Fig. 2. Time-lapse observation of -SMA-EGFP in SMA-FP-treated cells and determination of the morphological features of RLSs. (A) Cells were trypsinized and plated into observation chambers for 3 days in DMEM-FCS (2%). Before recording, cells were mounted on an inverted confocal microscope (LSM510, Carl Zeiss) and treated with SMA-FP. EGFP images were collected every 2 minutes for 90 minutes. RLSs appeared mainly in the submembrane area or along stress fibers (arrowhead); fluorescence in the -SMA-EGFP positive stress fibers (arrows) decreases over time. Scale bar, 20 µm. See also videos in supplementary material. (B) Length (a) and diameter (b) of RLSs were determined on digitized confocal images using Openlab 3.0.6 software. Their length varied from 2 to 20 µm and their diameter from 0.1 to 1.2 µm. (C) -SMA-EGFP transfected cells were either non-treated (Cont), or treated for 60 minutes with control peptide SKA-FP or SMA-FP. Reversibility of SMA-FP treatment was assessed by removing the peptide by washing cells with fresh medium for 60 minutes. The percentage of EGFP-positive cells displaying RLSs was estimated by counting a minimum of 200 cells. RLSs were visible in approximately 60% of the transfected cells and the effect of SMA-FP was reversible. Controls in the absence of SMA-FP or in the presence of SKA-FP were close to baseline. Bars represent s.e.m. (**P0.001 compared to control). (D) Non-transfected cells were treated with 5 µg/ml SMA-FP for 30 minutes, fixed and permeabilized with 1% paraformaldehyde for 10 minutes followed by methanol for 3 minutes and stained with anti-actin antibody (see Table 1). By using this detergent-free procedure, RLSs were also found in non-transfected cells. Scale bar, 20 µm.