Extended Data Fig. 4: Depletion of talin significantly impairs invasion into brain slices.
a-e, Talin 1 KO. The talin 1 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. 2 corresponds to clone 1). (a) Percentage of scramble control, talin 1 clone-1 KO and talin 1 clone-2 KO cells adhered to vessels after 24h of ex vivo culture (Scr: n=7, Tln1 KO: n=8, 4 experiments). (b) Left: representative western blot of talin 1 and talin 2 expression in scramble control, talin 1 clone-1 KO and talin 1 clone-2 KO cells. Right: quantification of all western blots (one per experiment, 4 experiments). (c) Violin plots of cell shape circularities for scramble control, talin 1 clone-1 KO and talin 1 clone-2 KO cells adhered to the vasculature (Scr: n=826, 7 slices; Tln1 KO c1: n=725, 8 slices; Tln1 KO c2: n=741, 8 slices; 4 experiments). (d) Violin plots of invasion depths for scramble control, talin 1 clone-1 KO and talin 1 clone-2 KO cells moving through the bulk brain tissue (Scr: n=425, 7 slices; Tln1 KO c1: n=521, 8 slices; Tln1 KO c2: n=721, 8 slices; 4 experiments). (e) Violin plots of invasion depths for scramble control, talin 1 clone-1 KO and talin 1 clone-2 KO cells moving along the vasculature (Scr: n=826, 7 slices; Tln1 KO c1: n=725, 8 slices; Tln1 KO c2: n=741, 8 slices; 4 experiments). f-i, Talin1+2 KO. (f) Percentage of scramble control, talin 2 KO and talin 1 and 2 KO cells adhered to vessels after 24h of ex vivo culture (n=8 slices, 4 experiments). (g) Left: representative western blot of talin 1 and talin 2 expression in scramble control, talin 2 KO and talin 1 and 2 KO cells. Right: quantification of all western blots (one per experiment, 4 experiments). (h) Violin plots of invasion depths for scramble control, talin 2 KO and talin 1 and 2 KO cells moving through the bulk brain tissue (Scr: n=560, Tln2 KO: n=509, Tln1+Tln2 KO: n=515, 8 slices, 4 experiments). (i) Violin plots of invasion depths for scramble control, talin 2 KO and talin 1 and 2 KO cells moving along the vasculature (Scr: n=717, Tln2 KO: n=640, Tln1+Tln2 KO: n=544, 8 slices, 4 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 all other graphs, statistical analysis was performed using a one-way ANOVA test followed by a post hoc Tukey’s test. Graphs show mean ± standard deviation. For western blot quantification, protein expression was normalized to loading control (β-actin), and to the expression on the scramble control condition.
