Chemicals and reagents

The following commercially available reagents were used for this study: DFO (HY-B0988, MCE), 740 Y-P (B5246, APExBIO), SC79 (HY-18749, MCE), and AS1842856 (B8219, APExBIO).

Animals

C57BL/6 male mice were housed in the pathogen-free barrier facility of Sun Yat-sen University in accordance with institutional guidelines and approved by the regional board. Mice were maintained in a temperature- and humidity-controlled animal facility with a 12-h light/dark cycle and free access to distilled water. The animals were kept in stainless steel mesh wire bottom cages with no bedding material and no exposure to feces. Mice were randomly assigned to one of the following five groups, with nine mice in each group: normal control (NC), iron deficiency (ID), ID + 740 Y-P (10 mg/kg per day by intraperitoneal injection for one month), ID + SC79 (20 mg/kg every 5 days by intraperitoneal injection for one month) and ID + AS1842856 (50 mg/kg per day by gavage administration for one month). All mice were approximately 21 days of age on day 0 of treatment. The mice were fed a control diet (Dyets, 110700, 37 ppm Fe) or an ID diet (Dyets, D115109, approximately 1 ppm Fe) for 8 weeks and were weighed 2–3 times per week from the first day on the diets up to and including the day of sacrifice.

Cell isolation and culture

The isolation and expansion of human MSCs followed established procedures. Bone marrow was collected from healthy donors, and MSCs were isolated through density gradient centrifugation. Subsequently, these cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 atmosphere. The culture medium was refreshed every 3 days. Upon reaching 90% confluence in the flasks, the MSCs were trypsinized using 0.25% trypsin containing 0.53 mM ethylenediaminetetraacetic acid (EDTA) and then reseeded in new flasks for expansion. Notably, all MSCs used in the experiments were utilized before they reached passage 2.

Quiescence and activation induction

For induction of cell quiescence in vitro, MSCs were seeded at a density of 5 × 104 per well in 6-well cell culture plates. Subsequently, the culture medium was replaced with DMEM without FBS. After three days of serum starvation culture, the MSCs were then stimulated with DMEM supplemented with 10% FBS with or without DFO. Unless otherwise specified, 20 µM DFO was used to stimulate MSCs for 24 h. The MSCs were collected and utilized in subsequent experiments.

CCK-8 assay

A total of 1 × 104 MSCs per well were seeded for use in a cytotoxicity assay. At the indicated time points, fresh culture medium supplemented with CCK-8 reagent was added (Dojindo, Japan). After 2 h of culture, the absorbance of the wells was measured at 450 nm. Medium without cells was used as a negative control.

Western blotting analysis

Total protein was extracted from the cells with RIPA buffer containing protease and phosphatase inhibitors. Twenty micrograms of total protein from each sample was resolved by 10% or 15% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE), transferred to a polyvinylidene difluoride membrane (Millipore), blocked in 5% nonfat milk and immunoblotted with various primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Anti-KDM4A (ab191433, 1:5000), anti-KDM4D (ab93694, 1:200) and anti-FTO (ab280081, 1:1000) antibodies were purchased from Abcam. Anti-Akt (4691, 1:1000), anti-phospho-Akt (4060, 1:2000), anti-Foxo1 (2880, 1:1000), anti-phospho-FoxO1 (9461, 1:1000), anti-PIK3R3 (9461, 1:1000) and anti-GAPDH (5174, 1:1000) antibodies were purchased from Cell Signaling Technology.

Quantitative real-time PCR

Quantitative real-time PCR (qRT–PCR) was performed as previously described [59]. Briefly, total RNA was isolated from MSCs using an RNA Quick Purification kit (ESscience) according to the manufacturer’s instructions and was transcribed into cDNA using a PrimeScript™ RT reagent kit (TaKaRa). qRT–PCR was then performed on a Light-Cycler® 480 PCR System (Roche) using SYBR® Premix Ex Taq™ (TaKaRa). Gene expression was calculated by the ΔΔCT method using GAPDH as the reference gene.

In vitro histone demethylation assay

The in vitro histone demethylation assay was performed as previously described [60]. In brief, 5 µg of calf thymus histones (Sigma; H9250) was incubated with 2–4 µg of KDM4D protein in demethylation buffer (comprised of 20 mM Tris-HCl, pH 7.3, 150 mM NaCl, 1 mM α-ketoglutarate, 50 µM FeSO4, and 2 mM ascorbic acid) at 37 °C overnight. Subsequently, the reaction mixtures were resolved on a gel and analyzed by Western blotting utilizing H3K9me3- and H3-specific antibodies.

RNA interference and lentiviral construction

To silence KDM4D expression in MSCs, three KDM4D siRNAs, along with a negative control siRNA, were designed and obtained from Guangzhou IGE Biotechnology, Ltd. Transfection was performed using Thermo Fisher’s Lipofectamine RNAi MAX reagent following the manufacturer’s guidelines. We verified the effectiveness of these siRNAs at both the RNA and protein levels and selected the most efficient siRNAs for subsequent experiments.

For KDM4D overexpression, lentiviruses were constructed by OBiO Technology, along with their corresponding vector controls. MSCs were exposed to lentiviruses at a concentration of 109 TU/mL with 5 µg/mL polybrene during transfection. The transfection process lasted for 24 h, maintaining a multiplicity of infection of 50. Subsequent experiments were conducted on the fourth day following transfection.

RNA sequencing and data analysis

RNA was extracted and used to construct a cDNA library. Subsequently, we conducted RNA sequencing on the BGISEQ-500 platform. The sequencing data were filtered with SOAPnuke (v1.5.2), and the resulting clean reads were mapped to the reference genome GCF_000001405.39_GRCh38.p13. The alignment of the clean reads to the reference coding gene set was carried out using Bowtie2 (v2.2.5), followed by the calculation of the expression level for each gene using RSEM (v1.2.12). For sequencing data analysis, tasks such as heatmap clustering, Venn diagram generation, and KEGG analysis were conducted utilizing BGI Dr. Tom 2.0.

CUT&Tag assay

The CUT&Tag assay was conducted using the NovoNGS CUT&Tag 3.0 High-Sensitivity Kit (catalog no. N259-YH01; NovoProtein). MSCs (1.0 × 105) were subjected to two washes with 0.5 mL of wash buffer. Subsequently, the cells were mixed with ConA beads and incubated with an anti-KDM4D antibody or an anti-H3K9me3 antibody overnight at 4 °C. Next, a goat anti-rabbit IgG antibody (NovoProtein) was diluted at a ratio of 1:500 and added to the sample. The sample was then incubated for 1 h at room temperature (RT). The cells were further washed and subsequently incubated with pAG-Tn5 for 1 h at RT. Afterward, MgCl2 was introduced to activate tagmentation for 1 h at 37 °C. The isolated DNA was processed using NovoNGS DNA Extract Beads and dissolved in TE buffer. DNA was amplified with N5 and N7 primers, which was enriched by PCR to create sequencing-ready libraries. Following PCR, the libraries were purified using NovoNGS DNA Clean Beads, and the library quality was assessed using an Agilent Bioanalyzer 2100 system.

CUT&Tag-seq data analysis

CUT&Tag-seq data analysis was conducted as previously described with slight modifications [61]. In summary, we filtered and trimmed the raw reads to eliminate adaptor sequences and low-quality reads using TrimGalore (version 0.6.6) with the following parameters: -q 20 –phred33 –stringency 3. Clean reads were then mapped to the human genome version hg38 using Bowtie2 (version 2.5.1) with the parameters -p 6 -q, retaining only the uniquely mapped reads. Peak calling was carried out using MACS2 with the following command: ‘macs2 -q 0.05–call-summits –nomodel –shift 100 –extsize 200 –keep-dup all’. To simulate the peaks called per input read, we utilized aligned and deduplicated BAM files without additional filtering. To normalize the CUT&Tag-seq data (in reads per kilobase per million [RPKM]), we used the bamCoverage command and generated heatmaps with the computeMatrix and plotHeatmap commands in deepTools (version 2.3.6.0). The normalized CUT&Tag-seq data were then visualized using the Integrative Genomics Viewer (IGV).

EdU incorporation analysis

To assess DNA synthesis, we used the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime, China) in accordance with the manufacturer’s protocol [62]. The working solution containing EdU was added to the cell culture medium and coincubated with MSCs for 2 h. After fixation and permeabilization, the MSCs were treated with the Click reaction solution for 30 min at room temperature in the absence of light. The cells were then stained with DAPI (Thermo Fisher, USA) for 10 min. All the resulting images were captured using an LSM 5 Exciter confocal imaging system (Carl Zeiss).

Immunofluorescence staining assay

In the cell immunofluorescence assay, MSCs were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100. After the cells were blocked with goat serum, they were incubated overnight at 4 °C with primary antibodies. Following washing, the cells were treated with a fluorescein-labeled secondary antibody (Alexa Fluor® 488; 1:1000) for 1 h and then stained with DAPI (Thermo Fisher) for 10 min.

For the tissue immunofluorescence assay, femurs were fixed in 4% paraformaldehyde and decalcified in a 15% EDTA solution. Subsequently, the samples were paraffin-embedded and sectioned at 5 μm. The sections were dewaxed and subjected to antigen retrieval by overnight heating at 60 °C. They were then permeabilized with 0.5% Triton X solution and blocked with 3% BSA. For immunostaining, the slides were incubated overnight at 4 °C with mouse anti-Ki67 antibody (CST) and rabbit anti-CD105 antibody (Abcam). Afterward, the sections were washed and incubated with Alexa Fluor 555-conjugated goat anti-rabbit IgG (Abcam) or Alexa Fluor 488-conjugated goat anti-mouse IgG (Abcam) for 1 h at room temperature. Subsequently, the sections were washed and further stained with DAPI (Beyotime) to label the nuclei. Finally, the samples were mounted and imaged using an LSM 5 Exciter confocal imaging system (Carl Zeiss).

Flow cytometry

To assess the quiescence-activation status, we collected MSCs using 0.25% trypsin and fixed them with precooled 75% ethanol. Subsequently the MSCs were permeabilized using the FIX & PERM™ Cell Permeabilization Kit (GAS004, Invitrogen, USA) according to the manufacturer’s instructions. The sections were then incubated with anti-Ki-67 eFluor® 450 for 1 h and propidium iodide (PI) for 10 min. Subsequently, we used a FACSCelesta Multicolor Flow Cytometer (BD, USA) for detection of the samples, and the analysis was conducted using FlowJo software version 10. For the evaluation of MSC activation in mice, femurs were collected from sacrificed mice. We trimmed both ends of the femurs with scissors and washed them with PBS using a syringe needle until they turned white. The collected cells were then fixed and permeabilized using FIX & PERM Cell Permeabilization Reagents (Thermo Fisher). Subsequent steps included staining with an eFluor450-conjugated anti-Ki-67 antibody (Thermo Fisher), a fluorescein isothiocyanate (FITC)-conjugated anti-CD45 antibody (BioLegend), phycoerythrin (PE)-conjugated anti-Sca-1 antibody (BioLegend), and allophycocyanin (APC)-conjugated anti-PDGFR-α antibody (BioLegend). Cells that were negative for CD45 and positive for both Sca-1 and PDGFR-α were identified as MSCs in the bone marrow. Ki67-positive cells were further measured to analyze MSC activation in mice.

Hematoxylin and eosin (H&E) staining

The femurs were initially fixed in 4% paraformaldehyde and then decalcified in a 15% EDTA solution. Next, the samples were paraffin-embedded and sectioned into 5 μm slices. The sections were subsequently dewaxed and stained with hematoxylin for 5 min. After a 10-minute PBS wash, the sections were subjected to eosin staining for 3 min. The sections were then dehydrated using ethanol and cleared with a dimethylbenzene solution. Finally, the slides were mounted and examined using a microscope.

Micro-CT analysis

To assess bone mass and microarchitecture across different groups, we utilized a micro-CT scanner, the Inveon MM system (Siemens). The image acquisition involved 360 rotational steps, with a pixel size of 8.82 μm, an 80 kV voltage, a 500 µA current, and an exposure time of 1,500 ms. Subsequently, we calculated key parameters, including BV/TV and Cb.Th. These calculations adhered to the guidelines provided by the American Society for Bone and Mineral Research.

Statistical analysis

Each experiment involved a minimum of three biological replicates. The data are presented as the mean ± standard deviation (SD). For comparisons between two groups, an unpaired Student’s t test was applied. In cases where more than two groups were compared, one-way ANOVA was utilized. GraphPad Prism 8.0 software was used for data analysis. Significance levels are denoted as *P < 0.05 and **P < 0.01.