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Figure 6 | Vascular Cell

Figure 6

From: Regulation of endothelial permeability and transendothelial migration of cancer cells by tropomyosin-1 phosphorylation

Figure 6

Phosphorylation of Tm1 at Ser283 is associated with a decrease of cancer cells migration through the endothelial barrier in response to H 2 O 2 . Exponentially growing HUVECs were transduced with CSII-Venus (empty vector; MOI of 30.8) or CSII-Venus-Tm1wt (Tm1 wild-type vector; MOI of 10.4) or CSII-Venus-Tm1S283A (non-phosphorylatable Tm1 mutant vector; MOI of 9.9) using lentivirus-mediated infection. These Tm1 constructs were insensitive to siRNA7Tm1 that was transfected 24 hours later in each condition. The next day, HUVECs were plated at a density of 1.0x105 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.

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