*2.3. Pathway Enrichment and Protein-Protein Interaction Cluster*

Firstly, pathway enrichment against the Reactome knowledgebase [30] was employed to get insight into the potential underlying regulation pathways. Nineteen of the total 35 DEGs were successfully enriched in 276 pathways among 21 clusters (Figure 4a and Table S2). Among them, 4 pathways, including "G0 and early G1" and "mitotic G1 phase and G1/S transition" pathways of the "cell cycle" cluster, "alanine metabolism" of the "metabolism" cluster, and "RUNX1 regulates expression of components of tight junctions" pathway of the "gene expression (Transcription)" cluster, display a false discovery rate (FDR) value less than 0.05 (Figure 4b), highlighting themselves as the

potential vital OA-responsive pathways in both cartilage and synovium. Noticeably, 16 other DEGs, including *Leucine-rich repeat-containing protein 15* (*LRRC15*), *Myocilin* (*MYOC*), *cytoskeleton-associated protein 2 like* (*CKAP2L*), *forkhead box I2* (*FOXI2*), *ALDH1L1 antisense RNA 2* (*ALDH1L1-AS2*), *IQ motif containing N* (*IQCN*), *transmembrane protein 211* (*TMEM211*), *anillin actin-binding protein* (*ANLN*), *abnormal spindle microtubule assembly* (*ASPM*), *PEAK family member 3* (*PEAK3*), *Interferon epsilon* (*IFNE*), *Chitinase-3-like protein 2* (*CHI3L2*), *Lymphokine-activated killer T-cell-originated protein kinase* (*PBK*), *V-set and immunoglobulin domain containing 4* (*VSIG4*), *long intergenic non-protein coding RNA 1411* (*LINC01411*), and *MIR31 host gene* (*MIR31HG*), were not picked by the pathway database, which indicates that our current understanding of OA pathogenesis is dreadfully inadequate.

**Figure 4.** Pathway enrichment of the 35 common osteoarthritis (OA)-responsive differential expressed genes (DEGs) in cartilage and synovium has been conducted against the Reactome database. (**a**) The diagram of the distribution of the identified pathways. (**b**) The list of the identified pathways with a false discovery rate (FDR) less than 0.05. *TOP2A*—*DNA topoisomerase 2-alpha*; *CDK1*—*cyclin-dependent kinase 1*; *GPT2*—*Glutamate pyruvate transaminase 2*/*Alanine aminotransferase 2*; *CLDN5*—*Claudin-5*.

Since only 19 of 35 (54.3%) common OA-responsive DEGs in cartilage and synovium were picked by Reactome knowledgebase, manually align these 35 DEGs was achieved for further functional clustering. As inflammation is the primary and tissue shared event during OA, we first checked the DEGs that could relate to immune regulation. Based on the functional information collected in the Uniprot database [31] (Table S3), 10 DEGs, including *Apolipoprotein* (*APOD*), *complement C1q subcomponent subunit B* (*C1QB*), *N-formyl peptide receptor 3* (*FPR3*), *histone cluster 1 H3 family member b* (*HIST1H3B*), *IFNE*, *Macrophage scavenger receptor type I and II* (*MSR1*), *PBK*, *Tumor necrosis factor-inducible gene 6 protein* (*TNFAIP6*), *Triggering receptor expressed on myeloid cell 1* (*TREM1*), and *VSIG4*, were categorized to the inflammation-modulating group (Table 1). Protein-protein interactions among APOD, C1QB, FPR3, VSIG4, and MSR1 have also been assembled by the STRING networks [32]. These interactions, albeit weak (such as "textmining" and "co-expression"), were gathered in the biological processes related to "regulation of immune system process", "response to stress", "regulation of inflammatory response", and "response to the stimulus" (Figure 5).






**Figure 5.** STRING database displayed the protein-protein interaction network among the 10 common differential expressed genes (DEGs) in cartilage and synovium that related to inflammation modulating. (**a**) the diagram generated from the STRING data based to demonstrate the potential interactions. (**b**) The list contains identified pathways with a false discovery rate (FDR) less than 0.05. *APOD*—*Apolipoprotein*; *C1QB*—*complement C1q subcomponent subunit B*; *FPR3*—*N-formyl peptide receptor 3*; *HIST1H3B*—*histone cluster 1 H3 family member b*; *IFNE*—*Interferon epsilon*; *MSR1*—*Macrophage scavenger receptor type I and II*; *PBK*—*Lymphokine-activated killer T-cell-originated protein kinase*; *TNFAIP6*—*Tumor necrosis factor-inducible gene 6 protein*; *TREM1*—*Triggering receptor expressed on myeloid cell 1*; *VSIG4*—*V-set and immunoglobulin domain containing 4*.

Additionally, 7 genes, including *MYOC*, *Amelotin* (*AMTN*), *CHI3L2*, *Prolyl endopeptidase FAP* (*Fibroblast activation protein alpha*; *FAP*), *LRRC15*, *MMP13*, and *TNFAIP6*, were grouped based on their functions related to "extracellular matrix (ECM) binding, formation, degradation" listed in the Uniprot database (Table 2). Although the STRING network could assemble no direct protein-protein interaction with statistical significance, several biological processes were detected, such as "fibronectin binding", "collagen binding", and "metalloendopeptidase activity" (Figure 6).




**Figure 6.** The clustering results from the STRING database by inputting 7 extracellular matrix related genes. (**a**) the diagram generated from the STRING data based to demonstrate the potential interactions. (**b**)The list contains identified pathways with a false discovery rate less than 0.05. *MYOC: Myocilin*, *AMTN: Amelotin*, *CHI3L2: Chitinase-3-like protein 2*, *FAP: Fibroblast activation protein alpha*, *LRRC15: Leucine-rich repeat-containing protein 15*, *MMP13: Matrix metallopeptidase-13*, *TNFAIP6: Tumor necrosis factor-inducible gene 6 protein.*

On the other hand, functions of the 3 DEGs whose OA-induced responses are different in synovium and cartilage were also examined. However, only *Phosphatase and actin regulator 3* (*PTACTR3*) could be found in the Uniprot database. As a protein expressed in the cell nucleus, PTACTR3 functions in "actin binding", "protein phosphatase 1 binding" and "protein phosphatase inhibitor activity" (Table S4). No known OA-related function of the other two noncoding DEGs, *RNA, U12 small nuclear* (*RNU12*), and *RNA, U6 small nuclear 2* (*RNU6-2*) again highlights the lack of sufficient knowledge related to OA.
