Fig 1.
Morphological characteristics of enthesis cells.
a, Enthesis cryocut section was stained for cells using SYTO® 13. Cells are depicted cyan, confocal reflection is depicted magenta. Scale bar corresponds to 150 μm. b, Scheme of cell arrangement observed at the enthesis. c, Cells within the interface are round shaped and are often arranged in pairs. Scale bar corresponds 50 μm. d, Cells residing within tendon are longitudinally arranged along the axis of tension in strings between tendon fibers. Scale bar corresponds 50 μm.
Fig 2.
Comparative transcriptome analysis of tendon, enthesis, and cartilage.
a, Experimental design of the transcriptomic study. Tendon (blue), interface region (orange), and cartilage (green) samples were excised, shock-frozen in liquid nitrogen, chemically treated to prevent RNA degradation and cryocut sectioned to enhance RNA extraction. High quality RNA was extracted using a refined protocol including proteinase K and DNase digests. cDNA libraries were produced and sequenced using next generation sequencing on an Illumina platform. Transcriptomes of enthesis, tendon, and cartilage were compared to each other. b, Global differences in the transcriptome of enthesis (E), tendon (T), and cartilage (C). c, Venn diagram of genes detected in the three tissues showing the number of overlapping and differentially expressed genes. Only genes that were annotated and had FPKM > 0 were considered, duplicates were not considered twice.
Fig 3.
Comparative transcriptome analysis of enthesis and tendon.
a, Venn diagram of differences between enthesis and tendon transcriptomes depicts the number of annotated genes that are only present in tendon (69), enthesis (35) or present in both tissues (13867). b, Volcano plot of all differentially enriched transcripts detected in the transcriptomes of tendon and enthesis. Statistically significant enriched genes were marked red. c, Top 10 genes with highest gene expression difference in tendon (blue) or enthesis (orange).
Table 1.
Top 10 genes with highest and lowest log2 fold change in enthesis/tendon transcriptome comparison.
Gene names and functions derive from GeneCards® and Ensembl.
Fig 4.
Protein-protein interaction networks of transcription factors and growth factors enriched in enthesis (a) or tendon (b). Protein-protein interactions were predicted using the database Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Line thickness indicates the strength of data support for protein-protein interaction.
Fig 5.
Comparative transcriptome analysis of enthesis and cartilage.
a, Venn diagram depicts the number of annotated genes that were only present in enthesis (70) or cartilage (100) or present in both tissues (13832). b, Volcano plot of all differentially enriched genes detected in the transcriptomes of enthesis (orange) and cartilage (green). Statistically significant enriched genes were marked red. c, List of genes with highest enrichment of gene expression in enthesis (orange) or cartilage (green). d, Protein-protein interaction network for transcription factors and growth factors enriched in cartilage compared to enthesis. Protein-protein interactions were predicted using the database Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Line thickness indicated strength of data support for protein-protein interaction.
Table 2.
Top 10 genes with highest and lowest log2 fold change in enthesis/cartilage transcriptome comparison.
Gene names and functions derive from GeneCards® and Ensembl.
Fig 6.
Comparative transcriptome analysis of tendon and cartilage.
a, Venn diagram depicts the number of annotated genes that are only expressed in tendon (99) or cartilage (95) or expressed in both tissues (13837). b, Volcano plot of all differentially enriched transcripts detected in the transcriptomes of tendon (blue) and cartilage (green). Statistically significant enriched genes were marked red. c, Top 10 genes with highest enrichment in tendon (blue) or cartilage (green).
Table 3.
Top 10 genes with highest and lowest log2 fold change in cartilage/tendon transcriptome comparison.
Gene names and functions derive from GeneCards® and Ensembl.
Fig 7.
Identified biomarkers of enthesis and tendon.
Biomarkers are presented which were statistically significant enriched in both the proteomics and transcriptomics data sets of enthesis (E) or tendon (T). Orange and blue bars represent enrichment in enthesis and tendon, respectively. Gray boxes correspond to enrichment in proteome and black boxes to enrichment in transcriptome. 13 genes were identified as tendon biomarkers (blue) and 24 genes as enthesis biomarkers (orange). Two genes (COL14A1 and ECM1) were identified that were enriched in the tendon proteome and conversely in the enthesis transcriptome.
Table 4.
Biomarkers that were at least twofold enriched in both transcriptome and proteome.
The biomarkers as well as the respective functions and log2 ratios are depicted. Log2 ratio differences of proteome and transcriptome data sets were calculated for each candidate.