Fig. 7. SiRNA₁₀₇ decreases the amount of MT1-MMP, suppresses proMMP-2 activation and abolishes pseudotube formation by HMECs cultured on matrigel. HMECs were cultured in six-well culture plates and transfected at 60% confluency with 25 nM siRNA₁₀₇ or scrambled siRNA₁₀₇ (ssiRNA₁₀₇) or oligofectamine alone (OL) for 72 hours. Then, the medium was replaced with fresh medium containing 2% (v/v) FCS and cells were incubated in the absence (-KE) or presence (+KE) of 10 µg/ml KE or 10^-7 M phorbol myristate acetate (PMA) for 24 hours. Total MT1-MMP (A) and proMMP-2 activation (B) was analyzed by ELISA and zymography analysis, respectively. (C,D) HMECs were transfected with 25 nM siRNA₁₀₇ or scrambled siRNA₁₀₇ (ssiRNA₁₀₇) for 72 hours and then cultured on matrigel for 24 hours in ECGM MV in the presence of 1 µg/ml KE. OF, HMECs incubated with oligofectamine alone. Non-transfected HMECs were cultured on matrigel in the absence (control) or presence of 1 µg/ml KE (+KE). (D) Tube formation was observed with a phase-contrast microscope equipped with a digital camera. Images were taken using a phase-contrast microscope with a x10 lens. (C) Semi-quantitative evaluation of pseudotubes was performed after white and black pixellization of images and was expressed as black pixels relative to total pixels from 20 selected fields. Data represent the mean±s.d. of four experiments. ^***P<0.001.