Fig. 1: Expression of PRRT2 in heterologous cells reduces cell motility and promotes cell aggregation.
From: Proline-rich transmembrane protein 2 (PRRT2) regulates the actin cytoskeleton during synaptogenesis
A Phase-contrast images of NIH 3T3 fibroblasts expressing either EGFP (control) or PRRT2-EGFP taken from timepoint 0 (30 min after scratching) and at the indicated time intervals (h) and showing the wound closure progression. Leading edges are labelled in black for easier visualization. Scale bar: 20 μm. B Quantification of the wounded area invaded at different time points, expressed as percentage of healing at time 0. % Healing: 7 h EGFP = 56.7 ± 12.9, PRRT2-EGFP = 30.3 ± 8.4; 9 h EGFP = 73.2 ± 12.7, PRRT2-EGFP = 39.2 ± 10.1; 11 h EGFP = 81.4 ± 11.2, PRRT2-EGFP = 48.3 ± 12.2; 13 h EGFP = 84.8 ± 8.1, PRRT2-EGFP = 55.6 ± 14.5; p < 0.01. Results represent the mean ± SEM of n = 3 independent experiments, with six movies/group in each experiment. Two-way ANOVA/Bonferroni’s tests; **p < 0.01. C Phase-contrast images denoting the empty area among cells treated as described in A. D Quantification of the area among cells expressed as cumulative area of empty spaces among cells measured in all the frames. Sum of the empty area (pixel2) EGFP = 2,539,468 ± 387,867, PRRT2-EGFP = 1,395,844 ± 209,285; p < 0.05. Data are expressed as means ± SEM of n = 18 movies from three independent experiments. Student’s t-test + Welch-correction; *p < 0.05. E Fluorescence images of HEK293T cells transfected with plasmids coding for either EGFP, PRRT2-EGFP or ∆N-PRRT2-EGFP. Cells were incubated in adhesion favouring conditions. At the end of the assay, cells were fixed and processed for immunofluorescence with β-catenin antibody (red) to label cell edges and Hoechst 33342 to label nuclei (blue). Scale bar: 50 μm. F Quantification of the cell number/cluster from the experiments shown in E. Median n. of cells/cluster: EGFP = 2.8, PRRT2-EGFP = 4.6, ∆N-PRRT2-EGFP = 3.1; p < 0.0001 EGFP vs PRRT2-EGFP, p < 0.001 PRRT2-EGFP vs ∆N-PRRT2-EGFP. Results are expressed as median ± interquartile range from n = total number of fields from three independent experiments carried out in triplicate (total number of fields: EGFP = 60, PRRT2-EGFP = 57, ΔN-PRRT2-EGFP = 62). Kruskal–Wallis/Dunn’s tests; ***p < 0.001, ****p < 0.0001.
