Figure 3.

Stable downregulation of Mut Col1a1 transcript in Brtl fibroblasts following transduction with LacZ-, E-Mut or F-Mut shRNA-expressing lentivirus. (a) Flow cytometry analysis of EGFP demonstrated a similar transduction efficiency for all shRNAs used. Percentages of EGFP positive cells are indicated. (b) Cell viability evaluation by MTT assay. Untransduced (diamond) and shLacZ (square) or F-Mut shRNA (triangle) transduced Brtl fibroblasts were tested at different time points. Each point was measured in triplicate and each assay was repeated in three independent experiments. No difference in proliferation was detected among the untransduced and transduced cells. (c) The relative expression of the total Col1a1 transcript and (d) WT and Mut Col1a1 transcripts were assessed in untransduced and transduced Brtl fibroblasts. Ratio of WT/Mut Col1a1 transcripts is shown. Although shRNA E-Mut and F-Mut significantly inhibited both WT and Mut allele, the WT/Brtl allele ratio was significantly increased only in cells transduced with F-Mut shRNA. The results are shown as mean±SD of a triplicate experiment (*P<0.05, referred to untransduced cells).