Fig. 2: Impairment of DC migration in constricted areas under Lamtor1 deficiency.

a Chemotaxis of WT (white bar) and Lamtor1^−/− (black bar) BMDCs toward the indicated concentrations of CCL19 (upper) or CCL21 (lower) in a Transwell assay system (pore size, 5 μm). Data are representative of three experiments. b–f Motility of DCs in response to CCL19 in 3D collagen matrices. Movement of WT (upper) and Lamtor1^–/– (lower) BMDCs in response to CCL19 (5 μg/ml) in type I collagen gels (2 mg/ml) in a Zigmond chamber were observed at 1-min intervals by time-lapse video imaging. Velocities of DCs were determined using the ImageJ manual tracking software. Consecutive images of DC locomotion: time (h:min) is shown above each panel. Scale bar, 30 μm (b). Mean velocity of the leading edge (c) and uropod (d), the maximum length of a single cell (e), and percentage of the immotile period of the uropod (f) (n = 40 WT cells and n = 40 KO cells). g MLC phosphorylation in WT and Lamtor1^−/− BMDCs. MLC phosphorylation was evaluated by western blotting with anti-p-MLC antibody (upper). Data are representative of three experiments. Protein concentration in SDS–PAGE gel bands was determined using ImageJ, and statistical analysis was performed (lower) (n = 3). h Localization of LAMP1⁺ lysosomes in non-polarized and polarized DCs. Lamtor1^−/− DCs were stained with anti-LAMP1 (green), phalloidin (blue), and DAPI (white), and visualized by confocal microscopy. Representative images of a non-polarized cell (left) and a polarized cell (right). Scale bar, 10 μm (left). Percentage of the region of interest (ROI) of perinuclear and peripheral regions of non-polarized cells and polarized cells (n = 30) (right). i Lamp1 protein level of WT and Lamtor1^−/− BMDCs. The amount of Lamp1 protein was evaluated by western blotting with anti-Lamp1 antibody (upper). Data are representative of three experiments. The protein concentration in SDS–PAGE gel bands was determined using ImageJ and statistical analysis was performed (lower) (n = 3). Statistical analyses were performed by two-sided Student’s t-test (a, g, i) [means ± s.d.; *p < 0.05, **p < 0.01, ***p < 0.005] or two-sided Mann–Whitney U test (c–f, h) [median; 25th and 75th percentiles; and minimum and maximum of a population excluding outliers; *p < 0.001, **p < 0.0001; NS, not statistically significant].