Figure 5
Figure 5. RNAi of cytohesin-1 strongly reduces chemotaxis of mature integrin(+/+) but not of integrin(−/−) BM-DCs in 3-dimensional collagen. Using 3D collagen type I gels, we analyzed the role of cytohesin-1 in BM-DC complex matrix migration. Quantification of time series after live cell imaging shows that directionality, y-forward migration index, and velocity of cytohesin-1 knockdown cells toward the chemokine CCL19 are strongly reduced (C,E,F). RNAi of RhoA results in a highly similar phenotype (D-F). Respective silencing efficiencies are shown by Western blot analysis (G,H). Quantification of 3D migration of integrin-deficient BM-DCs shows that directionality, y-forward migration index, and velocity of cytohesin-1 knockdown cells toward the chemokine CCL19 are strongly reduced in wild-type, that is, integrin(+/+) BM-DCs (J,M,N) but not in integrin(−/−) BM-DCs (L-N). Efficiency of cytohesin-1 RNAi was comparable in both cell types, as shown by Western blot analysis (O). For every 3D migration experiment, the tracks of 60 individual cells were monitored over a period of 3 hours by capturing digital images every 5 minutes. Cell directionality, forward migration index, and velocity were calculated and visualized as plots (A-D, I-L) and animated plots (Videos S1–S8) by analyzing the acquired data with the Chemotaxis and Migration tool plug-in (Ibidi) for ImageJ. Error bars indicate ± SD. ***P < .001; ns indicates not significant. The data are representative for 3 independent experiments.

RNAi of cytohesin-1 strongly reduces chemotaxis of mature integrin(+/+) but not of integrin(−/−) BM-DCs in 3-dimensional collagen. Using 3D collagen type I gels, we analyzed the role of cytohesin-1 in BM-DC complex matrix migration. Quantification of time series after live cell imaging shows that directionality, y-forward migration index, and velocity of cytohesin-1 knockdown cells toward the chemokine CCL19 are strongly reduced (C,E,F). RNAi of RhoA results in a highly similar phenotype (D-F). Respective silencing efficiencies are shown by Western blot analysis (G,H). Quantification of 3D migration of integrin-deficient BM-DCs shows that directionality, y-forward migration index, and velocity of cytohesin-1 knockdown cells toward the chemokine CCL19 are strongly reduced in wild-type, that is, integrin(+/+) BM-DCs (J,M,N) but not in integrin(−/−) BM-DCs (L-N). Efficiency of cytohesin-1 RNAi was comparable in both cell types, as shown by Western blot analysis (O). For every 3D migration experiment, the tracks of 60 individual cells were monitored over a period of 3 hours by capturing digital images every 5 minutes. Cell directionality, forward migration index, and velocity were calculated and visualized as plots (A-D, I-L) and animated plots (Videos S1Video 2. Animated chemotaxis plot appendant to Video 1 (MOV, 773 KB)Video 3. Mature BM-DC that were transfected with control-siRNA show high migratory capacity in 3-dimensional collagen, as well as directionality towards a diffusive chemotactic gradient (CCL19 on top) (MOV, 4.49 MB)Video 4. Animated chemotaxis plot appendant to Video 3 (MOV, 814 KB)Video 5. Mature BM-DC that were transfected with Cytohesin1-siRNA show in 3-dimensional collagen obvious deficiencies in directionality and migration velocity towards a diffusive chemotactic gradient (CCL19 on top) (MOV, 3.61 MB)Video 6. Animated chemotaxis plot appendant to Video 5 (MOV, 799 KB)Video 7. Mature BM-DC that were transfected with RhoA-siRNA show in 3-dimensional collagen obvious deficiencies in directionality and migration velocity towards a diffusive chemotactic gradient (CCL19 on top) (MOV, 4.79 MB)–S8) by analyzing the acquired data with the Chemotaxis and Migration tool plug-in (Ibidi) for ImageJ. Error bars indicate ± SD. ***P < .001; ns indicates not significant. The data are representative for 3 independent experiments.

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