Fluorescence Labeling of VSM Cells

For fluorescence labelling of 48 h VSM (25 μg/ml) treated cells, the cells were grown in Ibidi dishes (Ibidi GmbH, Martinsried, Germany), fixed in 4% paraformaldehyde (Santa Cruz, Dallas, USA), permeabilized with 0.1% Triton X-100 (Santa Cruz, Dallas, USA) and labelled with F-Actin antibody Phalloidin-Alexa 596 (Invitrogen, USA). Afterwards, a counter-staining with Hoechst (PanReacAppliChem, Darmstadt, Germany) was performed. The labelling procedure was also described previously [21 (link), 30 (link)]. The fluorescence microscopical images were captured on a confocal laser-scanning microscope Leica DMi8 (Leica, Wetzlar, Germany). For subsequent quantification of F-actin fibers (stress fibers induction), the confocal images were mathematical processed with the software FilaQuant (University of Rostock, Institute of Mathematics, Mathematical Optimization, Rostock, Germany) as described previously [22 , 33 (link)].