Figure 1

MiR-9 affects cell proliferation and survival during chondrogenesis of chick limb mesenchymal cells. (A) Changes in the phosphorylation levels of Akt, GSK, and JNK were analyzed by Western blotting. (B) Total RNA was purified from chondroprogenitors cultured with or without 5 μM JNK inhibitor and the expression of mir-9 was measured with real-time PCR. (C) Chondroprogenitor cells were treated with 100 nM of anti-mir-9 oligonucleotides (mir-9inhibitor). The expression of mir-9 was measured with real-time PCR (upper panel) and Precartilage condensation was analyzed by PA staining at day 3 and Alcian blue staining at day 5 of culture (lower panel). The data shown are representative of at least four independent experiments. The diameter of typical standard culture is 5 mm. (D) Total cell numbers were counted at 2 day of culture. (E) Apoptotic cells were analyzed by FACS analysis. (F) Changes in the cleaved form of caspase-3 were analyzed by Western blotting. Results of cell adhesion experiments were pooled from 5 replicate samples derived from 4 independent experiments. The mean is plotted and the error bars represent 95% CI (lower/upper limit). ***, statistically different from control cells (p < 0.001).