Fig. 1.

Efficient and specific Col1a2-siRNAs suppress collagen α2 chain protein expression in primary murine embryonic fibroblasts.

(A) The stability of Col1a2 silencing of the 2 most effective siRNAs (siRNA-3554 and – 3825) at 10 or 50 nM was evaluated 24, 48 and 72 h post transfection in MEFs plated 1 × 10⁴ cells/well. qPCR analyses showed Col1a2 relative expression. (B) The Col1a2 specificity was evaluated for the more promising molecule, siRNA-3554, analyzing the Col1a1 and Col1a2 expression by qPCR. (C) Representative images of SDS-Urea-PAGE analyses of ³H-proline labelled type I collagen extracted from medium and cell layer of transfected cells. The densitometric analyses of collagen type I α1 and α2 was performed and the α1/α2 ratio evaluated. In the collagen fraction extracted from cell layer a contribution of collagen type V in the α1(I) quantitation cannot be excluded. Not transfected cells (NT) and cells transfected with siRNA-LacZ were used as negative controls. A biological duplicate of the experiment was performed and each experiment was performed in triplicate. * p value <.05 vs siRNA-LacZ, # p value <.05 vs NT.