Cell culture and treatment
The hFOB1.19 and SaOS-2 osteoblast cell line were purchased from Procell Life Science & Technology. The cells were cultured according to the procedures of American Type Culture Collection (Manassas). hFOB1.19 cells were maintained in α-MEM (HyClone) supplemented with 10% foetal bovine serum (FBS, Biological Industries) and 0.3 g/L G418 (Sigma-Aldrich), whereas Saos-2 cells were maintained in McCoy5A medium (ATCC) supplemented with 15% of FBS. hFOB1.19 cells were cultured in a humidifed incubator with 5% CO2 at 33.4 °C and Saos-2 cells at 37 °C. The medium was replaced three times a week. Iron dextran (Fe, Sigma-Aldrich) was used to simulate an iron accumulation environment in vitro. Erastin (Sigma-Aldrich) was used as positive control for cell ferroptosis. Deferoxamine (DFO, Sigma-Aldrich) and Ferrostatin-1 (Ferr-1, Sigma-Aldrich) were used to inhibit Fe or erastin induced cell death. All samples were collected for relative assays.
Cell proliferation assay
hFOB1.19 osteoblast cells were seeded (103 cells per well) in 96-well plates. After cells were treated with erastin or Fe or/and Ferr-1 or/and DFO, a solution containing fresh medium (90 μl) and CCK-8 reactant (10 μl) (Dojindo) was added to each well, and cells were incubated at 37 °C for 1.5 h in the dark followed by addition of stop buﬀer. Absorbance at 450 nm was then measured using an enzyme-labelled instrument (Thermo). All operations were performed in accordance with the manufacturer's instructions.
The Hepc1-/- mice were used for endogenous iron accumulation mice model in this study . The mice were housed in the specific pathogen free barrier system with normal diet in the animal facility of Soochow University. Genotype was verified by PCR of tail DNA samples. 20-week-old C57BL/6 wild-type mice were administered intraperitoneal injection of 0.1 g kg−1 week−1 of ferric ammonium citrate (FAC, Sigma-Aldrich) for 8 weeks to establish exogenous iron accumulation model . The mouse tissues were collected at 28 weeks old. All animal experiments were approved by the institutional animal care and use committee of Soochow University.
Measurement of hepcidin, ferritin and MDA content
The levels of mouse serum hepcidin and ferritinwere detected using the following ELISA kits: hepcidin (ElabScoemce) and Ferritin (abcam). Human bone and mouse serum malondialdehyde (MDA) content was measured using a kit (Beyotime). All the procedures were performed according to the manufacturers' instructions.
Human bone samples
Patients (11 individuals) admitted to our hospital with hip fractures were divided into two groups as Fer <150 and Fer >150 μg/L in terms of ferritin level. Whole blood samples for ferritin and other clinical indicators were collected from patients within 24 hours of admission. Areal bone mineral density (BMD) in collum femoris was measured by dual-energy X-ray absorptiometry (DXA). All patients were enrolled if the following criteria were met: pertrochanteric fracture and indication of a surgical treatment. Patients who suffered from inflammation, metabolic endocrine diseases, and malignancy and those who received medicines affecting bone metabolism were excluded. Human bone samples were collected from patients who were admitted to our hospital with hip fractures and required a surgical intervention. Bone samples were collected during proximal femoral nails (PFN) for intertrochanteric femur fractures using a special instrument that is 6 mm in diameter. Fresh samples obtained during PFN were divided into two halves for micro-CT scanning, immunofluorescence, MDA content detection, and western blot. Iatrogenic bone loss did not occur from the surgery itself. Informed consent was obtained from each patient before the procedure, and this process was approved by the institutional review board of the Second Affiliated Hospital of Soochow University.
Physical examination data
A total of 214 individuals physical examination results were analysis for blood test. The physical examination information is from Physical Examination Center, the Second Affiliated Hospital of Soochow University.
Mouse femora and fresh human bone samples obtained during surgery were fixed in 4% paraformaldehyde for 48 hours, then scanned and analyzed by micro-CT (SkyScan 1174). The acquisition settings were as follows: X-ray voltage = 50 kV, X-ray = 800 μA, filter = 0.5 mm aluminum, rotation step = 0.7°, and image pixel size = 10.3 μm. After scanning, images were reconstructed using NRecon software. Parameters were performed using CTAn software, and 3D image reconstruction were performed using CTvox software. For the human bone samples, the volume of interest (VOI) was outlined by a rectangular box-sized 1.5 mm × 2.0 mm × 1.5 mm, whereas for mouse bone samples, the VOI (100 slices) was outlined starting from a point ~ 0.7 mm proximal to the distal growth plate and extending 1.0 mm toward the diaphysis. The following parameters of the trabecular bone were calculated: bone mineral density (BMD, g/cm3), the relevant parameters percent bone volume (BV/TV, %), trabecular thickness (Tb.Th, mm), trabecular number (Tb.N, /mm), trabecular separation (Tb.Sp, mm).
Bone immunofluorescence analysis
The human and mouse bone samples used for micro-computed tomography scanning were decalcified in 0.5 M EDTA solution for 4 weeks with the change of solution twice a week. The tissues were then dehydrated using 20% sucrose and 2% polyvinylpyrrolidone solution for 2 week at 4 °C and then embedded in O.C.T. compound (Tissue-Tek). Cryosections (10 μm) were prepared using freezing microtome (Leica, Wetzlar) for immunostaining. The bone sections were washed three times with 0.3% PBST (Triton X-100) and blocked with 5% bovine serum albumin in PBS for 1 hour. Sections were incubated with primary antibody rabbit-anti-human (mouse) Glutathione Peroxidase 4 (GPX4, abcam) at 4 °C overnight. After three washes with 0.3% PBST, the sections were then incubated with fluorescein-conjugated secondary antibody together with nuclear counterstaining dye (DAPI) at room temperature in the dark. After another three washes, slides were mounted with 50% glycerol. The processed sections were visualized and imaged using a Multiphoton Laser Scanning Microscope (Olympus).
Western blot assays
Human and mouse bone samples were ground into powder with the liquid nitrogen and then lysed using RIPA solution containing protease inhibitor (Roche) on ice for 30 min. Cells were washed with PBS and then lysed using RIPA solution containing protease inhibitor on ice for 30 min. Bone and cell lysates centrifuged, and the supernatants were then collected. The collected supernatants were separated by SDS-polyacrylamide gel electrophoresis and blotted onto transfer membranes (Immobilon). Membranes were then incubated with the following specific antibodies: GPX4 (abcam), NADPH oxidase 4 (NOX4, abcam). The membranes were visualized with a solution of 100 mM Tris-HCl, luminal, 30% H2O2, and coumaric acid.
Lipid peroxides and NADPH assays
For lipid peroxides detection, cells were incubated in the dark at 37℃ in serum-free medium containing 2 μM BODIPY 581/591 C11 sensor (Invtrogene) for cellular lipid peroxides measure, and 0.5 μM MitoPeDPP fluorescence sensor (Dojindo) for mitochondrial lipid peroxide content measure, and 500 nM Mito Tracker Deep Red FM mitochondria fluorescence sensor (Invtrogene) for mitochondria measure for 20 min. The cells were washed three times in serum-free medium to remove extracellular sensor and then incubated with Hoechst 33342 (Beyotime) at room temperature in the dark. Cells were immediately examined using a Confocal Microscope (Olympus).
SaOS-2 cells were cultured and treated in 35mm glass bottom culture dish (Cellvis). For NADPH detection, cells transfected with iNap1 sensor (iNap1 sensor was encoded fluorescent indicators for NADPH which was responsive only to NADPH concentrations and not to the NADPH/NADP+ ratio. iNap1 sensor used in this study was provided by profession Yi Yang) using Lipofectamine Max (Invitrogen) in Opti-MEM medium (Gibco), and follow the manufacturer′s protocol for transfection details. Replacement with fresh medium at 4 - 6 hours post transfection. After 24 - 36 hours, remove the medium, rinse the cells twice with 1 mL of PBS for imaging with Confocal Microscope (Olympus).
Mitochondria morphology analysis by transmission electron microscopy
SaOS-2 (hFOB1.19 used in Supplementary Fig. 1) cell pellets were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4. Transmission electron microscopy was performed using standard procedures by Nanjing Medical University Analysis and Testing Center.
Real-time PCR analysis
Total RNA was prepared from cells and mixed in 1 ml TRIzol. RNA was reversed transcribed with the PrimeScript Reverse Transcriptase Kit according to the manufacturer's protocol (Takara). cDNA (2 μg) was used for real-time quantitative PCR using SYBR Premix Ex Taq (Takara). The sequences of RT-PCR primers are listed in Supplementary Table 2.
hFOB1.19 cells were transiently transfected with NOX4 siRNA using GP-siRNA-mate Plus (Genepharma). The sequence of the NOX4 siRNA was as follows: siRNA NOX4 forward, 5′-CCUCAGCAUCUGUUCUUAATT-3′ and reverse, 5′-UUAAGAACAGAUGCUGAGGTT-3′ (GenePharma).
Whole-cell respiration analysis
Mitochondrial respiration was measured using the Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience) according to the manufacturer′s instructions. hFOB1.19 cells were seeded onto an XF24 microplate at 5000 cells/well. The cellular Oxygen Consumption Rate (OCR) was monitored in unbuffered assay medium (Sigma) supplemented with (in mM) 2 GlutaMAX (Gibco), 2.5 sodium pyruvate, and 25 glucose (pH 7.4 at 37 °C), following the sequential addition of oligomycin (1 μM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 500 nM), and rotenone (Rot, 1 μM) and antimycin A (AA, 1 μM) or in KHB buffer (in mM, 111.3 NaCl, 4.7 KCl, 2.0 MgSO4, 1.2 Na2HPO4, 2.5 glucose, 0.5 L-Carnitine). For fatty acid supported respiration, the 2.5 mM sodium pyruvate and 25 mM glucose were replaced with 200 μM palmitate. Basal respiration was calculate by subtracting the OCR in the presence of Rot and AA from that in the non-presence of oligomycin. Maximal respiration was calculated by subtracting the OCR in the presence of Rot and AA from that in the presence of FCCP. Spare respiratory capacity was calculated by subtracting the OCR in the presence of FCCP from that in the non-presence of oligomycin. ATP production was calculated by subtracting the OCR in the non-presence of oligomycin from that in the presence of oligomycin. Proton leak was calculated by subtracting the OCR in the presence of oligomycin from that in the presence of Rot. and AA.
2000 base pair (bp) in the sequence upstream regulatory region of NOX4 were PCRed from humangenomic DNA using the primers P-H-NOX4 forward: ccggtacctgagctcgctagcGAGATAGAGTCTTGCTCTGTCGCA, reverse: cagtaccggattgccaagcttGGAAGCCCGAAGGCCCGG and were cloned into pGL4.17 vector to form a new reporter plasmid NOX4-luc. The 2 mutation sites in the sequence upstream of NOX4 were designed as follows: from 5′-CAGAG-3′ to 5′-GTCTC-3′, respectively, and named as NOX4-M1-luc and NOX4-M2-luc. SaOS-2 cells were plated at 12-well plate and cultured with different treatments. Reporter plasmids were transfected using Lipofectamine Max (Invitrogen). The luciferase activity was measured using the luciferase activity assay kit (Promega).
Iron regulatory protein1 (IRP1) overexpression
Human IRP1 cDNA was subcloned into pcDNA3.1 between the KpnI (5′) and NotI (3′) sites. The pcDNA3.1 and pGL4.1 plasmid vectors were transfected into SaOS-2 cells and measured the luciferase activity.
Cleavage Under Targets and Tagmentation (CUT&Tag) assay
CUT&Tag assay was performed as described previously with modifications using CUT&Tag Kit . hFOB1.19 cells were treated with or without Fe for 24 h, and then washed with warm PBS twice. Then, 105 cells were washed with 500 μl of wash buffer, and centrifuged at 600 g for 3 min at room temperature. Cell pellets were resuspended with 100 μl of wash buffer. Concanavalin A-coated magnetic beads were added and incubated at room temperature for 10 min with slow rotation. Bead-bound cells were resuspended in 50μl of cold antibody buffer. Then, 1 μg of primary antibody (mouse monoclonal anti-IRP1 antibody, mouse monoclonal anti-IRP2 antibody, Santa Cruz) or normal mouse IgG was added and incubated at room temperature for 2 h with slow rotation. The primary antibody was removed using a magnet stand. Secondary antibody 0.5 μl was diluted in 50 μl of Dig-wash buffer and cells were incubated at room temperature for 1 h. Cells were washed three times with Dig-wash buffer to remove unbound antibodies. The Hyperactive pG-Tn5 Transposase adapter complex (TTE mix) was diluted in 100μl of Dig-300 buffer. Cells were incubated with 0.04 μM TTE mix at room temperature for 1 h. Cells were washed three times with Dig-300 buffer to remove unbound TTE mix. Cells were then resuspended in 300μl of tagmentation buffer and incubated at 37 °C for 1 h. To terminate tagmentation, 10 μl of 0.5 M EDTA, 3 μl of 10% SDS and 2.5 μl of 20 mg/ml Proteinase K were added to 300 μl of sample and incubated at 50 °C for 1 h. DNA was purified using phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation as well as RNase A treatment.
For library amplification, 24 μl of DNA was mixed with 1 μl of TruePrep Amplify Enzyme, 10 μl of 5× TruePrep Amplify Enzyme buffer and 5 μl of ddH2O, as well as 5 μl of P5 and P7 primers from TruePrep Index Kit V2 for Illumina (Vazyme). A total volume of 50 μl of sample was placed in a Thermocycler using the following program: 72 °C for 3 min, 98 °C for 30 s, 20 cycles of 98 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s, 72 °C for 5 min and hold at 4 °C.
To purify the PCR products, 60 μl 1.2× volumes of VAHTS DNA Clean Beads (Vazyme) were added and incubated at room temperature for 5 min. Libraries were washed twice with 80% ethanol and eluted in 22μl of ddH2O. Library DNA concentration was calculated with a Qubit dsDNA HS Assay kit (Invitrogen) and a Qubit Fluorimeter. Library DNA (20 μg) was used for real-time quantitative PCR using SYBR Premix Ex Taq (Takara) with forward primer, 5′-TCGCCCCTAGACAAAGGGG-3′ and reverse primer, 5′-TACCCAGAGCCGGGTTTTC-3′.
Data were presented as mean ± standard deviation (SD). Statistical analysis was carried out as described in each corresponding figure legend. For comparisons between two groups, we used two-tailed Student’s t tests. For comparisons among multiple groups, we used one-way analysis of variance (ANOVA). P values of 0.05 or less were considered statistically significant. Graphical presentation and statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, USA). All representative experiments were repeated at least three times.