Probes for INS

To understand the subcellular localization of RUNX2 and two lncRNAs, LINC02035 and LOC100130207, immunocytochemistry (for RUNX2 protein) and RNA _in situ_ hybridization assays (for both lncRNAs) were performed using human primary chondrocytes isolated from knee cartilage of OA patients. We confirmed that the RUNX2 protein was strongly detected in the nucleus of chondrocytes isolated from damaged cartilage (Figure 4A). The fractionated western blot results also showed that the RUNX2 protein was detected only in the nucleus of chondrocytes isolated from damaged cartilage (Figure 4B). To further understand the molecular mechanisms of the lncRNAs LINC02035 and LOC100130207, we performed an _in situ_ assay using primary chondrocytes derived from patients, because primary chondrocytes are a valuable model for studying OA pathogenesis. The results showed that both LINC02035 and LOC100130207 were highly expressed in chondrocytes isolated from the knee cartilage of patients with OA (Figure 4C). We then evaluated the mRNA levels and subcellular localization of both lncRNAs to elucidate their site of action using a commercially available kits in primary chondrocytes isolated from intact or damaged cartilage tissues. The results showed that both lncRNAs were more upregulated in primary chondrocytes isolated from damaged cartilage tissue than in intact cartilage tissue (Figure 4D). In primary chondrocytes, LINC02035 and LOC100130207 were merely detected in the cytoplasm of human primary chondrocytes and both lncRNAs were localized to nucleus (Figure 4E). Likewise, we also studied the subcellular localization of both lncRNAs in TC28a2 cells. The results showed that LINC02035 and LOC100130207 were evenly distributed in the nucleus and cytoplasm of normal chondrocytes (Figure 4F, left). However, both lncRNAs were preferentially localized to the nucleus and to a lesser extent to the cytoplasm after TC28a2 cells were treated with hypertrophic medium or TNF-α (Figure 4F, middle and right). To investigate whether RUNX2 is regulated at the post-translational level during hypertrophic changes in chondrocytes, human primary chondrocytes or TC28a2 cells were treated with the proteasome inhibitor MG132. The results showed that the protein level of RUNX2 was dose-dependently increased by MG132 treatment (Figure 4G-H), indicating that the upregulation of RUNX2 in osteoarthritic or hypertrophic chondrocytes occurs at the post-translational level. To examine whether both lncRNAs are involved in the stabilization of RUNX2 protein during hypertrophic differentiation and the inflammatory response in chondrocytes, IP was conducted to confirm the ubiquitination of RUNX2 protein. First, we investigated how the ubiquitination of RUNX2 protein is regulated during hypertrophic differentiation or the inflammatory response of chondrocytes, and as a result, it was confirmed that ubiquitination of RUNX2 was reduced by hypertrophic medium or TNF-α treatment (Figure 4I). However, ubiquitination of RUNX2 protein was clearly increased in TC28a2 cells transfected with siRNAs targeting LINC02035 or LOC100130207, even though the cells were treated with hypertrophic medium or TNF-α (Figure 4J-K). These results suggest that both lncRNAs upregulated during hypertrophic differentiation and the inflammatory response in chondrocytes contribute to the stabilization of the RUNX2 protein.

* Alfredo Dueñas Rey Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Víctor López-Soriano Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Zelia Corradi Radboudumc Department of Human Genetics, Nijmegen, Gelderland, Netherlands * Claire-Marie Dhaenens Univ. Lille, Inserm, CHU Lille, U1172 - LilNCog - Lille Neuroscience & Cognition, Lille, France * Manon Bouckaert Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Jasper Verwilt Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium OncoRNALab, Cancer Research Institute Ghent, Ghent, Belgium * Avril M Watson Newcastle University Faculty of Medical Sciences, Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom * Majlinda Lako Newcastle University Faculty of Medical Sciences, Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom * Eva D’haene Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Karla Alejandra Ruiz Ceja Telethon Institute of Genetics and Medicine, Napoli, Campania, Italy * Sandro Banfi Telethon Institute of Genetics and Medicine, Napoli, Campania, Italy * Miriam Bauwens Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Frans P Cremers Radboudumc Department of Human Genetics, Nijmegen, Gelderland, Netherlands * Steve Lefever Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Elfride De Baere Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Frauke Coppieters Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium