Fig 2: In situ PLA of lubricin with possible cartilage binding partners. In situ PLA was performed on OA cartilage biopsy cryosections with the following pairs. In situ PLA uses two primary antibodies targeting proteins of interest and secondary antibodies conjugated to DNA oligonucleotides (PLA probes). If the two proteins of interest are in a complex the two PLA probes will be in close enough proximity to bind and facilitate formation of a circular DNA molecule using two additional DNA oligonucleotides which then undergo rolling circle amplification to enhance the signal. Fluorescently (red) labelled complementary DNA is then added in order to visualise the DNA. (a) Lubricin (PA3-118) and COMP (16F12). Bound lubricin and COMP (red) were identified at the cartilage surface. A second layer was also apparent below the surface. (b) Lubricin (5C11) and fibronectin (ab32419). Bound lubricin and fibronectin (red) were predominately identified at the cartilage surface. Only when the PLA signal was thinner at the surface was signal observed just below the surface. (c) Lubricin (5C11) and collagen II (sc-7763). Bound lubricin and collagen II (red) were identified only at the surface with no PLA signal further into the tissue. PLA signal are red, DAPI staining in blue. For the 3 antibody pairs, 5 separate antibodies were used as anti-lubricin 5CII was used for 2 pairs. Negative controls were performed by omitting each primary antibody for all of the five antibodies used. (d) lubricin negative control (PA3-118), (e) COMP negative control (16F12), (f) lubricin negative control (5CII), (g) fibronectin negative control (ab32419) and (h) collagen II negative control (sc-7763).

Fig 3: Western blots of lubricin released from the cartilage surface by MMP-9. (a) Cartilage biopsy tissue pieces were incubated with pro- and activated MMP-9 and released proteins were separated by SDS-PAGE and analysed by Western blot with lubricin antibody mAb13. (b) Purified lubricin was also incubated with activated MMP-9. Lubricin was released from the cartilage biopsy pieces when the surface of the cartilage was digested by MMP enzymes which digest the proteins of the cartilage ECM.

Fig 4: Illustration of the cartilage surface showing protein interactions. Lubricin is blue with glycosylation shown in light blue, COMP is red shown as the pentameric structure, fibronectin is green shown as the dimeric structure and collagen II is purple shown as collagen II fibrils. The SF is shown in light straw, the cartilage in yellow and the cartilage surface in pink. COMP, collagen II and fibronectin adhere lubricin, primarily by the termini of lubricin, to the cartilage surface creating an exposed lubricin layer. The ECM proteins also bind to each other and are shown in the cartilage. Lubricin is also found in the SF.

Fig 5: Proteomics analysis of primary human synovial fibroblasts from non-OA and OA patients.a Workflow schematic of proteomics experimental design. Primary human synovial biopsies from non-OA and OA patients were isolated (n = 3 biological replicates, and 2 technical replicates). Primary synovial fibroblasts were sorted using CD90 (THY1). Non-OA primary fibroblasts were treated for 1 hour with PRG4 (blue), tryptase β + PRG4 1:100 enzyme to substrate ratio (magenta), tryptase β + buffer 1:100 (dark grey) or buffer (pale grey). Confluent OA primary fibroblasts were treated with buffer for 1 h (cyan). Next, cell were lysates and isotopically labeled with tandem mass tags (TMT) 6-plex and subjected to LC-MS/MS and analyzed using MaxQuant at 1% FDR. Graphic created using BioRender. b Dot plot analysis of raw and relative protein expression across the five conditions. The log2 ratio is shown as a gradient from blue to red and the log2 intensity of each protein is shown as the size of the circle. c, d Proteins identified by shotgun proteomics are represented as volcano plots. Log2 fold change using an interquartile boxplot analysis is represented on the x-axis and -log10 p value is represented on the y-axis. Two-sided analysis was performed, and it was adjusted for multiple comparisons. The complete list of proteins identified is shown in Supplementary Table 1. e Metascape48 analysis of different pathways between PRG4 and PRG4 + tryptase β. Accumulative hypergeometric p-values and enrichment factors were calculated and used for filtering as performed as a two-sided analysis. Remaining significant terms were then hierarchically clustered into a tree based on Kappa-statistical similarities among their gene’s memberships. Then, 0.3 kappa score was applied as the threshold to cast the tree into term clusters. f Heatmap of proteins and associated reactome pathways as determined by Metascape48. Interquartile box plot and -log10 adjusted p-value analysis was used for statistics. Data analysis was accomplished using the R software94. The plot was generated using the heatmap.2 function from the gplots package95.