Extended Data Fig. 7: Depletion of vinculin significantly impairs invasion into brain slices.
The vinculin CRISPR KO cell lines were established as clonal cell lines by single cell sorting after infection with the CRISPR lentivirus, and two cell lines were analysed (data shown in Fig. 5 corresponds to clone 2). (a) Percentage of scramble control, vinculin clone-1 KO and vinculin clone-2 KO cells adhered to vessels after 24h of ex vivo culture (n=5, 3 experiments). Statistical analysis: one-way ANOVA test followed by a post hoc Tukey’s test. Graphs show mean ± standard deviation. (b) Left: representative western blot of vinculin expression in scramble control, vinculin clone-1 KO and vinculin clone-2 KO cells. Right: quantification of all western blots (one per experiment, 3 experiments). (c) Violin plots of cell shape circularities for scramble control, vinculin clone-1 KO and vinculin clone-2 KO cells adhered to the vasculature (Scr: n=411, Vcl KO c1: n=453, Vcl KO c2: n=474, 5 slices, 3 experiments). (d) Violin plots of invasion depths for scramble control, vinculin clone-1 KO and vinculin clone-2 KO cells moving through the bulk brain tissue (Scr: n=242, Vcl KO c1: n=385, Vcl KO c2: n=411, 5 slices, 3 experiments). (e) Violin plots of invasion depths for scramble control, vinculin clone-1 KO and vinculin clone-2 KO cells moving along the vasculature (Scr: n=411, Vcl KO c1: n=453, Vcl KO c2: n=474, 5 slices, 3 experiments). For all violin plots, dots represent the median values per slice analysed. Statistical analysis: Kruskal–Wallis test followed by a post hoc Dunn’s test. Graphs show median ± interquartile range. For western blot quantification, protein expression was normalized to loading control (β-actin), and to the expression on the scramble control condition.
