Fig. 4: The regulation of circFOXO3 on osteoarthritis is FOXO3-dependent.

A RIP assay was performed to detect circFOXO3 levels in ATDC5 cells stably expressing FOXO3. (n = 3) ***p < 0.001. B Representative images of circFOXO3 (red) and FOXO3 (green) labeled fluorescence in situ hybridization (FISH) staining. Scale bar, 10 µm. C Cell viability determined by CCK-8 assay. (n = 3) **p < 0.01, ***p < 0.001, ****p < 0.0001. D, E Western blot analysis of Cleaved PARP, Cleaved caspase-3, Bax and Bcl2 when Lv-circFOXO3 and si-FOXO3 were co-transfected. The optical density analysis was performed from the results of three independent experiments of western blot samples. ***p < 0.001, ****p < 0.0001. F, G Western blot analysis of MMP13, ADAMTS5 and Collagen II when Lv-circFOXO3 and si-FOXO3 were co-transfected. The optical density analysis was performed from the results of three independent experiments of western blot samples. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. H TUNEL staining assay was conducted on the ATDC5 chondrocytes, as treated above. Scale bar, 200 µm. I Immunofluorescence staining of MMP13 and collagen II in ATDC5 chondrocytes as treated above. Scale bar, 25 µm. J Alcian blue staining of Lv-circFOXO3 and si-FOXO3 co-transfected ATDC5 chondrocytes.