Figure 5.

The knockdown of human Tm1 in endothelial cells is associated with an increase of transendothelial migration of cancer cells. Exponentially growing HUVECs were transfected or not with siRNA7Tm1 or with non-targeting negative control siRNA. The day after, HUVECs were plated at a density of 0.6×10⁵ per well in the upper part of a Boyden chamber and were cultivated for 3 days until the formation of a tight monolayer. Thereafter, H₂O₂ (250 μM) was added or not to the upper chamber for 30 minutes. Then, the medium was removed and replaced by migration buffer. HT-29 cells previously stained with calcein were added on the upper chamber at a density of 1.5×10⁵ cells per well. After 4 hours and a half, HT-29 cells that crossed the membrane of the Boyden chamber were counted in five different fields using a TE300 Nikon fluorescence microscope (20X). In each condition, the number of HT-29 trans-migrating cells was calculated from triplicate samples of a representative experiment (mean±SEM). p value was determined by using unpaired Student t test.