Animal models

C57BL/6 J mice were procured from Hunan SJA Laboratory Animal Company (Hunan, China). Pclaf-floxed mice were generated by inserting two flox sequences at the terminals of Pclaf exon 3. For genotyping, genomic DNA was extracted from tail tips, and the primers of genotyping for Pclaf-floxed mice were: Pclaf-L-loxp-F: CACATGATTCTGGGTTCAATCTCT; Pclaf-L-loxp-R: GACCTTATTCGTGCCACAACACAT; Pclaf-R-loxp-F2: TTGCAATCCTCTGCCTCGACT; Pclaf-R-loxp-R2: GTGGATTCGGACCAGTCTGA. Lyz2-Cre mice were purchased from the cyagen company. The primers for Lyz2-Cre mice were: Lyz2-cre-F1:CTTGGGCTGCCAGA ATTTCTC; Lyz2-cre-R1: CCCAGAAATGCCAGATTACG; Lyz2-cre-F2:CTTGGGC TGCCAGAATTTCTC; Lyz2-cre-R2:TTACAGTCGGCCAGGCTGAC. The myeloid-cell-specific Pclaf knockout mice were generated by interbreeding Pclaf-floxed mice and Lyz2-Cre mice. Pclaf-floxed littermates were utilized as controls.

All mice were maintained in a standard, specific pathogen-free facility at the Laboratory Animal Research Center of Central South University, with a controlled temperature (22–24 °C), a 12 h dark/light cycle (07:00 to 19:00 light on), and ad libitum access to standard food (Hunan SJA Laboratory Animal Company, China) and water. Environmental enrichments were provided to ensure their well-being. Mice were used for in-house mating to generate the required number of animals for experiments as indicated in the figure legends. Only male mice were used. All animal care protocols and experiments were reviewed and approved by the Animal Care and Use Committees of the Laboratory Animal Research Center at Xiangya Medical School of Central South University.

BMSCs isolation

Bone marrow cells were flushed from tibias and femurs with a 1 mL syringe by using ice-cold α-MEM, then dispelled into single cell and seeded in culture 10 cm dish. The adherent cells were then incubated with phycoerythrin (PE)-, FITC-, peridinin chlorophyll protein (PerCP)- and allophycocyanin (APC)-conjugated antibodies that recognized mouse Sca-1 (BioLegend, 108108, 1:100), CD29 (BioLegend, 102206, 1:100), CD45 (BioLegend, 103132, 1:100), and CD11b (BioLegend, 101226, 1:100) for 20 min at 4 °C. The Sca-1+ CD29+ CD45- CD11b- cells were sorted as BMSCs by FACS (BD Biosciences), and were used for the further study.

Bone marrow monocytes and macrophages isolation

Bone marrow cells were extracted from male mice tibias and femurs and cultured in α-MEM with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin overnight. The floating cells were then collected to obtain monocytes and macrophages through adding 50 ng/mL M-CSF (R&D Systems) in the culture medium. To obtain preosteoclasts and mature osteoclasts, the monocytes and macrophages were then incubated with 30 ng/mL M-CSF and 60 ng/mL RANKL (462-TEC-010, Novus Biologicals) for 8 days. Following this, Alizarin Red staining were performed as described previously.4 The cells were fixed with 4% paraformaldehyde and TRAP (G1492-4, solarbio) staining was performed to detect osteoclastic differentiation according to the manufacturer’s instructions.

Osteogenic differentiation assay

To induce osteoblastic differentiation, BMSCs were cultured in 6/12-well plates with culture medium consisting of α-MEM supplemented with 10% fetal bovine serum, 0.1 mmol/L dexamethasone, 10 mmol/L beta-glycerol phosphate, and 50 mmol/L ascorbate-2-phosphate, and cells were incubated for 21 days. Following this, Alizarin Red staining were performed as described previously.44 In brief, cells were fixed with 4% paraformaldehyde and stained them with 2% Alizarin red solution (Sigma-Aldrich). After images capture, 10% Hexadecylpyridinium chloride solution was added to the culture dish, and the supernatant was collected and measured OD value at 405 nm.

Adipogenic differentiation assay

To induce adipogenic differentiation of BMSCs in vitro, BMSCs treated with α-MEM containing 10% fetal bovine serum, 0.5 mmol/L 3-isobutyl-1-methylxanthine, 5 μg/mL insulin, and 1 μmol/L dexamethasone and cells were incubated for 10 days. Following this, cells were fixed with 4% paraformaldehyde and Oil Red O (Sigma-Aldrich) staining was performed to detect lipid in mature adipocytes.

Transwell experiments

BMSCs were isolated and induced for adipogenic differentiation for 10–14 days as described above. Then they were seeded at density of 105 cells per well in 12-well plate in α-MEM with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin. BMMs were acquired as described above. And they were transfected with Pclaf-OE and control plasmid for 24–48 h. Next, Transwell (Corning, 3401) permeable polycarbonate membrane 0.4 μm pore size Transwell inserts were added to wells intended for co-culture. Above BMSCs-differentiated BMAds were seeded to each Transwell inserts. After 48 h, BMAds were collected for further examinations.

BMAds isolation

Mature BMAds were isolated directly from bone marrow of mice according to previously described protocols.16,69 Briefly, femurs and tibias were harvested from the mice, and the bone ends were trimmed. The bones were then placed in a small microcentrifuge tube (0.6 mL) that had been cut open at the bottom. This assembly was inserted into a larger microcentrifuge tube (1.5 mL). Fresh bone marrow was extracted by quick centrifugation. The collected bone marrow was treated with the RBC lysing buffer, followed by centrifugation (3 000 r/min, 5 min, RT). The adipocytes, forming a top layer, were carefully collected from the tube and washed three times with PBS.

Cell transplantation

For the validation of adipocytes persistence, bone marrow adipocytes were isolated according to previously described protocols16 and labeled with HBAD-EGFP adenovirus. Afterwards, they were transplanted into femurs of 3-month-old mice. After 1 month, femurs were collected to detect the immunofluorescence intensity of EGFP to reflect the persistence of transplanted adipocytes.

For transplantation, bone marrow adipocytes were isolated from AAV-FABP4-ShAdgrl2, AAV-Scramble mice and C57BL/6 mice. After being treated with rPCLAF or PBS for 3 days, they were transplanted into 3-month-old mice. Recipient mice were anesthetized, and a longitudinal incision was made on the front of the right knee to expose the patellar tendon. A 27 G needle was carefully inserted through the patellar tendon, positioned between the condyles of the femur, and gradually advanced with a twisting motion until reaching a depth of 2–4 mm within the bone. Confirmation of successful penetration was indicated by slow but consistent bleeding. Cell suspensions containing BMAds (3 × 103) in 20 μL PBS were then slowly injected into the medullary space of the femur. The injection site was immediately sealed with bone wax, and the skin was sutured to complete the procedure.

Colony formation assay

To perform the colony formation assay, cells were detached and centrifuged to obtain a cell pellet. The pellet was resuspended, enumerated, and adjusted to a concentration of 1 × 105 cells/mL. This solution was then further diluted to a final concentration of 1 × 103 cells/mL. An appropriate amount of the cell suspension was plated in a 6-well plate containing 4 mL of culture medium. The cells were uniformly dispersed and incubated in a 5% CO2 incubator for 2–3 weeks until colonies could be seen with the naked eye. Once visible, the cell culture was terminated, and the culture medium was discarded. To fix the colonies in place, the cells were treated with methanol for 15 min and then stained with crystal violet for an additional 10 min. The number of colonies visible to the naked eye was counted, and the colony rate was calculated as (colony number/number of seeded cells) x 100%.

Recombinant PCLAF treatment

Human recombinant PCLAF (rPCLAF) was obtained from Sinobiological (Beijing, China, 10997-H07E). For animal studies, rPCLAF was administered intramedullary at a dose of 0.2 mg/kg/week bilaterally for one month. For cell experiments, rPCLAF was dissolved in PBS and applied at the indicated concentration and time.

Intramedullary injection of adeno-associated virus

Recombinant adeno-associated serotype 8 viruses with F4/80 promoter for Pclaf overexpression in BMMs (AAV-F4/80-Pclaf) was purchased from Company (Shanghai, China). The AAV of Fabp4 promoter driven Adgrl2 knockdown was generated by replacing the Adgrl2 transcript sequence with shRNAs target for Adgrl2. An AAV-empty vector served as the control.

The experimental procedure involved depilation of hair near the knee joints of mice followed by making an incision on the matching skin around the joint. Subsequently, microscissors were employed to separate the muscle tissue and tweezers were used to displace the tendon towards the left side. Thereafter, a 29-gauge insulin syringe was carefully inserted into the bone marrow cavity from the distal femur, and 5 μL of 1.8 ×1012 μg/mL was infused into the bone marrow cavity. Subsequently, the muscle and tendon were repositioned to their original state, and the skin was sutured using a continuous stitch.

ELISA

Bone marrow supernatants and bone marrow adipocytes supernatants were centrifuged at 12 000 r/min for 10 min to remove cellular debris. The ultrafiltration tubes are used for liquid concentration. Whole blood samples were centrifuged at 3 000 r/min for 10 min to get serum. For preparation of bone marrow supernatant, we exposed bone marrow of euthanized mice after cutting two ends of tibias and femurs and placed the samples for centrifugation for 15 min at 3 000 r/min and 4 °C to obtain bone marrow supernatants, which we then stored at −80 °C.

ELISA measurements were conducted using kits for PCLAF and CTX from CUSABIO (china), CCL2, TNF-α, IL-1b, IL-6, IL-8 form Multi Sciences LTD (Hangzhou, China), OPN, PINP from Abbexa (United Kingdom) according to the manufacturer’s instructions.

Human bone marrow samples

Human bone marrow samples were obtained from 20 male patients with bone fracture, with ages ranging from 50 to 70 years. Human bone marrow aspiration and collection were conducted by the Orthopedic Surgery Department at the Xiangya Hospital of Central South University. Prior to the study, all participants underwent a thorough screening process which included a detailed questionnaire, medical history review, and physical examination. Those with conditions that could affect bone metabolism, such as kidney or liver diseases, parathyroid or thyroid disorders, diabetes mellitus, hyperprolactinemia, oophorectomy, rheumatoid arthritis, ankylosing spondylitis, malabsorption syndromes, malignant tumors, hematological diseases, or previous pathological fractures within the past year were excluded from the study. Participants who had received treatment with glucocorticoids, estrogens, thyroid hormone, parathyroid hormone, fluoride, bisphosphonate, calcitonin, thiazide diuretics, barbiturates, or antiseizure medication were also excluded. The bone marrow aspiration and collection were performed during bone fracture surgery for the remaining participants.

Micro-CT analysis

The femur of mice were dissected, fixed for 24 h with 4% paraformaldehyde, and scanned using high-resolution micro-computed tomography (mCT) (Skyscan 1172, Bruker MicroCT, Kontich, Belgium).4,5,70 The parameters of trabecular bone in the metaphysis and cortical bone in the mid-diaphysis were analyzed using NRecon image reconstruction software version 1.6 (Bruker MicroCT), CTAn data-analysis software version 1.9 (Bruker MicroCT), and CTVol 3-dimensional model visualization software version 2.0 (Bruker MicroCT). The scanner settings were 50 kVp, 201 mA, and a resolution of 12.64 mm/pixel.

The analysis was performed as described previously.5 For the distal femur, the region of interest (ROI) analyzed was 5% of the femoral length, ranging from 0.1 mm below the growth plate, to determine Tb.BV/TV, Tb.N, Tb.Sp, Tb.Th. For cortical bone, cross sectional images of the mid-diaphysis of femur were used to perform 3-dimensional histomorphometric analysis of cortical bone. The ROI of cortical bone selected for analysis was of 10% of femoral length in mid-diaphysis of the femur to determine cortical thickness (Ct. Th).

β-gal staining

For cell senescence assay, BMSCs and bone marrow adipocytes were fixed and stained by a senescence β-galactosidase staining Kit (Cell Signaling Technology, 9860), according to the manufacturer’s instructions.

For femoral bones histological analysis, femoral bones were dissected and freshly fixed in 4% paraformaldehyde overnight, followed by a 14-day decalcification in 0.5 mol/L EDTA (pH 7.4). The samples were then dehydrated in 20% sucrose plus 2% polyvinylpyrrolidone solution for 24 h and embedded in OCT. Ten mm-thick coronal sections of the femurs were obtained for SA β-gal staining according to the manufacturer’s instructions.

Immunoprecipitation and co-immunoprecipitation

The membrane protein of bone marrow adipocyte was extracted using the Membrane and Cytosol Protein Extraction Kit (Beyotime, P0033) and were incubated with his labeled rPCLAF protein and his antibody overnight at 4 °C. Protein A/G Magnetic Beads (MedChemExpress, HY-K0202-1) was rinsed for three times and was then add into protein lysate mixture for another incubation for 2 h at room temperature. The resulting immunoprecipitants were separated using SDS-PAGE and then analyzed using MS.

To perform the immunoprecipitation, HEK293T cells were transfected with Flag-Adgrl2 plasmids and His-Pclaf plasmids respectively. The cell lysate was collected and immunoprecipitated using antibodies against His (TA150088, 1:2 000, 1:2 000, Origene) and Flag (TA50011-100, 1:100, Origene) respectively, followed by adsorption to protein G Sepharose. The resulting immunoprecipitants were separated using SDS-PAGE and blotted onto a PVDF (Millipore) membrane. The membrane was then incubated with antibodies against Flag (TA50011-100, 1:2 000, Origene) and His (TA150088, 1:2 000, Origene) respectively, and visualized using a chemiluminescence reagent (Thermo Fisher Scientific, 32106) and imaged using a ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad Laboratories, USA).

Single-cell RNA sequencing analysis

Previous published scRNA-seq data(GEO: GSE202710 and GSE137869)5,24 were re-analyzed here and the Cell Ranger software pipeline (version 5.0.0) provided by 10x Genomics was used to demultiplex cellular barcodes, map reads to the genome and transcriptome using the STAR aligner, and down-sample reads as required to generate normalized aggregate data across samples, producing a matrix of gene counts versus cells. Raw reads were processed with fastQC and fastp to remove low quality reads. Poly-A tails and adaptor sequences were removed by cutadapt. After quality control, reads were mapped to the reference genome Rnor_6.0 using STAR. Gene counts and UMI counts were acquired by featureCounts software. Expression matrix files for subsequent analyses were generated based on gene counts and UMI counts (Singleron Biotechnologies, Nanjing, China). (2) Quality control, dimension-reduction and clustering. Cells were filtered by gene counts below 500. Cells with over 10%itochondrial content were removed. After filtering, 23 736 cells were retained for the downstream analyses. We used functions from Seurat V3.1.2 (Satija et al. 2015) for dimension-reduction and clustering. All gene expression was normalized and scaled using NormalizeData and ScaleData. Top 2 000 variable genes were selected by FindVariableFeautres for PCA analysisWe processed the unique molecular identifier (UMI) count matrix using the R package Seurat (version 3.1.1). violin plots displaying the expression of PCLAF were generated by Seurat V3.1.2 DotPlot/Vlnplot.

In previous raw data processing (GEO: GSE202710),24 we scored each cluster by the normalized expressions of the following canonical markers: Neutrophils (Lcn2, Camp, Retnlg, Csf3r), Granulocytes macrophages progenitor cells (GMPs) (Elane, Mpo, Prtn3), Macrophages (S100a4, Ccl9, Cd300e, Cd68), Dendritic cells (Cd74, Irf8), Plasma cells (Cd79b, Jchain, Mzb1), B cells (Cd79b, Cd19), Late pro-B cell (Dntt, Rag1, Cd79b, Cd19), Pro-B cells (Cd19, Cd79b, Ezh2), Erythroblast (Hba-a1,Hbb-bs), T cells/NK (Cd3d, Cd8a, Nkg7), Basophils (Ms4a2, Cd63), MSCs(Lepr, Cxcl12, Kitl, Angpt1), MPPs(CD34hi, CD45hi, CD38lo, CD224hi).

In raw data processing(GEO: GSE137869),5 to assign one of the 13 cell types to each cluster, we scored each cluster by the normalized expressions of the following canonical markers: Neutrophils (Lcn2, Camp, Retnlg, Csf3r), Pro-neutrophils (Pglyrp1, Lcn2, Camp, Mki67), Granulocytes macrophages progenitor cells (GMPs) (Elane, Mpo, Prtn3), Macrophages (Lyz2,Cd68), Plasma cells (Cd79b, Jchain, Mzb1), B cells (Cd79b, Cd19), Late pro-B cell (Dntt, Rag1, Cd79b, Cd19), Pro-B cells (Cd19, Cd79b, Ezh2), Proerythroblast (Hbb, Tfrc, Mki67), Erythroblast (Hbb, Tfrc), T cells (Cd3d, Cd8a, Nkg7), Basophils (Ms4a2, Cd63), Megakaryocytes (Pf4). Within Macrophages, we used the following markers for subtype identification: Cluster 1 (Vcan, Slpi, Chi3l1), Cluster 2 (Ccne2, Elane, Nkg7, Ctsg), Cluster 3 (Map2k3, Lgals1, S100a9, S100a8), Cluster 4 (P2ry10, Cd74, RT1-Da, Gngt2), Cluster 5 (Top2a, Ube2c, Histlh2an, H2afx).

Mass spectrometry analysis

For mass spectrometry analysis, membrane protein of bone marrow adipocytes extraction was carried out as previously described in ref.16. After digestion, samples were desalted using C18 cartridges (Empore SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL, Sigma), concentrated using vacuum centrifugation, and reconstituted in 40 mL of 0.1% (v/v) formic acid. TMT reagent was used to label 100 mg of peptide mixture from each sample, according to the manufacturer’s instructions (Thermo Scientific). LC-MS/MS analysis was performed using a Q Exactive mass spectrometer (Thermo Scientific) coupled to Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific) for 90 min. The peptides were loaded onto a reverse phase trap column (Thermo Scientific Acclaim PepMap100, 100 mm × 2 cm, nanoViper C18) connected to the C18-reversed phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 mm inner diameter, 3 mm resin) in buffer A (0.1% Formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nL/min controlled by IntelliFlow technology. The mass spectrometer was operated in positive ion mode. MS data was acquired using a data-dependent top 10 method that dynamically chose the most abundant precursor ions from the survey scan (300–1 800 m/z) for HCD fragmentation. AGC target was set to 3e6, and maximum inject time to 10 ms. Dynamic exclusion duration was 40.0 s. Survey scans were acquired at a resolution of 70 000 at m/z 200, and resolution for HCD spectra was set to 17 500 at m/z 200, and isolation width was 2 m/z. Normalized collision energy was 30 eV, and the underfill ratio was defined as 0.1%. Peptide recognition mode was enabled. The MS raw data for each sample were searched using the MASCOT engine (Matrix Science, London, UK; version 2.2) embedded into Proteome Discoverer 1.4 software for identification and quantitation analysis. Significance was assessed with t tests. The differentially expressed peptides were subsequently filtered for median fold-change > 1.3 and P-value < 0.05 (Student’s t test). The mass spectrometry proteomics data will be deposited in a public repository upon acceptance.

RNA-sequencing

RNAs were extracted from bone marrow adipocytes from adipogenic differentiation derived BMSCs which treated with rPCLAF or PBS using RNeasy Mini Kit (QIAGEN, 74014) following manufacturer’s instruction. The index-coded samples were clustered using the HiSeq PE Cluster Kit v4-cBot-HS (Illumina) on a cBot cluster generation system following the manufacturer’s instructions. Subsequently, the libraries were sequenced on an Illumina platform, and 150 bp paired-end reads were produced. The accession number for the RNA sequencing data will be deposited in a public repository upon acceptance.

Sample demultiplexing and conversion to FASTQ files was performed using Illumina’s fastq software with all default options. Reads from FASTQ files of the cultured cells were aligned to mouse genome using the STAR aligner (v2.4.2). Uniquely mapped reads were used for gene expression estimates as transcripts per million reads (TPMs). Kruskal–Wallis tests were used for identifying genes differentially expressed among sample groups. Pathway analysis was performed for functional annotation of the ASIGs in the dataset using established tools available online (EnrichR). Heatmap was formed using established online tool.

Histological analysis

Femurs were dissected and fixed overnight with 10% paraformaldehyde at 4 °C. The samples were then decalcified using 10% EDTA (pH 7.4) for 21 days at 4 °C.

For frozen section, the samples were dehydrated in a solution consisting of 20% sucrose and 2% polyvinylpyrrolidone for a duration of 24 h and then embedded in OCT. Coronal sections of the femurs, with a thickness of ten micrometers, were obtained for SA β-gal staining by utilizing staining kit from Cell Signaling Technology (Danvers, MA). The oil O red staining was also performed according to the manufacturer’s instructions.

For paraffin sections, the samples were then embedded in paraffin. Paraffin sections with a thickness of 6 μm were prepared and stained with TRAP (Sigma-Aldrich) to quantify the number and surface of osteoclasts. For immunohistochemical staining, bone sections were treated with 0.05% trypsin at 37 °C for 15 min for antigen retrieval, followed by overnight incubation with a primary antibody against Osteocalcin (Takara, M173) at 4 °C. The HRP-streptavidin detection system (Dako) was used to detect immunoactivity, followed by counterstaining with hematoxylin (Sigma). Bone sections were incubated overnight with primary antibodies: F4/80 (123110, 1:200, BioLegend), perilipin (9349, 1:200, Cell Signaling Technology), PCLAF (sc-390515, 1:200, Santa Cruz), γH2AX (9718, 1:200, Cell Signaling Technology), p21 (sc-166630, 1:50, Santa Cruz), Osteocalcin (M173, 1:200, Takara), Leptin (BAF497, 1:200, R&D system), Ctsk (57056, 1:300, Cell Signaling Technology). Fluorescence-conjugated secondary antibodies (Jackson- ImmunoResearch, 1:200) were used to detect fluorescent signals.

Calcein double-labeling

Mice were intraperitoneally injected with calcein (10 mg/kg, Sigma) 10 days and 3 days before euthanasia. After separation, the femur was immersed in 70% ethanol and embedded in methyl methacrylate. The samples were cut into 5 mm with a hard tissue cutter and examined under a fluorescence microscope to evaluate the mineral attachment rate using Image-Pro Plus 6.0. MAR is the distance between two labels divided by the time between labels.

Western blot analysis

Western blot analysis was conducted as previously described in refs. 24,71. Briefly, cells were lysed in RIPA lysis buffer and harvested with a rubber policeman. Samples (20 mg protein) were loaded into pre-cast electrophoresis gels (Bio-Rad), separated by SDS-PAGE and electro-transferred onto a PVDF membrane. After blocking with 5% non-fat milk, the blots were incubated with primary antibodies overnight at 4 °C and then with secondary antibodies at room temperature for 1 h.

The primary antibodies used were AKT (4685, 1:1 000, Cell Signaling Technology), phospho-AKT (4060, 1:1 000, Cell Signaling Technology), PCLAF (sc-390515, 1:500, Santa Cruz), mTOR (2983, 1:1 000, Cell Signaling Technology), phospho-mTOR (5536, 1:1 000, Abcam), ADGRL2 (140830, 1:1 000, Abcam). The protein bands were visualized using a chemiluminescence reagent (Thermo Fisher Scientific, 32106).

RNA isolation and qRT-PCR analysis

Total RNA was extracted using TRIzol (Sigma, T9424) and converted into cDNA using the PrimeScript RT Reagent Kit (Takara, PR037A). RNA of BMAds was extracted using Total RNA kits (Omega, R1034) according to the manufacturer’s instructions. Real-time reverse transcription PCR was performed using the ABI QuantStudio 3 system. Amplification reactions were prepared in 25 μL reaction volumes with SYBR Green, cDNA, and amplification primers. The primer sequences used are listed in Table S1.

PCLAF neutralizing antibody

Five male Balb/c mice (2-3 months, each weighing about 16–20 g) were immunized with a dose of 50 μg/mouse with the immunogen PCLAF (MVRTKADSVPGTYRKVVAARAPRKVLGSSTSATNSTSVSSRKAENKYAGGNPVCVRPTPKWQKGIGEFFRLSPKDSEKENQIPEEAGSSGLGKAKRKACPLQPDHTNDEKE) mixed with an equal volume of adjuvant (complete/incomplete Freund’s adjuvant). The adjuvant was purchased from BD Company (263910). After mixed together, they were injected subcutaneously at multiple points in the abdomen, with the second immunization at an interval of 2 weeks and the third immunization at another interval of 3 weeks. After 3 days of enhanced immunization, the spleen was taken for hybridoma fusion. One week after the last immunization, 50–60 mL of blood was drawn from the orbital vein clusters of mice. After standing at 4 °C overnight, the upper serum was separated by centrifugation for detection. A suitable coating buffer was used to dilute the amount of the detected protein to 5 mg/mL, and then 100 mL was added to each well of the 96-well plate and coated overnight at 4 °C. The plate was washed once with washing buffer at 200 mL/well. Then it was sealed with a 300 mL/hole sealing plate and buffered for 1 h at room temperature. The plate was washed twice with washing buffer, and then the sample (gradient diluted sample and sample diluent 100 mL/well) was added, and the antibody (100 mL/well to 96-well plate) was detected at room temperature for 2 h. Wash the plate and add the reaction buffer to 200 mL/ well, and place at room temperature for 12 min. Finally, 50 mL/well termination buffer was added to stop the reaction, and the microplate analyzer with a detection wavelength of 450 nm was used for detection. All spleen cells of immunized mice were mixed with mouse myeloma cells at a ratio of 1:1, and hybrid cells were obtained by electrofusion. The antigen protein was coated, and the cell supernatant was determined by ELISA. The positive wells were selected and cloned by dilution until the hybridoma cell line stably obtained the secreted monoclonal antibody. After filtration, a total of 6 hybridoma cells were obtained. Subsequently, the hybridoma cell clone numbered MM01 was selected for antibody preparation. The 1 mL hybridoma cells were transferred to a 100 mL culture flask, and a certain amount of medium was added regularly for cell amplification. The cells were cultured for 10–12 days. The protein A affinity chromatography column was washed with ultrapure water, and then the equilibrium buffer was balanced. The supernatant of the treated hybridoma cells was loaded onto an affinity chromatography column. After loading, the cells were washed with equilibrium buffer. The elution buffer was eluted and the elution peak was collected. After neutralization, Tris buffer was used to desalt to PBS7.4 to obtain PCLAF-Nab for in vivo and in vitro experiments.

Quantification and statistical analysis

The GraphPad Prism 8 software (GraphPad Software) was used for statistical analysis. The datas downloaded from GTEx program were analized with R version 4.0.2. Cell-based experiments were repeated at least twice, while at least three mice were used for each group in animal experiments unless otherwise specified. Data is presented as mean ± SD. Data showed a continuous normal distribution. Two-tailed Student’s t test was used for comparisons between two groups, and one- or two- way ANOVA were used for comparisons among multiple groups. Pearson’s Correlation analysis was used for correlation coefficient. Statistically significant differences were denoted as follows: * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001.