Fig 2: Reverse degradomics identification of CMA1 substrates. A. Volcano plot of significant peptides (upper left quadrant, matrisome-derived peptides are in red). B. iceLogo plot of CMA1 cleavage site preference. The arrow indicates the cleaved peptide bond. C. Top ten CMA1 targets. The number of peptides for each target is indicated at the top of each bar. D. Venn diagram showing the overlap between the CMA1, cartilage and synovial fluid degradomes (matrisome-derived peptides only are shown, since CMA1 is a secreted protease). E. Molecules having the most cleavage site overlaps in the cartilage degradome and CMA1 degradome are shown.

Fig 3: Chymase reduces the metabolic activity and mitochondrial respiration of primary HLFs without affecting viability or mitochondrial protein abundance. (A) Oxygen consumption rate (OCR) profiles of primary HLFs, untreated or treated with 5 nM chymase for 24 h. Sequential injections include Oligomycin A (ATP synthase inhibitor), FCCP (protonophore), and a Rotenone/Antimycin A mixture (Complex I/III inhibitors). Data represent means ± SD. (B) Quantification of basal respiration in HLFs following 24-hour treatment with 5 nM chymase. Data are presented as means + SD, pooled from three independent experiments. *P < 0.1 (paired Student’s t-test). (C) Quantification of ATP-linked respiration in HLFs following 24-hour treatment with 5 nM chymase. Data are presented as means + SD, pooled from three independent experiments. (D) Quantification of maximal respiration in HLFs following 24-hour treatment with 5 nM chymase. Data are presented as means + SD, pooled from three independent experiments. (E) Extracellular acidification rate (ECAR) profiles of primary HLFs, untreated or treated with 5 nM chymase for 24 h. Sequential injections include Oligomycin A, FCCP, and a Rotenone/Antimycin A mixture (Complex I/III inhibitors). Data represent means ± SD. (F) Flow cytometric assessment of HLF viability and apoptosis after 24-hour treatment with 5 nM chymase, stained with Annexin V and DRAQ7. Populations include viable (Annexin V−/DRAQ7−), early apoptotic (Annexin V+/DRAQ7−), and late apoptotic/necrotic (Annexin V+/DRAQ7+) cells. Data are shown as means ± SEM. (G) Quantification of EdU-positive proliferating HLFs after 24-hour treatment with 5 nM chymase, relative to untreated cells. Data are presented as means ± SEM. (H) Quantification of HLF metabolic activity assessed by an independent PrestoBlue assay following 24-hour treatment with 5 nM chymase. Data are presented as means ± SEM. ****P ≤ 0.0001. (I) Representative Western blots showing the protein levels of subunits for oxidative phosphorylation (OXPHOS) complexes (Complex I, II, III, IV, V) in primary HLFs after 24-hour treatment with 5 nM chymase. (J) Total protein levels of COXIV, MTCO1, and MTCO2 are shown. GAPDH serves as a loading control

Fig 4: Chymase modulates intracellular kinase phosphorylation and heat shock protein levels in primary HLFs. (A) Representative Proteome Profiler Phospho-Kinase Array membranes showing phosphorylation profiles in whole cell lysates from untreated primary HLFs and HLFs treated with 5 nM chymase for 24 h. A scheme is provided for target identification. Blue dotted boxes highlight targets with decreased phosphorylation after chymase treatment, while red dotted boxes indicate targets with increased phosphorylation. (B-C) Western blot analysis of total and phosphorylated forms of CREB (p-CREB Ser133), c-Jun (p-c-Jun Ser63), and HSP27 (p-HSP27 Ser15, Ser78, Ser82), as well as total HSP60, in primary HLFs treated with 5 nM chymase for 24 h. (D) AmpliSeq transcriptomic analysis of heat shock protein (HSP) gene expression in HLFs after 24 h of 5 nM chymase treatment. HSPB1 (HSP27), HSPD1 (HSP60), and HSPH1 (HSP105) gene expression levels were not significantly affected by chymase treatment. (E) Confocal images showing intracellular Alexa Fluor 488-labelled chymase (white arrows) in HLFs. (F) Immunofluorescence detection of unlabelled chymase (white arrows) within HLFs using anti-CMA1 and Alexa Fluor 488-conjugated secondary antibody. Nuclei, blue (Hoechst 33342); F-actin, red (phalloidin); chymase, green

Fig 5: Chymase reduces the migratory capacity of primary HLFs. (A) Representative phase-contrast micrographs illustrating the cell-free area at indicated time points (0, 6, 24, 48 h) in untreated and chymase-treated HLFs. (B) Quantification of relative cell-free area over time. Data are presented as mean values ± SEM, pooled from three independent experiments. **P ≤ 0.01; ***P ≤ 0.001