Figure S2.
Perturbation of the MT cytoskeleton affects DCs migration and subcellular Lfc localization. (a) Non-treated control or nocodazole-treated cells migrating under agarose toward a CCL19 gradient were fixed and stained for endogenous distribution of α-tubulin and F-actin. Blue line indicates position of the nucleus; red line, cell outline. Scale bar, 10 µm. (b) In situ migration of endogenous DCs on a mouse ear sheet. Z-projections of separated ear sheets upon control conditions or nocodazole (Noco.) treatment. Lymphatic vessels were stained for Lyve-1 and DCs for MHC-II. Right panel: Mean distance from lymphatic vessels of endogenous DCs was determined 48 h after ear separation. Per condition, four mouse ears with two fields of view were analyzed. Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Annotation above columns indicates results of unpaired Student’s t test; **, P ≤ 0.01. Scale bar, 100 µm. (c) Nocodazole-treated DC migrating in a collagen gel toward a soluble CCL19 gradient. Yellow line outlines cell shape. Red arrowheads indicate the loss of cellular coherence. Scale bar, 100 µm. (d) Levels of active RhoA upon MT depolymerization with nocodazole determined by luminometry. RhoA activity levels were normalized to nocodazole-treated samples. Plotted is mean ± SD from N = 3 experiments. Annotation above columns indicates results of unpaired Student’s t test; ****, P ≤ 0.0001. (e) Levels of MLC phosphorylation determined by Western blot analysis. Cells were treated with the indicated compounds (DMSO, nocodazole [Noco.], or Y27632 together with nocodazole [Y./N.]). Right panel: The mean fluorescence intensity of phospho-MLC (pMLC) was normalized to the GAPDH signal and shown as fold increase relative to DMSO control ± SD. Blots are representative of n = 3 experiments. r.u., relative units. (f) Co-localization of Lfc-GFP on α-tubulin (a.-tub.) structures. An Lfc-GFP–expressing cell was fixed while migrating under agarose and stained for α-tubulin distribution. Scale bar, 10 µm. (g) Polarized distribution of Lfc-GFP in trailing edges and retracting protrusions. A double-reporter cell expressing Lfc-GFP and EB3-mcherry was followed while migrating under agarose. Purple arrowheads denote trailing edge, and orange arrowheads highlight retracting protrusion followed by cell repolarization. Scale bar, 10 µm. (h) Lfc-GFP distribution upon nocodazole treatment. A nocodazole-treated double–fluorescent reporter cell was followed while migrating under agarose. Note the absence of filamentous structures in both channels and the diffuse signal distribution of Lfc-GFP. Scale bar, 10 µm.

Perturbation of the MT cytoskeleton affects DCs migration and subcellular Lfc localization. (a) Non-treated control or nocodazole-treated cells migrating under agarose toward a CCL19 gradient were fixed and stained for endogenous distribution of α-tubulin and F-actin. Blue line indicates position of the nucleus; red line, cell outline. Scale bar, 10 µm. (b) In situ migration of endogenous DCs on a mouse ear sheet. Z-projections of separated ear sheets upon control conditions or nocodazole (Noco.) treatment. Lymphatic vessels were stained for Lyve-1 and DCs for MHC-II. Right panel: Mean distance from lymphatic vessels of endogenous DCs was determined 48 h after ear separation. Per condition, four mouse ears with two fields of view were analyzed. Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Annotation above columns indicates results of unpaired Student’s t test; **, P ≤ 0.01. Scale bar, 100 µm. (c) Nocodazole-treated DC migrating in a collagen gel toward a soluble CCL19 gradient. Yellow line outlines cell shape. Red arrowheads indicate the loss of cellular coherence. Scale bar, 100 µm. (d) Levels of active RhoA upon MT depolymerization with nocodazole determined by luminometry. RhoA activity levels were normalized to nocodazole-treated samples. Plotted is mean ± SD from N = 3 experiments. Annotation above columns indicates results of unpaired Student’s t test; ****, P ≤ 0.0001. (e) Levels of MLC phosphorylation determined by Western blot analysis. Cells were treated with the indicated compounds (DMSO, nocodazole [Noco.], or Y27632 together with nocodazole [Y./N.]). Right panel: The mean fluorescence intensity of phospho-MLC (pMLC) was normalized to the GAPDH signal and shown as fold increase relative to DMSO control ± SD. Blots are representative of n = 3 experiments. r.u., relative units. (f) Co-localization of Lfc-GFP on α-tubulin (a.-tub.) structures. An Lfc-GFP–expressing cell was fixed while migrating under agarose and stained for α-tubulin distribution. Scale bar, 10 µm. (g) Polarized distribution of Lfc-GFP in trailing edges and retracting protrusions. A double-reporter cell expressing Lfc-GFP and EB3-mcherry was followed while migrating under agarose. Purple arrowheads denote trailing edge, and orange arrowheads highlight retracting protrusion followed by cell repolarization. Scale bar, 10 µm. (h) Lfc-GFP distribution upon nocodazole treatment. A nocodazole-treated double–fluorescent reporter cell was followed while migrating under agarose. Note the absence of filamentous structures in both channels and the diffuse signal distribution of Lfc-GFP. Scale bar, 10 µm.

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