Supplementary Material for: NCOA3 Loss Disrupts Molecular Signature of Chondrocytes and Promotes Posttraumatic Osteoarthritis Progression

<b><i>Background/Aims:</i></b> Osteoarthritis (OA) is the most common joint disease. Recently, a novel variant near the nuclear receptor coactivator 3 (<i>NCOA3</i>) has been identified in association with greater risk of developing OA. However, how NCOA3 is regulated in chondrocytes and involved in OA pathogenesis remain elusive. <b><i>Methods:</i></b> The expression and DNA methylation of <i>NCOA3</i> in knee OA cartilage and <i>in vitro</i> dedifferentiated chondrocytes with or without rs6094710 SNP were analyzed by qRT-PCR, immunoblotting, methylation-specific PCR and bisulfite sequencing. NCOA3 was depleted by siRNA or shRNA or inhibited by a chemical inhibitor to assess its role in chondrocyte dedifferentiation or OA pathogenesis in posttraumatic OA animal model established by cruciate ligament transection surgery. <b><i>Results:</i></b> We found that compared with normal counterparts, samples with rs6094710 SNP failed to upregulate NCOA3. Further evidence associated this phenotype with DNMT1-mediated hypermethylation in gene promoter region. Moreover, we showed that NCOA3 maintained the molecular signature of chondrocytes dedifferentiating <i>in vitro</i> or exposed to IL-1β, nevertheless, NCOA3 appeared dispensable for preventing OA initiation, since NCOA3 loss did not trigger OA in young mice. Instead, NCOA3 loss promoted posttraumatic OA progression, and in parallel, enhanced NF-κB activation. Finally, the promoted posttraumatic OA progression was significantly retarded when administrated with NF-κB pathway inhibitor, suggesting that NCOA3 lose promotes posttraumatic OA at least partially by enhancing NF-κB activation. <b><i>Conclusion:</i></b> Thus, our findings indicate a critical role of NCOA3 in chondrocytes, and imply that manipulating NCOA3 might present a potential therapeutic approach to interfere OA progression.