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) in a concentration of 5% CO2 and 37℃ incubator. Ferric ammonium citrate (FAC) and Deferoxamine (DFO) were purchased from Sigma (St. Louis, MO, USA).
Cell viability assay
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 ((100 μM)) and PBS were added to different groups respectively. Then the 96-well plate was assessed at 450 nm.
Plate colony formation
Cells were collected and resuspended in RPMI 1640 medium containing 100 μM FAC and 100 μM DFO, respectively. Then, the cells were transferred to 6-well plates with the density of 500 cells per well and incubated for 14 days. At last, colonies were stained and counted.
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 a co-culture of 100 μM FAC and DFO for 24 hours, cells were stained and photoed by Olympus FSX100 microscope (Olympus, Tokyo, Japan).
Soft-agar colony formation assay
Two percent agar solution was made at a proper temperature combined with culture medium containing 100 μM FAC or 100 μM DFO, respectively. Then the mixed medium was added into 24-well plate, and moved to 4 ℃ refrigerator immediately until it turns into a solid state. 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). Diluted (1:6) Matrigel (BD Bioscience, San Diego, CA, USA) were used to detect the invasion ability of the cells. After 24 hours incubation, the cells on the upper surface (non-migrated or non-invasion cells) were gently removed. The cells on the opposite surface (migrated or invasion cells) were counted and imaged.
Wound healing assay
SAOS-2 and U2OS cells were cultured 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).
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. 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).
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.
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.
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.
Cell transfection
SAOS-2 and U2OS cells were transfected with shRNA. Human SLC25A37 shRNA and human SLC25A28 shRNA sequences were designed by Biomics Biotech (Shanghai, China). We selected the most effectively one according to the results of qPCR and Western blot. The most effective sequence of SLC25A37 shRNA and human SLC25A28 shRNA are listed in supplementary Table 2. Lentivirus medium of shRNA was generated by HEK293T cells. Then it was used to infect the two cell lines. 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.
Mouse xenograft tumor model
The animal experiments in this study was approved by the Animal Ethics Committee of Shanghai Pudong Hospital, in accordance with the National Institutes of Health (NIH) Guide for the animal treatments of Laboratory Animals. 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.
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.