Filamin B Regulates Chondrocyte Proliferation and Differentiation through Cdk1 Signaling
Figure 1
Reduced proliferative capacity and enhanced differentiation in FlnB knockdown proliferating chondrocytes.
(A) Fluorescent immunocytochemistry performed on FlnB knockdown ATDC5 (FlnBsh2) chondrocytes shows downregulation of both proliferating chondrocyte markers, Sox9 and Col2a1 in the FlnB knockdown ATDC5 cells. The immunostaining also shows down-regulation of the prehypertrophic markers, Pthr1 and Ihh in the FlnB knocked down cells and increased expression for the hypertrophic marker Col10a1 and chondrocyte differentiation marker Runx2 in FlnBsh2 proliferating chondrocytes. (B) Western blot analyses of FlnBsh2 chondrocytes show a similar increased differentiation of the ATDC5 chondrocytes following FlnB knockdown compared to control. Results are graphically summarized to the right. (C) Alkaline phosphatase assay. Alkaline phosphatase activity was decreased in FlnB knockdown ATDC5 cells. (D) FlnBsh2 chondrocytes undergo a slower growth rate compared to normal ATDC5 cells. Quantification reveals an approximate 64% reduction in the proliferation rate by 5 days in culture. Cells are plated at a density of 1×105 and quantified at daily intervals. (E) Statistical analyses (n≥3 independent samples per experiment) show that the numbers of cells per unit area positively labeled for BrdU, Ki67, and PH3 (red) FlnBsh2 proliferating chondrocytes are decreased by 57%, 46%, and 45%, respectively when compared to control ATDC5 cells. * = p<0.05, ** = p<0.01, *** = p<0.001. Scale bars = 50 µm.