Fig. 6. Subcellular distribution of siRNA₁₀₇ and time- and concentration-dependent inhibition of MT1-MMP expression in HMECs transfected with siRNA₁₀₇. (A) HMECs were cultured to 60% subconfluency in ECGM MV and then transfected with Cy3-labeled siRNA₁₀₇ for 72 hours. (a,d) Confocal microscopy analysis of scrambled siRNA₁₀₇ (ssiRNA₁₀₇) and siRNA₁₀₇, respectively. (b,e) Phase-contrast microscopy analysis of scrambled siRNA₁₀₇ and siRNA₁₀₇, respectively. (c,f) Overlays of fluorescence and phase-contrast photomicrographs. Perinuclear distribution of siRNA₁₀₇ and nucleus membrane is indicated by the arrows. (B) HMECs were cultured in ECGM MV to 60% subconfluency and then transfected with various concentrations of siRNA₁₀₇ or scrambled siRNA₁₀₇ (ssiRNA₁₀₇) for several periods in the presence of oligofectamine as a transfecting reagent. MT1-MMP expression was determined by semi-quantitative RT-PCR and expressed relative to GAPDH expression. PCR products were resolved by 1% (w/v) agarose gel electrophoresis. Control cells (C) were incubated with oligofectamine alone. The X174/HaeIII markers (M) were used to evaluate the DNA fragment length. (C,D) Time- and concentration-dependent inhibition of MT1-MMP expression by siRNA. Semi-quantitative evaluation of MT1-MMP expression was performed by fluorometric scanning of the gel and computerized using BioProfile software (Vilbert-Lourmat, Marne la Vallée, France). Data represent the mean±s.d. of four experiments. NS, not significant; ^*P<0.05; ^**P<0.01; ^***P<0.001.