Figure 2

miR-9 targets PRTG and inhibits chondrogenic differentiation. (A) Cells were cultured at a density of 2 × 10⁷ cells/ml and treated with miR-inhibitor, JNK inhibitor or in the combination of JNK inhibitor and miR-9 precursor (miR-9). Changes in the protein level of PRTG were analyzed at day 2 of culture. (B) Luciferase reporter gene assays of cells expressing the construct containing the human PRTG-3′-UTR or mutated seed sequence (mut 2436–2465, mut3067-3102) of putative targets (upper panel) in the absence or presence of miR-9. (C) Cells were electroporated with PRTG, incubated with either miR-9 precursor or miR-9 inhibitor, and changes in the protein level of PRTG were analyzed at day 2 of culture. (D) Mouse chondrogenitors were cultured at a density of 2 × 10⁷ cells/ml, sectioned, and stained with anti-PRTG antibody at day 6, 9, 12, 15, 18, 21 days of culture. (E) The expressions of PRTG, type X collagen (Col X), and miR-9 were measured with real-time PCR. Apoptotic cells were analyzed by FACS analysis. One-way analysis of variance (ANOVA) with Tukey post hoc comparisons of groups was used to test for significant effects. The mean is plotted and the error bars represent 95% CI (lower/upper limit). ***, statistically different from control cells (p < 0.001).