Fig 2: Proinflammatory cytokines down-regulated ERAD gene expression in mouse primary chondrocytes, and impaired ERAD function in chondrocytes promoted OA-like cartilage damage.(A) Mouse primary chondrocytes were treated with proinflammatory cytokines IL-1ß, TNF-a, or IL-17, and ERAD gene expression was analyzed by qPCR. Proinflammatory cytokines significantly reduced ERAD gene expression (n = 4). (B) Sel1L expression in articular chondrocytes was depleted by IA injection of Ad-Sel1L-shRNA, and articular cartilage integrity was analyzed by Safranin-O staining. GAG levels were reduced in Sel1L-depleted cartilage. Scale bars, 100 µm (top), 50 µm (bottom). (C) Osteoarthritic cartilage destruction was measured by OARSI grade and osteophyte formation after IA injection of Ad-Sel1L-shRNA. n = 8. (D) Expression levels of cartilage anabolic factors Col2a1, Acan, and Sox9 in Sel1L-depleted mouse articular cartilages were visualized by immunohistochemical staining. Sel1L depletion decreased Col2a1, Acan, and Sox9 expression. Scale bars, 50 µm. (E) Measurement of SEL1L, ACAN and SOX9 IHC density in Sel1L-depleted mouse articular cartilages. n = 5. Statistical analysis was performed using the Student’s t test. ***(P < 0.001), **(0.001 < P < 0.01), *(0.01 < P < 0.05), ns (0.05 < P).

Fig 3: Inhibition of ERAD function causes abnormal collagen accumulation in the ER lumen.(A) Collagen deposition in chondrogenic micromasses was detected by immunofluorescence staining upon ERAD gene depletion. Depletion of ERAD genes caused severe reduction in collagen deposition compared to control micromasses. The Sel1L-depleted micromass showed a severe phenotype. Scale bar, 1 mm (B) The areas of collagen-positive micromass cultures shown in (A) were measured and plotted (n = 4). Areas of collagen positivity for Derl2- and Edem1-knockdown micromasses were significantly reduced. Sel1L depletion caused the most severe phenotypes, whereas Hrd1-depleted micromasses displayed mild reductions in collagen deposition. (C) Staining intensity of collagen-positive micromass cultures shown in (A) were measured and plotted (n = 4). Statistical analysis was performed using one-way ANOVA. ***(P < 0.001), *(0.01 < P < 0.05), ns (0.05 < P). (D) High-resolution images of micromasses shown in (A) (top). Red represents Col2a1, and green represents the ER marker calnexin. In control micromasses, collagen was secreted and deposited in the ECM. However, in ERAD-depleted micromasses, most of the collagen signal was detected in the ER lumen. Scale bars, 5 µm. (E) TUNEL staining revealed increased cell death in ERAD-depleted micromasses. The bottom panels show the merged images with DAPI staining. Scale bars, 20 µm.