Fig. 2

PAR-3 knockdown impaired astrocyte migration and adhesion, but not Golgi polarization induced by Thy-1. a Immunoblotting for PAR-3 isoforms in cells transfected with PAR-3 siRNA compared to control siRNA. Actin was the loading control. Migration was evaluated using the wound-healing assay. Evaluated were DI TNC1 cells with silenced PAR-3 or control, stimulated or not (NS) with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL). Pictures show overlapping images of wound closure at time zero (red) and after 24 h (green). Dotted line shows the wound-closure boundary. Graph shows wound closure in relative units. b DI TNC1 astrocytes, transfected as in (a), were stimulated with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL) for 7 h. After fixation, cells were stained for nucleus (DAPI) and Golgi apparatus using an anti-giantin antibody. Golgi (red) positioning within an angle of 120° in front of the nucleus (blue), facing the wound, was considered polarized (1). Non-polarized cells are labelled as (2). Arrow shows the direction of migration. One hundred cells were monitored per condition. Graph shows the percentage of polarized cells. Scale bar = 10 μm. c DI TNC1 astrocytes, transfected as in (a), were stimulated for 15 min. FAs were visualized by immunofluorescence using an anti-vinculin antibody (red) and DAPI for nuclei (blue). Scale bar = 10 μm. Image analyses were performed with the Analyze Particles Tool in the Fiji image processing software [44]. Values in the graphs represent the mean ± s.e.m. from 3 independent experiments (n = 3) determined from at least 50 cells in each experimental condition per experiment. Significant differences are indicated as *P < 0.05