Cell culture

Mouse osteoblast-like (MC3T3-E1) cells (CRL-2593, American Type Culture Collection, Manassas, Virginia, USA) were cultured in alpha-Minimum Essential Medium. As a model cell line, 293T cells exhibit good transfection efficiency and are better suited for mechanism validation and protein interaction experiments [18]. 293T cells (CL-0005; Procell Life Science & Technology, Wuhan, China) were cultured in Dulbecco’s Modified Eagle’s medium. Cells were incubated with 10% fetal bovine serum and penicillin-streptomycin in an incubator containing 5% CO2 at 37℃. The cells were then cultured in 96-well plates for further treatment. For the establishment of an in vitro osteoporosis model, MC3T3-E1 cells were cultured with D-galactose (D-gal) (25.5 mM) in alpha-Minimum Essential Medium, as previously described [19]. All genetic recombination experiments were approved by Kunshan First People’s Hospital.

Cell transfection

Short hairpin RNA (shRNA) targeting FSP1 (shFSP1), ATF4 (shATF4), and YBX1 (shYBX1) (see Table 1 for details), negative control shRNA (shNC), ATF4 overexpression plasmid (ATF4), and negative control plasmid (NC) were obtained from Fenghbio (Changsha, China). MC3T3-E1 cells were seeded in 24-well plates at a density of 2 × 105 cells/well and transfected with 100 nM shRNA for 48 h to knockdown gene expression using 0.75 µL Lipofectamine 3000 (L3000075, Invitrogen, Carlsbad, CA). After incubation, the cells were treated with D-gal, which induced impaired osteogenesis for subsequent analyses.

Table 1 The shRNA sequencesCell viability assay

Cell Counting Kit-8 (CCK-8) assay kits (Sigma-Aldrich, St. Louis, MO, USA) were used to determine cell viability with or without the ferroptosis inhibitor ferrostatin-1 (Fer-1) (S7243, Selleck Chemicals, TX, United States) or deferoxamine (DFO) (D0160000, Sigma-Aldrich) in MC3T3-E1 cells, with dimethyl sulfoxide (DMSO) as a negative control. Briefly, MC3T3-E1 cells were plated in 96-well plates at a density of 5 × 103 cells/well for 24 h. Fer-1 (1 mg/mL) or DFO (1 mg/mL) was then added, followed by the addition of 10 µL of CCK-8 solution (5 mg/mL) for 2 h at 37 °C. Optical density at 450 nm was measured using a microplate analyzer.

Western blot

The cells were lysed in RIPA buffer containing phenylmethylsulfonyl fluoride and protease inhibitors. After centrifugation at 12,000 rpm, the supernatant was collected, and protein concentrations were measured using a BCA assay kit (Beyotime, Jiangsu, China). A total of 10 µg of protein was loaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis for electrotransfer, followed by blocking with 5% milk and incubation with primary antibodies. The primary antibodies used were ATF4 (ab85049, Abcam, Cambridge, MA, UK), FSP1 (ab197896, Abcam), YBX1 (ab76149, Abcam), and β-actin (ab8227, Abcam). The following day, the membranes were incubated with secondary antibodies (ab288151, Abcam) and visualized using a chemiluminescent horse radish peroxidase (HRP) substrate (ECL).

RNA pulldown

Specific ATF4 biotinylated probes (5’-CTTTCCTCTTCCCTCCCGCAGGGCTTG-3’) and antisense ATF4 biotinylated probes (5’-GAAAGGAGAAGGGAGGGCGTCCCGAAC-3’) were purchased from Roche (11685597910). After incubation with the biotin-labeled probe and Streptavidin Magnetic Beads for 4 h at 4℃ (following the manufacturer’s instructions), the complexes were pulled down using a magnetic stand. A RNeasy Mini Kit (Qiagen, Hilden, Germany), coupled with magnetic beads, was used to purify and extract RNA. Finally, western blot analysis was performed to detect YBX1 protein in the isolated complex.

Luciferase assay

To explore the effects of ATF4 on FSP1 promoter activity, wild-type (WT) or mutated (Mut) FSP1 promoter sequences (please see Table 2 for the sequences and location), constructed by Cyagen Bioscience (Suzhou, China), were inserted into a psiCHECK-2 vector (Promega, Madison, WI, USA). A total of 2 × 105 293T and MC3T3-E1 cells were transfected with the established luciferase reporter plasmids together with 100 nM shNC or shATF4 using Lipofectamine 3000 transfection reagent (Invitrogen). After incubation for 48 h, the luciferase activity of ATF4 on the FSP1 promoter was measured using a Dual-Luciferase Reporter Assay Kit (Promega).

Table 2 The mutant variants sequence of promoters of FSP1 and ATF4

To explore the influence of YBX1 on the m5C locus of ATF4 mRNA (Gene ID: 11911; NM_001287180.1), the fragment of ATF4 mRNA, which was acquired from NCBI database (https://www.ncbi.nlm.nih.gov/home/develop/), including the wild-type (ATF4-WT) or mutant (ATF4-Mut) binding sites with m5C locus (please see Table 2 for the sequences and location), constructed by Cyagen Bioscience (Suzhou, China), were inserted into the psiCHECK-2 vector (Promega). A total of 2 × 105 cells/well of 293T and MC3T3-E1 cells were transfected with the established luciferase reporter plasmids together with ATF4-WT or ATF4-Mut in combination with 100 nM shNC or shYBX1 using Lipofectamine 3000 transfection reagent (Invitrogen). After 48 h of transfection, the luciferase activity of YBX1 on ATF4 mRNA was determined using a dual-luciferase reporter assay kit.

Measurement of lipid ROS and cellular Fe2+

C11-BODIPY 581/591 fluorescent dye (D3861, Life Technologies, Waltham, CA, USA) was used to detect lipid ROS. MC3T3-E1 cells were incubated with 5 µM of C11-BODIPY, followed by incubation for an additional 20 min at 37 °C. MC3T3-E1 cells were then collected by trypsinization, resuspended in 400 µL of PBS, filtered through a 35 μm nylon mesh filter, and subjected to flow cytometry analysis with a 488 nm laser for excitation (BD Biosciences GmbH).

Iron assay kits (ab83366; Abcam) were used to detect cellular Fe2+. Briefly, iron reducer and assay buffer were mixed and incubated with the cells. After incubation with the iron probe, the absorbance was measured using a colorimetric microplate reader (optical density: 593 nm).

RNA stability assay

After shYBX1 transfection for 24 h in the MC3T3-E1 cells, the cells were treated with 5 µg/mL actinomycin D (HY-17559, MedChemExpress, Monmouth Junction, NJ, USA) or DMSO as a control. Subsequently, the remaining RNA was extracted for further RT-qPCR analysis.

RT-qPCR

RNA from the cells was extracted using TRIzol reagent (15596-026, Invitrogen) and reverse-transcribed into cDNA using a reagent kit (Takara Bio, Tokyo, Japan). RT-qPCR was then conducted using SYBR Green Master Mix (Takara Bio, Japan) on a QuantStudio v5 system (Thermo Fisher, Waltham, MA, USA). The comparative threshold cycle (2−ΔΔCt) method was used to analyze the data. The primer sequences are listed in Table 3.

Table 3 RT-qPCR primers information used in the manuscriptRNA immunoprecipitation (RIP)

The Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17700, Sigma-Aldrich) was used for RIP analysis. MC3T3-E1 cells were lysed with RIP buffer to cross-link protein-RNA complexes. After centrifugation, the cell nuclei were collected and resuspended in RIP buffer for mechanical shearing of the chromatin. YBX1 antibodies (ab76149; Abcam, Cambridge, UK) were added to the supernatant with magnetic beads. After elution, the precipitated ATF4 mRNA was analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) using the primers listed in Table 3.

Promoter prediction

The FSP1 (Gene ID: 20198; NM_001410571.1) promoter, that is, the location of the FSP1 transcription start site at 2000 bp upstream and 100 bp downstream (-2000 bp/100 bp), was obtained from the UCSC Genome Browser (https://genome-asia.ucsc.edu/). Through the JASPAR database (https://jaspar.elixir.no/), the binding sites of ATF4 on the FSP1 promoter region were analyzed. Finally, the potential binding sites (BS1 and BS2) of ATF4 on the FSP1 promoter were predicted, and the BS1 and BS2 primers are listed in Table 4.

Table 4 The primer of FSP1 promoters used in ChIP-qPCRChromatin immunoprecipitation (ChIP)

Following the standard ChIP protocol, MC3T3-E1 cells were fixed with 1% formaldehyde, and the supernatant was collected after centrifugation for immunoprecipitation with an anti-ATF4 antibody (ab85049, Abcam) or an IgG antibody (ab205718, Abcam). After precipitation of endogenous DNA-protein complexes with Sepharose and extraction of DNA fragments, the binding of FSP1 promoter fragments to ATF4 was detected by agarose gel electrophoresis.

Statistical analysis

All data are based on three independent experiments and are presented as mean ± standard deviation (SD). SPSS 20.0 was used to analyze statistical significance, which was determined using one-way analysis of variance, followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant (* P < 0.05, ** P < 0.01, and *** P < 0.001).