The cGAS/STING signaling pathway is activated by 29-kDa FN-f in primary chondrocytes. (A) The expression of cGAS and STING was increased in osteoarthritis (OA) compared to normal cartilage. The relative expression of cGAS and STING in human normal and OA cartilage was measured using SYBR Green-based real-time polymerase chain reaction (qPCR). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. ***P < 0.001 and ****P < 0.0001 vs. normal cartilage. Data are presented as the mean ± standard deviation (SD) based on duplicate experiments using cartilages derived from different donors (normal cartilage, n = 6 and OA cartilage, n = 8). (B, C) The expression of γH2AX, cGAS, and STING in 29-kDa FN-f-, IL-1β-, and etoposide (ET)-treated chondrocytes. Chondrocytes were treated with 29-kDa FN-f (300 nM), IL-1β (1 ng/ml), and etoposide (200 μM) for (B) 24 h and (C) 3, 5, and 7 days. Cell lysates were subjected to western blot analysis. (D) Activation of γH2AX by 29-kDa FN-f. After treatment of primary chondrocytes with 29-kDa FN-f for 5 days, the cellular level of γH2AX was determined using fluorescence microscopy. Nuclei were stained with DAPI.