Fig 2: ER retention and activation of ER stress/UPR by mutant COMP.a Wild-type, V66E, and R718W mutant COMP were expressed in primary tendon cells or RCS cells. Cell lysates and culture medium were respectively collected. Immunoblotting results indicate poor secretion of V66E mutant COMP by primary tendon cells but not RCS cells. The secretion of R718W-COMP is impaired in both types of cells. RCS rat chondrosarcoma cells, FBN fibronectin. The secretion ratios of different COMP from three experiments are quantified and summarized in the right panel, two-tailed t test, ***p = 0.00095 (V66E) and *p = 0.0161 (R718W) for primary tendon cells, p = 0.107 (V66E) and *p = 0.0108 (R718W) for RCS cells, error bars are ± SEM. b–d Normal (n = 2) and Family 1 patients’ TCL tissues (n = 3) were analyzed. b Immunofluorescent staining indicates strong co-localization of COMP (green) and endoplasmic reticulum (ER, recognized by KDEL antibody, red) in patients’ TCL cells. DAPI stains cell nucleus (blue). c Transmission electron microscopy (TEM) shows distended and fragmented ER in CTS patients’ TCL cells (red arrows). d Immunohistochemical staining shows upregulation of BIP, ATF4, and CHOP in patients’ TCLs, indicating activation of ER stress/unfolded protein response (UPR) in CTS patients. Arrows point to the perinuclear staining of the ER-resident BIP. The boxed areas (black box) in the figures are magnified and shown in the insets (white box). Source data are provided as a Source Data file.

Fig 3: Phenotypic analysis of the CTS mouse model.The COMP knock-in mouse model carries the mouse mutation corresponding to human p.V66E (n = 3 per genotype for each following analysis). a The H&E staining of 20-week-old mouse carpal tunnel shows comparable TCL, which is outlined by blue dotted lines, in wild-type and COMP mutants. The flexor tendons tend to be bigger in COMP mutants. b The statistical results of the cross-sectional area of flexor tendons (upper and lower parts, respectively). In total, 23 sections from three wild-type mice, 15 sections from four COMPVE/+ mice, and 28 sections from four COMPVE/VE mice were analyzed, two-tailed t test, upper **p = 0.002, 0.0034; lower **p = 0.0011, ***p = 0.0007, error bars are ± SEM. TCL transverse carpal ligament, N median nerve, B blood vessel, FT flexor tendon, CB carpal bone. c H&E staining of 20-month-old mouse carpal tunnel shows enlarged TCL in COMP mutants, which is outlined by blue dotted lines. a-SMA staining in 20-month-old mouse carpal tunnels (d) and Achilles tendons (e). The fibrosis is increased in COMPVE mutants. Vascular structures are observed in mutants (red arrows). Source data are provided as a Source Data file.

Fig 4: Compromised tendon-healing potential in the CTS mouse model.a The number of Scx-positive cells (green) in Achilles tendon is significantly decreased in 30-week-old COMP-mutant mice (n = 3 per genotype, 12 slides per genotype were analyzed, two-tailed t test, ***p = 0.0001 (COMPVE/+) and 6.22 × 10-5 (COMPVE/VE), error bars are ± SEM). b Primary tendon cells were isolated from 4-week-old Achilles tendons and cultured in vitro. Colony assay of isolated tendon cells shows fewer colonies formed by COMP-mutant mice (n = 3 per genotype, two-tailed t test, *p = 0.0107, **p = 0.002, error bars are ± SEM). See Supplementary Fig. 16a for representative pictures. c Schematic and results of Achilles tendon transection injury experiments on neonatal mice (n = 5 per genotype). Left Achilles tendon was transected at P5 (postnatal 5 days) and the right side was kept intact as a control. Tendon tissues were collected at P19 for analysis. H&E staining (lower panel) shows disorganization and poor recovery of injured Achilles tendons in mutant mice. d Immunohistochemical staining of injured Achilles tendons at P19 indicates increases of adipocytes (perilipin), fibrosis (a-SMA), and inflammation (CD11b) in mutant mice. e Schematic showing the contribution of different factors to the development of CTS, either familial or sporadic. The width of the arrow reflects the approximate extent of the contribution. Source data are provided as a Source Data file.

Fig 5: Impaired COMP complex formation and secretion in a COMP knock-in mouse model.Primary tendon cells and chondrocytes were isolated from 6- to 8-week-old wild-type and mutant mice and cultured in vitro. Cell lysates and culture medium were respectively collected. a Reduced secretion of endogenous mutant COMP by primary tendon cells but not primary chondrocytes. FBN fibronectin. The secretion ratios of different COMP from three experiments are quantified and summarized in the right panel, two-tailed t test, *p = 0.0187 (COMPVE/+) and 0.0174 (COMPVE/VE) for primary tendon cells, p = 0.688 (COMPVE/+) and 0.795 (COMPVE/VE) for primary chondrocytes, error bars are ± SEM. b Native gel electrophoresis of primary tendon cells and chondrocytes reveals significant amounts of monomer or small protein complex in COMPVE mutants. The major difference between tenocytes and chondrocytes is the retention of a big protein complex (white box) and a monomer in the tendon cell lysates. c The sucrose density-gradient ultracentrifugation of culture medium of primary tendon cells and chondrocytes also indicates lower molecular weight of protein complex containing the mutant COMP. d Immunoblotting of Bip and Atf4 in primary tendon cells and chondrocytes indicates increased ER stress/UPR in tenocytes. The protein levels from three experiments are quantified in the right panel, two-tailed t test, *p = 0.030 (COMPVE/+) and 0.041 (COMPVE/VE) for Bip, *p = 0.0245 (COMPVE/+) and 0.0358 (COMPVE/VE) for Atf4, error bars are ± SEM. Source data are provided as a Source Data file.