Fig. 10. Loss of Nesprin-2 Giant affects fibroblast migration and cell polarity. (A-F) In contrast to WT fibroblasts (A-C), wounds generated by scratching remain unclosed in Nesprin-2-Giant-deficient fibroblasts (D-F). Fibroblasts of passage 2 were used and KO fibroblasts were verified to be negative for K56-382 staining prior to usage. A representative experiment is shown from three separate experiments. Scale bars: 200 µm. (G) Mutant cells exhibit a statistically significant reduced migration velocity. Mean values and s.d. were calculated from three different primary WT and five different mutant cell cultures, which were assayed in triplicate experiments. (H,I) Indirect immunofluorescence examination of WT and Nesprin-2-Giant-deficient cell monolayers 6-hours post-wounding, using antibodies against Golgi (GM130; row H) and MTOC (-tubulin; row I). The broken lines indicate the migrating cell front, which was visualized by FITC-phalloidin counterstaining. Note that both the Golgi complex and MTOC are not positioned towards the wound edge in the mutants. (H',I') Statistical evaluation of the representative experiments shown in H,I indicates a defective cell polarization in Nesprin-2-Giant mutants. Cells with Golgi structures (500 cells counted for each cell type) and MTOCs (300 cells counted for each cell type) positioned within a 120° sector facing the wound edge were assessed as polarized. Error bars denote s.d. from three different experiments. The statistical significance (P values; Student's t-test) is also indicated. Scale bar: 10 µm.