Chondrogenic differentiation is impacted by siRNA-mediated reductions in Snd1 (p100) in micromass assays.

(A) Mesenchymal cell condensation is unaffected after transfection of the dissociated limb mesenchyme with a scrambled siRNA control. (C and E) After condensation, the scrambled control transfected limb mesenchyme are competent to differentiate into cartilage nodules that stain positively for the cartilage marker alcian blue. (B,D,F) Parallel assays transfecting the same limb mesenchyme with a Snd1 siRNA cocktail severely impacts mesenchymal cell condensation producing few pre-cartilaginous condensations that can differentiate into mature cartilage nodules that stain positively with alcian blue. (G) Quantitation of the Snd1 levels 24H after transfection with the Snd1 siRNA cocktail or scrambled siRNA controls reveal a seventy percent reduction in endogenous Snd1 levels compared to parallel transfections using the scrambled siRNA controls. Values represent the average relative expression calculated from six independent replicates. Bars represent the standard deviation of the mean from the six independent assays. A Student’s t-test was used to determine significance of the averaged Snd1 expression values compared to the scrambled siRNA controls. A significant difference is indicated by an asterisk.