Cell lines and reagents
Human osteosarcoma cell line SAOS-2 and U2OS were obtained from American Type Culture Collection (ATCC, USA). Both cell lines were cultured in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) in a concentration of 5% CO2 and 37℃ incubator. Ferric ammonium citrate (FAC) and Deferoxamine (DFO) were purchased from Sigma (St. Louis, MO, USA).
Mouse xenograft tumor model
The animal experiments in this study was approved by the Animal Ethics Committee of Zhejiang University School of Medicine and in accordance with the National Institutes of Health (NIH) Guide for the animal treatments of Laboratory Animals. Twelve BALB/c nude mice were randomly divided into 2 groups. A number of 2 * 106 SAOS-2 cells resuspended in 200 µl FBS-free RPMI 1640 medium was injected subcutaneously into the arms of nude mice (6-week-old, female). Tumor volumes were measured every two days. Deferoxamine (DFO) 16mg / kg or normal saline (NS) was injected intraperitoneally for 2 weeks. Deferoxamine (DFO) was purchased from Sigma (St. Louis, MO, USA). After 2 weeks, all animals were sacrificed.
Cell viability assay and cell cycle analysis
Cell Counting Kit-8 (CCK-8; Beyotime, Shanghai, China) was obtained from Beyotime and be used to detect the viability of two cell line according to the manufactories’ protocol. FAC (100 μM), DFO ((100μM)) and PBS were added to different groups respectively. Then the 96-well plate was assessed at 450 nm 24 hours, 48 hours and 72 hours after incubation. For the cell cycle assay, a total of 1 x 106 cells nearly 80% confluent from 6-well plate were collected and fixed in 70% ethanol for 24 hours at 4 ℃ like previously reported 27. Cells were then washed in Phosphate Buffer Saline (PBS) and stained with propidium iodide (PI) before being analyzed by flow cytometry (BD Biosciences). G1, S and G2/M phases were calculated by Modfit Software (Verity Software House, Inc.).
Plate colony formation
Cells were collected and resuspended in RPMI 1640 medium containing 100μM FAC and 100μM DFO, respectively. Then, the cells were transferred to 6-well plates at the density of 500 cells per well and incubated for 14 days. The culture medium in different groups were replaced every 2 days until the end of this experiment. Finally, colonies were stained by 1% Giemsa stain solution (Solarbio, Beijing, China) for 30 min. All of the colonies were counted and quantified.
EdU cell proliferation assay
Edu Cell Proliferation Kit (Beyotime, Shanghai, China) was used to detect the proliferation of two cell lines visually according to the manufactories’ protocol. After being stimulated by 100 μM FAC or DFO for 24 hours respectively, cells were stained and captured by Olympus FSX100 microscope (Olympus, Tokyo, Japan).
Soft-agar colony formation assay
Two percent agar solution was made in room temperature and sterilized by pressure cooker in 120℃ for 6 hours, then it was stored in 4℃ in the refrigerator. Microwave oven was used to heat the solid agar until it was become liquid at 37℃. Two percent of agar solution mixed with culture medium containing 100 μM FAC or 100 μM DFO was made respectively. Then the mixed medium was added into 24-well plate, and moved to 4 ℃ refrigerator immediately until it turns into solid. Then cell suspension containing each type of cell was added on to the top of solidified agar (500 / 50μL). Thereafter, 2 % agar solution containing each conditional medium (1:6 v/v) along with Matrigel (1:30 v/v). After 2 weeks of culture, colony numbers were counted.
Trans-well assay
Trans-well chambers (Corning Costar, Cambridge, MA, USA) were used to detect the migration and invasion ability of cells. In brief, cells were cultured in FBS-free medium overnight. Then it was transferred to the upper chambers (105 cells / well). Culture medium containing 100μM was added into the lower chamber, followed by a 24-hour incubation. The next day, cells on the upper surface (non-migrated cells) were gently removed by small sticks. The cells on the opposite surface (migrated cells) were counted and imaged. For the invasion assay, diluted (1:6) Matrigel (BD Bioscience, San Diego, CA, USA) was used to cover the surface of upper chamber before cells being planted on it. Other steps were the same as migration assay.
Wound healing-scratch assay
SAOS-2 and U2OS cells were
d in 12-well plates. After cells were confluence, wound area was made in cell monolayer by pipette tips. FBS-free RPMI 1640 medium containing 100 μM FAC or 100 μM DFO were added and incubated for 24 hours. The wound closure was captured and the percentage of arear was evaluated by ImageJ software (USA).
Seahorse XF24 respirometry assay
Seahorse Bioscience Extracellular Flux Analyzer (XF24, Seahorse Bioscience Inc., North Billerica, MA, USA) was used to detect the oxygen consuming rate (OCR), and extracellular acid rate (ECAR). Mito Stress Test Kit from Agilent was used according to the manufacturer’s protocol. Briefly, 1*106 cells were seeded in the 24-well plate in conditional culture medium and incubated overnight. Then the cells were washed with XF media (1% FBS) then cultured in a CO2-free incubator at 37°C for 2 h. ECAR and OCR measurements were performed. OCR and ECAR were measured in a typical 8 minutes cycle of mix (2 to 4 min), dwell (2 min), and measure (2 to 4 min).
Mitochondrial extraction
Mitochondrial isolation and extraction were performed according to the manufacturers’ protocol. Mitochondria/Cytosol Fractionation Kits (ab65320, Abcam). Briefly, cells were harvested after culturing in different conditional medium for 24 hours. A number of 5*107 cells was centrifuged at 600 x g for 5 minutes at 4 ℃. Cells were resuspended with cytosol extraction buffer mix after washing with cold PBS. Then the cells were incubated 10 minutes and performed the task with grinder on ice.
Iron assay
Iron assay was performed according to the manufacturers’ protocol of Iron Assay Kit (ab83366, Abcam) as previously described 21. In brief, samples were incubated for 30 minutes at 25 ℃, followed by an incubation of 60 min with iron probe at 25℃. Then all the samples were moved to microplate reader.
Generation of RNAi stable cell lines
Human SLC25A37 shRNA and human SLC25A28 shRNA sequences were designed by Biomics Biotech (Shanghai, China). Non-specific shRNA (NS) was used as control. Briefly, HEK293T cells were transfected by lentivirus-shRNA. After 48 hours of incubation, culture medium containing lentivirus was used to infect SAOS-2 and U2OS cells lines. Lipofectamine 3000 was used in the transfection procedure. The transfection procedure was performed according to the manufacturers’ protocol of Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Puromycin (2μg/ml) was used to screen stable cell lines. Knockdown of SLC25A37 and SLC25A28 were confirmed by qPCR and Western blot. The most effective sequence of SLC25A37 shRNA and human SLC25A28 shRNA are listed in supplementary Table 2.
ROS production detection
Dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime, Shanghai, People's Republic of China) was used to detect ROS production according to the manufacturers’ protocol. Briefly, 5 * 105 cells were planted in the 6-well plate in different conditional culture medium (100 μM FAC or 100 μM DFO) for 24 h. at the day of measurement, then the culture medium was removed. Next, FBS-free medium with DCFH-DA was added to the dish and then incubated for 20 minutes. The fluorescence intensity of cells was detected by microplate reader.
TCGA database and analysis
The correlation of mitochondrion-related genes and Warburg genes was analyzed by GEPIA web tools (http://gepia.cancer-pku.cn/) based on the TCGA database.
Western blot analysis
Cells were collected after stimulated with 100 μM FAC or 100 μM DFO for 24 hours. Cellular proteins were extracted by RIPA lysis buffer containing protease and phosphatase inhibitors. SDS-PAGE was used to separate the proteins. After running process, gels were transferred to PVDF membranes and immersed in primary antibodies. The next day, membranes were incubated with secondary antibodies and be visualized by chemiluminescence detection kit (Beyotime). Slc25a28 antibody (ab90170 , 1:100) was from Abcam. antibodies specific for SLC25A37/ Mitoferrin1 (26469-1-AP, 1:100) and Glut1 (66290-1-Ig , 1:100) were purchased from Proteintech. Anti-phospho-AMPK (Thr172) antibody (#2535S, 1:100), Anti-AMPKα Antibody (#2532, 1:100), anti-p70-S6K (9202S, 1:100), anti-phospo-p70-S6K (Thr389) (9234S, 1:100), anti-Hexokinase 2 (2867S, 1:100), anti-phospho-4EBP1 (Thr70) (13396, 1:100) and anti-4EBP1 (9644s, 1:100) were from Cell Signaling Technology. Anti-PCNA (2586S, 1:100) was from Cell Signaling Technology. The anti-GAPDH antibody (BM1623, 1:1000), anti-β-actin antibody (BM0627, 1:1000), anti-α-tubulin antibody (BM1452, 1:1000), anti-rabbit IgG-HRP antibody (BA1054, 1:5000), and anti-mouse IgG-HRP antibody (BA1050, 1:5000) were purchased from Boster Biological Technology (Wuhan, China).
RNA extraction and qRT-PCR
Cells treated with 100 μM FAC or 100 μM DFO for 24 hours were collected for RNA extraction. RNeasy Mini Kit (Qiagen, Valencia, USA) was used to extract the RNA. Then it was reverse-transcribed into cDNA. The mRNA expression levels were assessed by qRT-PCR system (Applied Biosystems, Foster, CA, USA). The primers we used are listed in supplementary Table 1.
Statistical analysis
All experimental data was presented as the mean ±SD (n ≥ 3). GraphPad Prism (version 7, GraphPad Software, La Jolla, CA, USA) was used to analyse the data. Student's t-test was used between treated and control group. One-way ANOVA was used for multiple groups. LSD-t test was applied when data needed to be compared with control in multiple groups. P<0.05 was considered to be significant.