Figure 4. DC migration relies on... | Rockefeller University Press

$DC migration relies on membrane-bound galectin-9 fraction. (A) moDCs were transfected with LGALS9 or a NT siRNA. 48 h after transfection, gal9 KD cells were treated with 1 µg/ml recombinant galectin-9 (rGal9) protein (gal9 KD + rGal9) or nothing as a negative control for 24–48 h. Surface only (left) and total (right) galectin-9 depletion and treatment with exogenous protein were confirmed by flow cytometry 48 h after treatment. Gray population = WT; orange population = gal9 KD; black population = gal9 KD + rGal9; dotted line = isotype control. Numbers in the inset indicate gMFI. (B and C) moDCs treated as per (A) were embedded in 3D collagen matrices followed by time-lapse microscopy imaging to individually track cell migration. At least 20 cells were analyzed for each donor and transfection or treatment. (B) Violin plot showing average cell velocity of six individual donors (black symbols). Lines connect paired donors. (C) Average MSD ± SEM of five individual donors. (D) Individual trajectories of WT, gal9 KD, and gal9 KD + rGal9 moDCs. End points of tracks are indicated by red dots. Data show a representative donor out of six analyzed. (E) WT, gal9 KD, and gal9 KD moDCs treated with rGal9 for either 3, 24, or 48 h were embedded in a 3D collagen matrix, and their velocity was calculated. The graph depicts average velocity ± SEM for one representative donor out of 4 analyzed. (F) WT and gal9 KD moDCs were treated with rGal9 for 3 h prior to being embedded into 3D collagen matrices, and cell velocity was determined. Data show mean cell velocity ± SEM of three independent experiments (gray symbols). One-way ANOVA with a Bonferroni posttest correction (B, E, and F) or a two-way ANOVA (C) was conducted between WT, gal9 KD, and gal9 KD + rGal9 moDC conditions. n.s., P > 0.05; *P < 0.05; **P < 0.01. gMFI, geometrical mean fluorescence intensity.$

Figure 4.

DC migration relies on membrane-bound galectin-9 fraction. (A) moDCs were transfected with LGALS9 or a NT siRNA. 48 h after transfection, gal9 KD cells were treated with 1 µg/ml recombinant galectin-9 (rGal9) protein (gal9 KD + rGal9) or nothing as a negative control for 24–48 h. Surface only (left) and total (right) galectin-9 depletion and treatment with exogenous protein were confirmed by flow cytometry 48 h after treatment. Gray population = WT; orange population = gal9 KD; black population = gal9 KD + rGal9; dotted line = isotype control. Numbers in the inset indicate gMFI. (B and C) moDCs treated as per (A) were embedded in 3D collagen matrices followed by time-lapse microscopy imaging to individually track cell migration. At least 20 cells were analyzed for each donor and transfection or treatment. (B) Violin plot showing average cell velocity of six individual donors (black symbols). Lines connect paired donors. (C) Average MSD ± SEM of five individual donors. (D) Individual trajectories of WT, gal9 KD, and gal9 KD + rGal9 moDCs. End points of tracks are indicated by red dots. Data show a representative donor out of six analyzed. (E) WT, gal9 KD, and gal9 KD moDCs treated with rGal9 for either 3, 24, or 48 h were embedded in a 3D collagen matrix, and their velocity was calculated. The graph depicts average velocity ± SEM for one representative donor out of 4 analyzed. (F) WT and gal9 KD moDCs were treated with rGal9 for 3 h prior to being embedded into 3D collagen matrices, and cell velocity was determined. Data show mean cell velocity ± SEM of three independent experiments (gray symbols). One-way ANOVA with a Bonferroni posttest correction (B, E, and F) or a two-way ANOVA (C) was conducted between WT, gal9 KD, and gal9 KD + rGal9 moDC conditions. n.s., P > 0.05; *P < 0.05; **P < 0.01. gMFI, geometrical mean fluorescence intensity.

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