Chondrogenic differentiation is impacted by siRNA-mediated reductions in LncRNA-HIT 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 two siRNAs specific for LncRNA-HIT result in a loss in cell condensation (panel B) as well as severe reductions in the formation of cartilage nodules that stain with alcian blue (panels D and F). (G) Quantitation of the LncRNA-HIT levels 24H after transfection with the LncRNA-HIT siRNAs or scrambled siRNAs reveal an eighty percent reduction in endogenous LncRNA-HIT levels compared to parallel transfections using a scrambled siRNA control. 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 LncRNA-HIT expression values compared to the scrambled LncRNA-HIT siRNA control. A significant difference is indicated by an asterisk.