Figure 3

The knockdown of human Tm1 induces an increase in endothelial permeability in response to H ₂ O ₂ . A) HUVECs were electroporated or not with Hs_TPM1_7 siRNA (siRNA7Tm1) or non-targeting silencer negative control siRNA. Three days later, proteins were extracted, were separated by SDS-PAGE, and were transferred onto nitrocellulose membrane. Tm1 level was revealed by western blot using anti-tropomyosin mouse monoclonal antibody (upper panel). Tubulin-α level was measured as loading control (lower panel). The siRNA7Tm1-mediated knockdown of Tm1 has been evaluated as 80% following densitometric quantification of the bands. A representative blot is shown. B) HUVECs transfected as in A using X-tremeGene HP Transfection Reagent were plated at a density of 0.6×10⁵ per well in the upper part of a Boyden chamber and were grown for 3 days until the formation of a tight monolayer. Passage of FITC-labelled dextran through the monolayer of HUVECs was measured after exposure to H₂O₂ (250 μM) for 30 minutes. The data represent permeability expressed as the mean fold increases in treated cells (± SEM) relative to untreated cells (n=3 for each conditions). p value was determined by using unpaired Student t test C) HUVECs were transfected or not (a-c) with siRNAs that specifically target human Tm1 mRNA (j-o) or with non-targeting negative control siRNA (d-i). The next day, HUVECs were plated at a density of 2×10⁵ cells on gelatin-coated microscope cover glasses into 35 mm Petri dish for each condition and were grown for 2 days until the formation of a tight monolayer. Thereafter, cells were treated or not with H₂O₂ (250 μM) for 30 minutes (g-i and m-o). Then, cells were fixed, were permeabilized, and were stained for F-actin using Alexa 488-phalloidin (a, d, g, j and m), and for Tm1 using anti-tropomyosin mouse monoclonal antibody (b, e, h, k and n). A merged picture is shown for each condition (c, f, i, l and o). A representative field for each condition was captured using a Nikon Eclipse 600 fluorescence microscope (40X).