Galectin-9 regulates uropod contractility. (A) Time-lapse sequence of representative WT, gal9 KD, and gal9 KD+rGal9 moDCs migrating in a 3D collagen gel. Scale bar = 50 µm. Scale bar = 10 µm. (B and C) Mean ± SEM duration (min) of uropod presence (B) and cell elongation factor (C) in WT, gal9 KD, and gal9 KD + rGal9 moDCs for four independent donors (black dots). At least 20 cells were analyzed for each donor and condition. (D) Velocity of the front or rear in WT and gal9 KD moDCs. Graphs depict the mean ± SEM for four independent donors (individual cells represented by black symbols). (E) Total lysates from WT, gal9 KD, and gal9 KD +rGal9 moDCs were subjected to western blot and galectin-9 and total RhoA expression analyzed. Tubulin was used as a loading control. Immunoblot is representative of four independent donors. (F) Levels of active (GTP-bound) RhoA in WT and gal9 KD moDCs detected by immunoblotting. Rhotekin beads alone were used as a negative control (lanes 2 and 3). (G) Quantification of data shown in F. The graph depicts relative active RhoA content in galectin-9–depleted DCs (gal9 KD) compared with relevant WT control for three independent experiments. (H) WT or gal9 KD moDCs were embedded in 3D collagen matrices prior to being treated with a RhoA activator or PBS as a negative control. The graph depicts mean cell velocity ± SEM for four independent donors (black symbols). At least 20 cells were analyzed for each donor and condition. (I) Individual trajectory plots of WT and gal9 KD DCs untreated or treated with the RhoA activator of one representative donor out of four analyzed. End track points are indicated by red dots. The black line indicates the overall movement in x and y direction (µm). One-way ANOVA test with a Bonferroni posttest correction was conducted to test significance. n.s., P > 0.05; *P < 0.05; **P < 0.01. Source data are available for this figure: SourceData F5.
Figure 5.

Galectin-9 regulates uropod contractility. (A) Time-lapse sequence of representative WT, gal9 KD, and gal9 KD+rGal9 moDCs migrating in a 3D collagen gel. Scale bar = 50 µm. Scale bar = 10 µm. (B and C) Mean ± SEM duration (min) of uropod presence (B) and cell elongation factor (C) in WT, gal9 KD, and gal9 KD + rGal9 moDCs for four independent donors (black dots). At least 20 cells were analyzed for each donor and condition. (D) Velocity of the front or rear in WT and gal9 KD moDCs. Graphs depict the mean ± SEM for four independent donors (individual cells represented by black symbols). (E) Total lysates from WT, gal9 KD, and gal9 KD +rGal9 moDCs were subjected to western blot and galectin-9 and total RhoA expression analyzed. Tubulin was used as a loading control. Immunoblot is representative of four independent donors. (F) Levels of active (GTP-bound) RhoA in WT and gal9 KD moDCs detected by immunoblotting. Rhotekin beads alone were used as a negative control (lanes 2 and 3). (G) Quantification of data shown in F. The graph depicts relative active RhoA content in galectin-9–depleted DCs (gal9 KD) compared with relevant WT control for three independent experiments. (H) WT or gal9 KD moDCs were embedded in 3D collagen matrices prior to being treated with a RhoA activator or PBS as a negative control. The graph depicts mean cell velocity ± SEM for four independent donors (black symbols). At least 20 cells were analyzed for each donor and condition. (I) Individual trajectory plots of WT and gal9 KD DCs untreated or treated with the RhoA activator of one representative donor out of four analyzed. End track points are indicated by red dots. The black line indicates the overall movement in x and y direction (µm). One-way ANOVA test with a Bonferroni posttest correction was conducted to test significance. n.s., P > 0.05; *P < 0.05; **P < 0.01. Source data are available for this figure: SourceData F5.

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