General information

Dataset overview

Data name: eLife_Primary_data.zip

A detailed description of the experimental design and specific methodology can be found in the relevant publication (see below).

This dataset contains the underlying quantitative data and original images used to generate the figures and statistical analyses presented in the study. The study investigates the mechanism of obesity-related inflammation in osteoarthritis (OA) synovitis, specifically focusing on the role of GAS6 in macrophage efferocytosis. Sequencing data have been deposited in GEO under accession code GSE53986.

The data were derived from three main experimental approaches:

1. Human Clinical Samples: Synovial tissue, fluid, and cartilage obtained from patients with/without obesity undergoing arthroscopic treatment or total knee arthroplasty.
2. Animal Models: Destabilization of the medial meniscus (DMM) induced OA models in C57BL/6 (standard diet) and Apoe-deficient (Apoe-/-) mice (high-fat diet), including intra-articular injection treatments with recombinant mouse GAS6 or R428.
3. In Vitro Experiments: Efferocytosis assays, apoptosis induction, and molecular biology assays (RT-qPCR, ELISA, IF) using RAW264.7 cells, BMDM cells, and primary chondrocytes.

Data collection and processing involved:

• Histological Analysis: OARSI grading and synovitis scoring were performed on stained tissue sections.
• Image Analysis: Immunofluorescence (IF) and TUNEL assay images were captured using an Olympus BX43 microscope and analyzed using ImageJ software.
• Flow Cytometry: Macrophage phagocytic capacity was quantified via flow cytometry.
• Statistical Analysis: All raw data were processed and analyzed using GraphPad Prism software.

Notes for files

Directory structure and file naming

The dataset is organized into 5 directories, each corresponding to a main figure in the publication (e.g., Figure_1, Figure_2).

Within each directory, files are categorized into:

1. Statistical Data: An Excel file named Figure X-Statistical Data.xlsx containing the source data for graphs and statistical analyses.
2. Original Images: Raw microscopy images (histology, immunofluorescence) or blots used to generate the figure panels. Image files are named to reflect the experimental condition (e.g., HE_Normal individuals.png).

Data formats

1. Statistical Data (.xlsx)
Each Excel file contains multiple sheets (tabs), where each sheet corresponds to a specific sub-panel in the figure.

• Sheet Names: Follow the pattern Figure [X][Panel] [Variable Name].
  • Example: Figure 1C Synovitis Score, Figure 1D F4-80 positive cells.
• Data Organization: * Rows represent individual samples/animals.
  • Columns represent experimental groups (e.g., "Normal", "Obese", "Control", "DMM").
  • Cells contain quantitative values (e.g., OARSI scores, cell counts per field, fluorescence intensity, relative mRNA expression).
• Missing Data: Missing values are recorded as empty cells or N/A.

2. Image Files (.tif, .jpg, etc.)

• Microscopy images: Provide raw or minimally processed images for histology (Safranin O-fast green, H&E) and Immunofluorescence (IF).
• Scale bars: Scale information is either embedded in the image metadata or detailed in the figure legends of the related publication.

Abbreviations used

• DMM: Destabilization of the medial meniscus
• GAS6: Growth arrest-specific 6
• rmGAS6: Recombinant mouse GAS6
• OA: Osteoarthritis
• IF: Immunofluorescence
• IHC: Immunohistochemistry
• ACs: Apoptotic cells
• M1: Pro-inflammatory macrophages
• HE: Hematoxylin and Eosin staining
• SFO: Safranin O-fast green staining
• Col2: Collagen Type II
• MMP13: Matrix Metallopeptidase 13
• ACN: Aggrecan
• C57: C57BL/6

Dataset_Root
├── Figure_1
├── Figure_2
├── Figure_3
├── Figure_4
└── Figure_5

Sub-directories

Figure_1

Figure_1/
├── A
├── B\
├── Figure 1-figure supplement 1.csv\
└── Figure 1-Statistical data.xlsx

Contains underlying data for Figure 1: Synovial hyperplasia and macrophage polarization in obese OA patients.

Sub-directories: A and B Contains the original microscopy images corresponding to Figure 1A and Figure 1B.

Figure 1A Images (Histology)

• Content: "SFO" panels show human articular cartilage stained with Safranin O and Fast Green; "HE" panels show synovial tissue stained with Hematoxylin and Eosin (H&E).
• Experimental Groups: Normal individuals, OA patients without obesity, Obese individuals, and OA patients with obesity.
• File Naming: Files are named to reflect the staining and experimental group, e.g., HE_Normal individuals, SFO_Normal OA patients.
• Scale bars: 200 µm (Cartilage), 50 µm (Synovium).

Figure 1B Images (Immunofluorescence)

• Content: Immunofluorescence staining of synovial tissues to identify macrophage polarization.
• Markers & Channels:
  • F4/80 (Green): Total macrophages
  • iNOS (Red): M1 macrophage marker
  • CD206: M2 macrophage marker
• File Naming: Files are named by marker and experimental group, e.g., F480_Normal OA patients, INOS_Normal OA patients, CD206_Normal individuals.
• Scale bar: 50 µm.

Figure 1-figure supplement 1

Contains data for Figure 1—figure supplement 1: Blood lipids in obesity-related patients and general patients.

• Data Structure:
  • Columns: Experimental groups (Normal individuals, OA patients without obesity, Obese individuals, OA patients with obesity).
  • Rows: Clinical parameters including Total cholesterol (TC), Triglyceride (TG), and BMI index.

Figure 1-Statistical Data.xlsx

Contains the raw quantitative data used for the statistical graphs in Figure 1C and Figure 1D.

• Sheet: Figure 1C Synovitis Score
  • Description: Quantification of synovitis severity based on histological scoring (Krenn score).
    Corresponds to Panel C.
• Sheet: Figure 1D F4-80 positive cells
  • Description: Quantification of F4/80 positive macrophages as a proportion of the total lining cell population.
    Corresponds to Panel D.
• Sheet: Figure 1D INOS positive cells
  • Description: Quantification of iNOS positive (M1) macrophages.
    Corresponds to Panel D.
• Sheet: Figure 1D CD206 positive cells
  • Description: Quantification of CD206 positive (M2) macrophages.
    Corresponds to Panel D.

Figure_2

Figure_2/
├── A
├── D\
├── F\
├── Figure 2-figure supplement 1
├── Figure 2-figure supplement 2
├── Table
│     ├── Table2.csv
│     ├── Table3.csv
│     └── Table4.csv
└── Figure 2-Statistical data.xlsx

Contains underlying data for Figure 2: Cartilage loss, synovial hyperplasia, and macrophage polarization in Apoe−/− OA.

• Sub-directories: A, D, F, Figure2 figure supplement1, Figure2 figure supplement2
  • Contains the original microscopy images corresponding to the panels in Figure 2 and its figure supplements.
• Naming Convention:
  File names denote the tissue type (C = Cartilage; S = Synovium), staining method, and experimental group.
• File Naming:
  Files are named by marker and experimental group, or named to reflect the staining and experimental group, e.g.,
  CD206_Ctrl_Apoe, INOS_Ctrl_Apoe, C_HE_OA_Apoe.

Figure 2A, 2D, 2F Images

• Histology (Panel A):
  Safranin O and Fast Green staining (Cartilage, Scale bar: 200 µm) and H&E staining (Synovium, Scale bar: 50 µm) of controls and DMM mice.
• IHC (Panel D):
  Immunohistochemical staining for Aggrecan (Cartilage) and MMP-13 (Cartilage and Synovium).
  Scale bar: 50 µm.
• IF (Panel F):
  Immunofluorescence for iNOS (M1 marker) and CD206 (M2 marker) in synovial tissues.
  Scale bar: 50 µm.

Figure 2–figure supplement 1 Images (4 weeks post-surgery)

• Content:
  H&E staining of cartilage and synovial tissue in controls and DMM mice at 4 weeks post-surgery.
• Location:
  Directory Figure2 figure supplement1.

Figure 2–figure supplement 2 Images (4 weeks post-surgery)

• Content:
  Immunofluorescence of iNOS and CD206 in synovial tissues at 4 weeks post-surgery.
• Location:
  Directory Figure2 figure supplement2.

Sub-directory: Table

Contains source tables describing the animal model specifications.

• Table 2:
  Comparison of specifications and energy of ordinary feed and high-fat feed.
• Table 3:
  Lipid status of APOE−/− obese mice and C57BL/6 mice.
• Table 4:
  Weight gain after feeding for 8 weeks.

Figure 2 Statistical Data.xlsx

Contains the raw quantitative data used for the statistical graphs in Figure 2 and its supplements.

• Main Figure 2 Sheets:
  • Figure 2B OARSI score:
    Quantitative analysis of cartilage destruction based on the OARSI scale.
  • Figure 2C Krenn synovitis score:
    Synovitis scores for joints corresponding to Panel A.
  • Figure2E Cartilage_posivitve & Figure2E-Synovium_posotive:
    Quantification of MMP-13 positive cells in cartilage and synovium.
  • Figure 2G inos-positive & Figure2G CD206-positive:
    Quantification of M1 and M2 macrophage proportions in the synovial lining cell population.
• Supplementary Figure Sheets (4 weeks post-surgery):
  • Figure2 FigureS1B-OARSI:
    OARSI scores at 4 weeks.
  • Figure2 FigureS1B-Krenns score:
    Synovitis scores at 4 weeks.
  • Figure2 FigureS2-INOS positive:
    iNOS positive cell quantification at 4 weeks.
  • Figure2 FigureS2-CD206 positive:
    CD206 positive cell quantification at 4 weeks.

Figure_3

Figure_3/
├── A
├── C\
├── F\
├── Figure 3- figure supplement 2 primary blots
├── Figure 3- figure supplement 3
├── Figure 3- figure supplement 1.tif
├── Figure 3- figure supplement 1-souce data 1.csv
└── Figure 3-Statistical data.xlsx

Contains underlying data for Figure 3: Loss of GAS6 expression in synovium of obese OA patients and Apoe−/− OA mice, along with supplementary data regarding macrophage polarization and AXL receptor expression.

• Sub-directories: A, C, F, Figure 3- figure supplement 3
  • Contains the original immunofluorescence (IF) images corresponding to the panels in Figure 3 and Figure 3—figure supplement 3.
• File Naming:
  Files are named by marker and experimental group, or named to reflect the staining and experimental group, e.g.,
  GAS6_Normal individuals, GAS6_OA_Apoe, LPS-24h.

Figure 3A, 3C, 3F Images (GAS6 Expression)

• Content:
  Immunofluorescence staining to visualize GAS6 expression in macrophages.
• Samples:
  • Human (Panel A): Normal, Non-obese OA, Obese, Obese OA
  • Mouse (Panel C): C57BL/6 and Apoe−/− mice (Control vs. DMM)
  • Cells (Panel F): RAW264.7 cells treated with LPS (24/48 hr)
• Scale bar:
  50 µm

Figure 3—figure supplement 3 Images (AXL Expression)

• Content:
  Immunofluorescence staining for the GAS6 receptor AXL.
• Samples:
  • Human (Panel A): Normal, Non-obese OA, Obese, Obese OA
  • Mouse (Panel C): C57BL/6 and Apoe−/− mice (Control vs. DMM)

Sub-directory: Figure 3- figure supplement 2 primary blots

Contains raw, uncropped Western Blot images corresponding to Figure 3—figure supplement 2B.

• Target Proteins:
  • GAS6
  • iNOS (file labeled as NOS2): M1 macrophage marker
  • CD86: M1 macrophage marker
  • GAPDH: Loading control
• Experimental Conditions:
  RAW264.7 cells after LPS or IL-4 stimulation.

Figure 3 Statistical Data.xlsx

Contains the raw quantitative data used for the statistical graphs.

• Main Figure 3 Sheets:
  • Figure3B HomoF480-GAS6-positive:
    Quantification of F4/80-GAS6+ macrophages in human synovium (n = 6).
  • Figure3D miceF480-GAS6-positive:
    Quantification of F4/80-GAS6+ macrophages in mouse synovium (n = 6).
  • Figure3E GAS6 consentration:
    ELISA results for GAS6 in synovial fluid of non-obese and obese OA patients (n = 13 per group).
  • Figure3G F480-GAS6 positive:
    Quantification of GAS6+ RAW264.7 cells after LPS treatment (n = 6).
• Supplementary Figure Sheets:
  • Figure3 FigureS2A gas6 mRNA:
    Relative mRNA expression of GAS6 in LPS-treated cells (n = 3).
  • Figure3 FigureS2C mRNA level:
    Relative mRNA expression of inflammatory cytokines (IL-1β, IL-6, TNF-α) under LPS/rmGAS6/R428 treatments (n = 6).
  • Figure3 FigureS3-homo f480 axl:
    Quantification of f480-AXL+ macrophages in human synovium (n = 6).
  • Figure3 FigureS2-Mus f480-axl:
    Quantification of f480-AXL+ macrophages in mouse synovium (n = 6).
• Note:
  Data related to Figure 3—figure supplement 1 (GSE53986 analysis) is derived from the public dataset GSE53986.

Figure_4

Figure_4/
├── A
├── C\
├── E\
├── H\
├── Figure 4- figure supplement 1
├── Figure 4- figure supplement 2
├── Figure 4-figure supplement 3 primary blots
└── Figure 4-Statistical data.xlsx

Contains underlying data for Figure 4: Accumulation of apoptotic cells in OA and impaired phagocytic ability of M1-polarized macrophages, along with mechanistic studies on MerTK expression and rescue experiments.

• Sub-directories: A, C, E, H, Figure 4-figure supplement 1, Figure 4-figure supplement 2
  • Contains the original immunofluorescence (IF) images and flow cytometry plots.
• File Naming:
  Files are named by marker and experimental group, or named to reflect the staining and experimental group, e.g.,
  Caspase3_Normal individuals, Caspase3_Ctrl_C57, LPS+rmGAS6+R428.

In Vivo Apoptosis & Receptors (Panels A, C & Figure 4- figure supplement 1)

• Apoptosis Markers:
  Caspase-3 and TUNEL staining in synovial tissues.
• Receptor Staining:
  Double staining for F4/80 (Red) and MER (Green) to evaluate MerTK expression on macrophages.
• Samples:
  • Human synovial tissue: Normal individuals, Obese individuals, Normal OA patients, Obese OA patients
  • Mouse synovial tissue: Ctrl_C57, Ctrl_Apoe, OA_C57, OA_Apoe,
• File Examples:
  Caspase3_Normal individuals, TUNEL_OA_Apoe, MER_Ctrl_Apoe
• Scale bar:
  50 µm

In Vitro Efferocytosis Assays (Panels E, H & Figure 4- figure supplement 2)

• Methodology:
  Macrophage phagocytosis assay.
• Staining:
  F4/80 (Red): Macrophages
  CFSE (Green): Apoptotic Thymocytes. Co-localization indicates successful engulfment.
• Cells:
  • BMDMs (Bone Marrow-Derived Macrophages) and RAW264.7 cells
• Treatments:
  Files labeled with conditions like LPS+rmGAS6, LPS+rmGAS6+R428.
• Flow Cytometry:
  Includes fluorescence-intensity distribution plots.

Sub-directory: Figure 4-figure supplement 3 primary blots

Contains raw, uncropped Western Blot images corresponding to Figure 4—figure supplement 3.

• Target Proteins:
  • CD86 & iNOS: M1 macrophage markers
  • GAPDH: Loading control
• Condition:
  RAW264.7 cells after R428 (Axl inhibitor) stimulation.

Figure 4-Statistical Data.xlsx

Contains the raw quantitative data used for the statistical graphs.

• In Vivo Quantification (Apoptosis):
  • Figure4B-Caspase3 positive / Figure4B-TUNEL positive:
    Quantification in human synovium (n = 6).
  • Figure4D-Caspase3 positive / Figure4D-TUNEL positive:
    Quantification in mouse synovium (n = 6).
• In Vitro Quantification (Efferocytosis & mRNA):
  • Figure 4F effero index:
    Phagocytic ability of BMDMs from Apoe−/− vs C57BL/6 mice (n = 5).
  • Figure 4G mRNA:
    Relative expression of iNOS and Arg1 after LPS/rmGAS6/IL-4 stimulation.
  • Figure 4J effero index:
    Phagocytosis index in RAW264.7 cells quantified by microscopy (n = 6).
  • Figure 4K effero index:
    Phagocytosis index quantified by Flow Cytometry (n = 5).
  • Figure4 FigureS2B effero index:
    Rescue effect of rmGAS6 on BMDM efferocytosis (n = 5).
• Receptor Expression (Supplement 1):
  • Figure4 FigureS1B homo f480 mer:
    Quantification of F4/80-MER+ cells in human synovium (n = 6).
  • Figure4 FigureS1D mice f480 mer:
    Quantification of F4/80-MER+ cells in mouse synovium (n = 6).

Figure_5

Figure_5/
├── A
├── C\
├── Figure 5-Figure supplement 1 primary blots
├── Figure 5-Figure supplement 2.png
├── Figure 5-Figure supplement 3 primary blots
└── Figure 5-Statistical data.xlsx

Contains underlying data for Figure 5: GAS6 restored osteoarthritis (OA) cartilage loss and decreased apoptotic cell accumulation, focusing on the therapeutic effects of rmGAS6 and the underlying molecular mechanisms in chondrocytes.

• Sub-directories: A, C
  • Contains original microscopy images evaluating the therapeutic outcome of intra-articular treatments at 8 weeks post-surgery.

Histology (Panel A)

• Knee Cartilage:
  Safranin O and Fast Green staining (Scale bar: 200 µm).
• Synovial Tissue:
  H&E staining (Scale bar: 50 µm).
• Treatment Groups:
  DMM mice, DMM + R428 (inhibitor), and Apoe−/− + rmGAS6 (recombinant protein).
• Naming Examples:
  SFO_C57_OA_R428, HE_Apoe_OA_rmGAS6.

Apoptosis (Panel C)

• Content:
  Immunofluorescence staining for Caspase-3 and TUNEL in synovial tissues.
• Scale bar:
  50 µm.
• Naming Examples:
  Caspase3_Apoe_OA_rmGAS6, TUNEL_OA_R428.

Figure 5-Figure supplement 2

• Content:
  Images of human tibial plateau cartilage explants stained with Safranin O.
• Groups:
  Control (Con) vs. Recombinant human GAS6 (rhGAS6).

Sub-directories: Figure 5-Figure supplement 1 & 3 primary blots

Contains raw, uncropped Western Blot images reflecting cartilage health and cellular senescence.

• Biological Context:
  Primary chondrocytes treated with BMDM-derived supernatant (co-cultured with apoptotic cells) or direct rhGAS6 treatment.
• Target Proteins:
  • Matrix Markers: COL2, MMP13
  • Senescence Markers: p16, p21
  • Loading Control: GAPDH

Figure 5 Statistical Data.xlsx

Contains the raw quantitative data for therapeutic evaluation and molecular assays.

• In Vivo Evaluation (Panels B & D):
  • Figure 5B-OARSI score:
    Quantitative analysis of cartilage protection.
  • Figure 5B-Krenns score:
    Quantitative analysis of synovitis reduction.
  • Figure 5D-Caspase3 positive:
    Quantification of apoptosis in the synovial lining.
  • Figure 5D-TUNEL positive:
    Quantification of DNA fragmentation in the synovial lining.

Human subjects data

We confirm that written informed consent was obtained from all human participants involved in this study. The consent process explicitly included permission for the collection of biological samples and the publication of resulting de-identified data in the public domain.
De-identification Method:To protect participant privacy, all direct personal identifiers (including names, medical record numbers, dates of birth, and contact information) have been removed from the dataset.
1）Coding: Samples were assigned unique, non-identifiable alphanumeric codes (e.g., “HE_Normal_OA_patients”, "SFO_Obese individuals") or grouped by clinical phenotype (e.g., "Obese OA"). The key linking these codes to original patient identities is stored separately and securely by the investigators and is not included in this submission.
2) Imaging Data: The images provided are high-magnification microscopic histology and immunofluorescence fields. They do not contain any facial features or other biometric markers that could be used to re-identify individuals.
3）Quantitative Data: Clinical data (e.g., age, BMI) presented in the dataset has been aggregated or de-identified to prevent deduction of participant identities.