Figure 8.

FOXO1 mediates p21 expression and chondrogenic differentiation via TGFβ1 signaling. A, relative mRNA levels of p21, p27, and cyclin G₂ after incubation with TGFβ1 (10 ng/ml) for 24 h. Gene expression at each stage is shown relative to the level in cells incubated without TGFβ1; n = 4. B, ATDC5 cells were incubated with TGFβ1 (10 ng/ml) for 24 h after transfection with siControl or siFOXO1. Relative mRNA levels of p21 were measured by qRT-PCR. Gene expression is given relative to the level in cells transfected with siControl; n = 4. C, ATDC5 cells were incubated with or without TGFβ1 (10 ng/ml) for 24 h after transfection with siControl or siFOXO1. Levels of FOXO1 and p21 protein were determined by Western blotting. D, nascent RNA was extracted from ATDC5 cells incubated with EU (0.5 mm) for 1 h after incubation with AS18428568 (1 μm) for 30 min following TGFβ1 treatment (10 ng/ml) for 24 h. Expression of nascent p21 RNA was measured by qRT-PCR. Gene expression is shown relative to the level in cells incubated without AS18428568; n = 3. E, scheme of a FOXO-binding site in the p21 promoter region. ChIP assay was performed using anti-FOXO1 antibody or control IgG in ATDC5 cells incubated with or without TGFβ1 (5 ng/ml) for 24 h. Semi-quantitative RT-PCR was performed using primers for the p21 promoter (from −2304 to −2020 bp) and negative control (from −2968 to −2636 bp). F, ATDC5 cells were differentiated for 2 weeks after incubation with or without UC2288 (2.5 μm) for 24 h. Relative mRNA levels of Foxo1, p21, Sox9, Col2a1, Acan, and Col10a1 were measured by qRT-PCR. Gene expression at each stage is shown relative to the level on day 0 in cells incubated without UC2288; n = 4. Data are presented as mean ± S.D. Statistical analysis was performed using Wilcoxon's rank-sum test. *, p < 0.05.