Figure 1. Galectin-9 depletion reduces... | Rockefeller University Press

$Galectin-9 depletion reduces DC chemotactic migration. (A) Schematic representation of the transwell migration assay used. WT or gal9 KD DCs were seeded on a collagen gel in the top chamber of a transwell (5-µm porous membrane) and subjected to a CCL21 chemokine gradient. Migratory cells were collected after 24 h and quantified. (B) Day 3 moDCs were transfected with either a LGALS9 siRNA or a NT siRNA as a negative control, matured at day 6 for 48 h, and subjected to a transwell migration assay as per (A). Representative flow graphs from the migratory moDC fraction are shown. (C) Quantification of donors (black symbols) shown in B. Data show the percentage of moDCs that migrated relative to input, and lines connect matched WT and gal9 KD DCs. (D) Mature WT or gal9 KD moDCs were stained with CellTrace Far Red, seeded on top of a collagen layer overlaying a transwell chamber, and subjected to a CCL21 chemokine gradient. Cells were left to migrate for 24 h after which collagen gels were fixed and imaged by confocal microscopy. (E) Quantification of area fraction signal in WT and gal9 KD DC containing collagens over 10 z-planes from images shown in D from three independent donors (black symbols). (F) Schematic representation of the glass migration chamber used to analyze chemotaxis. WT or galectin-9–depleted moDCs were embedded in a collagen gel at the top of a glass chamber, and medium containing CCL21 was placed below. (G) Schematic of the migratory angle distribution in WT and gal9 KD DCs in the absence of any chemoattractant (- CCL21) or when subjected to CCL21 (+CCL21). Only WT cells are shown in the −CCL21 condition. (H) Overall angle to y axis (degrees) was computed for the overall displacement vector of WT or gal9 KD DCs subjected to CCL21 or nothing as a negative control. Each data point represents 1 cell track pooled from four independent donors with 50 cells analyzed per donor. Horizontal lines represent means for untreated negative control (gray), WT (black), or gal9 KD DCs (red). Plots show the bootstrapped distribution (red) and 95% confidence interval (line segments) of the difference in average angle (gal9 KD-WT). Unpaired Student’s t test was conducted to compare WT and gal9 KD cells. *P < 0.05.$

Figure 1.

Galectin-9 depletion reduces DC chemotactic migration. (A) Schematic representation of the transwell migration assay used. WT or gal9 KD DCs were seeded on a collagen gel in the top chamber of a transwell (5-µm porous membrane) and subjected to a CCL21 chemokine gradient. Migratory cells were collected after 24 h and quantified. (B) Day 3 moDCs were transfected with either a LGALS9 siRNA or a NT siRNA as a negative control, matured at day 6 for 48 h, and subjected to a transwell migration assay as per (A). Representative flow graphs from the migratory moDC fraction are shown. (C) Quantification of donors (black symbols) shown in B. Data show the percentage of moDCs that migrated relative to input, and lines connect matched WT and gal9 KD DCs. (D) Mature WT or gal9 KD moDCs were stained with CellTrace Far Red, seeded on top of a collagen layer overlaying a transwell chamber, and subjected to a CCL21 chemokine gradient. Cells were left to migrate for 24 h after which collagen gels were fixed and imaged by confocal microscopy. (E) Quantification of area fraction signal in WT and gal9 KD DC containing collagens over 10 z-planes from images shown in D from three independent donors (black symbols). (F) Schematic representation of the glass migration chamber used to analyze chemotaxis. WT or galectin-9–depleted moDCs were embedded in a collagen gel at the top of a glass chamber, and medium containing CCL21 was placed below. (G) Schematic of the migratory angle distribution in WT and gal9 KD DCs in the absence of any chemoattractant (- CCL21) or when subjected to CCL21 (+CCL21). Only WT cells are shown in the −CCL21 condition. (H) Overall angle to y axis (degrees) was computed for the overall displacement vector of WT or gal9 KD DCs subjected to CCL21 or nothing as a negative control. Each data point represents 1 cell track pooled from four independent donors with 50 cells analyzed per donor. Horizontal lines represent means for untreated negative control (gray), WT (black), or gal9 KD DCs (red). Plots show the bootstrapped distribution (red) and 95% confidence interval (line segments) of the difference in average angle (gal9 KD-WT). Unpaired Student’s t test was conducted to compare WT and gal9 KD cells. *P < 0.05.

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