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Figure 3 | Cell Communication and Signaling

Figure 3

From: MicroRNA-9 regulates survival of chondroblasts and cartilage integrity by targeting protogenin

Figure 3

PRTG induced apoptotic death of chondroprogenitors. (A) Cells were electroporated with PRTG/pCAGGS (PRTG) construct in the absence or presence of miR-9 precursor (miR-9) and electroporation efficiency was confirmed by immunoblotting (left upper panel). Precartilage condensation and chondrogenic differentiation were analyzed by PA staining at day 3 and Alcian blue staining at day 5 of culture, respectively (left lower panel) and chondrogenesis was quantified by measuring the absorbance of bound Alcian blue at 600 nm at day3 and day5 of culture (right panel). (B) Cells were electroporated with PRTG construct and the number of viable cells was determined at 1, 2, and 3 day of culture (left panel), treated with JNK inhibitor or miR-9, electroporated with PRTG or PRTG siRNA, or in the combination of JNK inhibitor and PRTG siRNA or PRTG and miR-9 and the number of viable cells were determined at day 2 and 3 of culture (right panel). (C) Apoptotic cells were analyzed by FACS analysis (left panel) and changes in the cleaved form of caspase-3 were analyzed by Western blotting (right panel). (D) Cells were treated with JNK inhibitor in the combination of miR-9 or PRTG-specific siRNA, or introduced with miR-9 in the combination of PRTG. Precartilage condensation and chondrogenic differentiation were analyzed by PA staining at day 3. The diameter of typical standard culture is 5 mm. (E) HH stage 18 chick embryos (wing bud) were treated with JNK inhibitor in the presence or absence of miR-9 precursor and incubated for additional 2 days (HH stage23). The number of embryos used for each experiment is represented as a table (upper panel), and a representative image of each limb is shown (lower panel). The mean is plotted and the error bars represent 95% CI (lower/upper limit). *, statistically different from control cells (p < 0.001).

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