Fig. 1

αII-spectrin depletion in endothelial cells modifies their shape and actin cytoskeleton. a Western blotting of αII-spectrin in HMEC-1 cells and HUVECs. Lysates (20 μg) of cells transfected with either siRNAs targeting αII-spectrin (Sp siRNA) or non-relevant siRNA (Nr siRNA) were analyzed 72 h after transfection. αII-spectrin and lamin A/C (used as a loading control) levels were checked using polyclonal antibodies. Knockdown of αII-spectrin was efficient with two siRNAs (see the Experimental procedure section). Residual expression of αII-spectrin in Sp siRNA-transfected cells was about 20 to 30%. The transfection efficiency in the cells was about 95% (as evaluated by flow cytometry, data not shown). b Analysis of cell morphology and actin cytoskeleton. HMEC-1 cells and HUVECs were transfected with siRNAs targeting either αII-spectrin (Sp siRNA) or non-relevant siRNA (Nr siRNA). Cells were labelled 72 h after transfection with polyclonal antibodies directed against αII-spectrin (labelled green in HUVECs and red in HMEC-1 cells) and phalloïdin toxin detecting actin (red in HUVECs and green in HMEC-1 cells). Sp siRNA transfection induces a decreased labelling of αII-spectrin that accumulates into some aggregates (arrows). Spectrin depletion also modifies the actin architecture in endothelial cells: depleted HUVECs and HMEC-1 cells present a marked phenotype with disorganization of stress fibers, patches and aggregates (arrows). Scale bar = 20 μm