FIG. 5.

siRNA knockdown of MX1 and FHL1 inhibits osteogenesis. MSC were treated with negative control siRNA, MX1 siRNA #1, MX1 siRNA #2, FHL1 siRNA #1, and FHL1 siRNA #2 and were cultured for 14 days under osteogenic differentiation media. (A) Knockdown of MX1 and FHL1 by siRNA was confirmed by RT-PCR at 14 days of induction. (B) siRNA targeted knockdown of MX1 and FHL1 protein was confirmed by western blot analysis. (C) Representative images of Alizarin red stained mineral (x40) showing a decrease in hydroxyl appetite mineral MX1 and FHL1 siRNA-treated OB. (D) Extracellular calcium quantitated and normalized to DNA per triplicate well revealed significant decrease in calcium in siRNA-treated OB (n = 3 donors). (E) RT-PCR reveals a significant decrease in osteogenic marker RUNX2, OP, and OC in MX1 and FHL1 siRNA knockdown OB. Mean values ± SEM, n = 3 independent MSC donors (*P < 0.05, 1-way ANOVA with Dunnett's multiple comparisons test). OP, osteopontin. Color images available online at www.liebertpub.com/scd