Figure 4

Phosphorylation of Tm1 at Ser283 regulates endothelial permeability. Exponentially growing HUVECs were transduced or not with CSII-Venus (empty vector; MOI of 30.2) or CSII-Venus-Tm1wt (Tm1 wild-type vector; MOI of 7.59) or CSII-Venus-Tm1S283A (non-phosphorylatable Tm1 mutant vector; MOI of 6.66) using lentivirus-mediated infection. These Tm1 constructs are insensitive to siRNA7Tm1. After 24 hours, infected HUVECs were transfected with siRNA7Tm1. In A) HUVECs were plated at a density of 1x10⁵ cells per well in the upper part of a Boyden chamber and were cultivated for 3 days until the formation of a tight monolayer. Thereafter, NaF (1 mM for 1 hour) was added to the incubation medium (upper chamber) to inhibit phosphatases and to allow the accumulation of phosphorylated tropomyosin-1. Then, H₂O₂ (250 μM for 30 or 60 minutes) was added to the upper chamber in the presence of 1 mg/ml FITC-labelled dextran. Passage of FITC-labelled dextran through the monolayer of HUVECs was measured immediately after exposure of HUVECs to H₂O₂. The data represent permeability expressed as the mean fold increase (± SEM) relative to untreated cells (n=4 for each condition obtained from a representative experiment out of 4). p value was determined by using unpaired Student t test. In B) HUVECs were plated at a density of 2×10⁵ cells on gelatin-coated microscope cover glasses into 35 mm Petri dish per condition and were cultivated for 2 days until the formation of a tight monolayer. Thereafter, cells were treated with H₂O₂ (250 μM) for 30 minutes (d-f, j-l and p-r) or were left untreated (a-c, 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, m and p), and for Tm1 using anti-tropomyosin monoclonal mouse antibody(b, e, h, k, n and q). A merged picture is shown for each condition (c, f, i, l, o, and r). A representative field for each condition was captured using a Nikon Eclipse 600 fluorescence microscope (40X).