells were cultured at 37°C and 5% CO2 in complete media consisting of DMEM (Dulbecco’s modified Eagle medium) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), 100 units/mL of penicillin (Sigma, St. Louis, MO, USA), 100 μg/mL streptomycin, and 1% glutamine (Sigma).
The DRP1 knockdown lentiviral vector (Lv-DRP1-OE) and DRP1 overexpressing lentiviral vector (Lv-shDRP1) were provided by Guangzhou Land Bio Co, Lit (Guangzhou, China). The constructed vectors were confirmed by qPCR and western blotting. Finally, the expression and interference efficiency of the target genes were verified by RT-PCR.
Imatinib (IM, Cell Signaling Technology, Danvers, MA, USA ) was dissolved in DMSO at a stock concentration of 10 nM and stored at -20°C. Mdivi-1 (Abcam, Cambridge, UK) was dissolved in DMSO (Cell Signaling Technology) at a concentration of 50 mM.
Cells were seeded onto six-well plates and grown to 70% confluence. MitoTracker-DeepRed dye (Invitrogen, California, USA) or autophagy detection reagent (Abcam) was used to label the mitochondria or autophagosomes of leukemic cells, at a final concentration of 250 nM, for 30 min at 37°C in a 5% CO2 incubator before harvesting the cells for confocal imaging. Before placing onto slides, labeled cells were washed three times with ice-cold FACS buffer and three times with cold PBS (Gibco), and then suspended at a final concentration of 5×103 cells/mL in ice-cold PBS. The slides were air-dried in the dark at 37°C for 5 min, fixed with 100% methanol (Sigma) at -20°C for 10 min, air-dried in the dark at 37°C for 5 min, and mounted onto slides. The slides were imaged using an Olympus FV3000 confocal microscope (Olympus, Tokyo, Japan).
Transmission electron microscopy
The leukemic cells were stained with uranyl acetate and lead citrate on a microtome (Leica Ultracut; Leica, Wetzlar, Germany) and analyzed with a transmission electron microscope (Hitachi H-7650; Hitachi, Tokyo, Japan) at an accelerating voltage of 60 kV. Micrographs were taken at 7000× or 20000× magnification. Quantification of mitochondrial volume was performed by drawing a contour of the cross-section of each mitochondrion and then calculating the area of the enclosed region using ImageJ software, version 1.50i.
An apoptosis assay was performed using the annexin V-FITC and PI apoptosis detection kit (Dojingdo, Kumamoto, Japan) according to the manufacturer’s protocol. After 48 h treatment with imatinib and/or Mdivi-1, the cells were analyzed using a FACSCalibur flow cytometer (BD Bioscience, San Jose, CA, USA).
Protein lysate preparation and western blotting
Cells were harvested and washed in ice-cold PBS before being lysed in 1× RIPA buffer. The RIPA-lysed samples were quantified using a BCA protein assay kit (ThermoFisher Scientific, Waltham, MA, USA). For western blotting, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes, and sequentially incubated overnight with primary antibodies at 4°C. The PVDF membranes were then incubated with secondary antibodies at 37°C for 2 h and subjected to imaging. Before the analysis of DRP1, the cytosolic and mitochondrial sub-cellular fractions were separated using a mitochondria isolation kit (Beyotime Biotechnology, Shanghai, China). Western blotting was conducted using the following primary antibodies: rabbit anti-human DRP1, cytochrome c, Cleaved-Caspase-9, Cleaved-Caspase-3, p-AKT(Ser473), p-AKT(Thr308), mTOR(Ser2448), P62, LCI/II, CDK5, CAMK2, p-AMPK, and β-actin, which were obtained from Cell Signaling Technology.
Oxygen consumption rate (OCR) measurements
KBM5, KBM5-STI, KBM5-shDRP1, or KBM5-DRP1-overexpressing (KBM5-DRP1-OE) cells were seeded at a density of 3×105 cells per well on Seahorse Biosciences 24-well culture plates (Seahorse Bioscience, Shanghai, China) coated with Cell-Tak solution (1 mg/mL, ACRO Biosystems, Newark, DE, USA) before the beginning of the assay. After serum shock synchronization, the cell medium was exchanged with 500 μL of the testing medium [glucose-free RPMI-1640 medium (Gibco) containing 2% fetal calf serum and 2 mM sodium pyruvate, pH 7.4]. Prior to the measurements, the microplates were incubated in a CO2-free incubator at 37°C for 1 h. Oligomycin (Seahorse Bioscience), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Seahorse Bioscience), and a mixture of rotenone and antimycin A (Seahorse Bioscience) were used for OCR analysis. The compounds were serially injected into the cells to measure mitochondrial respiration.
Total cellular ATP content was determined using a luminescent ATP detection kit (PerkinElmer Life Sciences, Boston, MA, USA), according to the manufacturer’s instructions. The luminescence intensity was measured with a Victor 3 luminometer (PerkinElmer).
Nude mouse xenograft experiment
Six-week-old BALB/c nude mice were purchased from the Laboratory Animal Center of Southern Medical University and housed in the pathogen-free mouse facility of Guangzhou Medical University. All procedures for animal experiments were approved by the Ethics Committee of the Third Affiliated Hospital of Guangzhou Medical University (GD2019-022). Mice were subcutaneously injected with PBS suspensions of KBM5, KBM5-shDRP1, or KBM5-DRP1-OE cells at a density of 1×108 cells/mL. Ten days after injection, mice were randomly divided into eight groups (KBM5, KBM5+IM, KBM5-shDRP1+IM, KBM5-DRP1-OE+IM, KBM5-STI, KBM5-STI+IM, KBM5-STI+Mdivi-1, or KBM5-STI+IM+Mdivi-1). Mice were administered with either IM (100 mg/kg/d) and/or Mdivi-1 (30mg/kg/d) daily when the tumor volume reached nearly 200 mm3. Tumor volumes were measured every 3 days for 25 days. The mice were sacrificed and the tumors were harvested 25 days later.
All experiments were repeated at least three times. Data represent mean ± SEM. GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) was used for all statistical analyses, and P > 0.05 was considered significant. Comparisons between two groups were performed using Student’s t-test (two-tailed) and comparisons between multiple groups were performed by one-way ANOVA. Survival analysis was performed using the Kaplan-Meier method.