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Galectin-9 depletion impairs chemokine-driven DC migration independently of cell maturation. (A and B) Galectin-9 expression was assessed by western blot (A) or flow cytometry (B) 48 h in WT or gal9 KD moDCs. Immunoblot is representative of four independent experiments. In B, numbers in the inset indicate gMFI. Cells were gated based on their forward-side scatter after which cell clusters were gated out and only single cells were analyzed. WT, light gray population; gal9 KD moDCs, orange population. A black dotted line represents isotype control values. (C) CCR7 surface expression in moDCs treated as per (B). Graph is a representative donor out of three analyzed, and numbers in the inset indicate gMFI. (D and E) WT or gal9 KD moDCs were matured and levels of HLA-DR, CD80, CD86, and <t>CD83</t> analyzed by flow cytometry. The graph depicts the mean ± SEM surface expression (gMFI) of four independent donors. (F) WT or gal9 KD moDCs were placed in the upper chamber of a transwell system and subjected to a CCL21 chemokine gradient. Migrated cells were collected at the bottom chamber 1, 2, and 4 h after seeding and measured by flow cytometry. Graphs show a representative donor of four independent donors analyzed. (G) Quantification of data shown in F. Graph shows mean percentage ± SEM of migratory DCs relative to the total number of seeded cells for each time point and genotype. An unpaired t test between WT and gal9 KD DCs was performed. (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 graph corresponds to one independent donor (labeled donor 1–4; 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). n.s., P > 0.05; *P < 0.05. gMFI, geometrical mean fluorescence intensity. Source data are available for this figure: .
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Galectin-9 depletion impairs chemokine-driven DC migration independently of cell maturation. (A and B) Galectin-9 expression was assessed by western blot (A) or flow cytometry (B) 48 h in WT or gal9 KD moDCs. Immunoblot is representative of four independent experiments. In B, numbers in the inset indicate gMFI. Cells were gated based on their forward-side scatter after which cell clusters were gated out and only single cells were analyzed. WT, light gray population; gal9 KD moDCs, orange population. A black dotted line represents isotype control values. (C) CCR7 surface expression in moDCs treated as per (B). Graph is a representative donor out of three analyzed, and numbers in the inset indicate gMFI. (D and E) WT or gal9 KD moDCs were matured and levels of HLA-DR, CD80, CD86, and <t>CD83</t> analyzed by flow cytometry. The graph depicts the mean ± SEM surface expression (gMFI) of four independent donors. (F) WT or gal9 KD moDCs were placed in the upper chamber of a transwell system and subjected to a CCL21 chemokine gradient. Migrated cells were collected at the bottom chamber 1, 2, and 4 h after seeding and measured by flow cytometry. Graphs show a representative donor of four independent donors analyzed. (G) Quantification of data shown in F. Graph shows mean percentage ± SEM of migratory DCs relative to the total number of seeded cells for each time point and genotype. An unpaired t test between WT and gal9 KD DCs was performed. (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 graph corresponds to one independent donor (labeled donor 1–4; 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). n.s., P > 0.05; *P < 0.05. gMFI, geometrical mean fluorescence intensity. Source data are available for this figure: .
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Galectin-9 depletion impairs chemokine-driven DC migration independently of cell maturation. (A and B) Galectin-9 expression was assessed by western blot (A) or flow cytometry (B) 48 h in WT or gal9 KD moDCs. Immunoblot is representative of four independent experiments. In B, numbers in the inset indicate gMFI. Cells were gated based on their forward-side scatter after which cell clusters were gated out and only single cells were analyzed. WT, light gray population; gal9 KD moDCs, orange population. A black dotted line represents isotype control values. (C) CCR7 surface expression in moDCs treated as per (B). Graph is a representative donor out of three analyzed, and numbers in the inset indicate gMFI. (D and E) WT or gal9 KD moDCs were matured and levels of HLA-DR, CD80, CD86, and <t>CD83</t> analyzed by flow cytometry. The graph depicts the mean ± SEM surface expression (gMFI) of four independent donors. (F) WT or gal9 KD moDCs were placed in the upper chamber of a transwell system and subjected to a CCL21 chemokine gradient. Migrated cells were collected at the bottom chamber 1, 2, and 4 h after seeding and measured by flow cytometry. Graphs show a representative donor of four independent donors analyzed. (G) Quantification of data shown in F. Graph shows mean percentage ± SEM of migratory DCs relative to the total number of seeded cells for each time point and genotype. An unpaired t test between WT and gal9 KD DCs was performed. (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 graph corresponds to one independent donor (labeled donor 1–4; 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). n.s., P > 0.05; *P < 0.05. gMFI, geometrical mean fluorescence intensity. Source data are available for this figure: .
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Galectin-9 depletion impairs chemokine-driven DC migration independently of cell maturation. (A and B) Galectin-9 expression was assessed by western blot (A) or flow cytometry (B) 48 h in WT or gal9 KD moDCs. Immunoblot is representative of four independent experiments. In B, numbers in the inset indicate gMFI. Cells were gated based on their forward-side scatter after which cell clusters were gated out and only single cells were analyzed. WT, light gray population; gal9 KD moDCs, orange population. A black dotted line represents isotype control values. (C) CCR7 surface expression in moDCs treated as per (B). Graph is a representative donor out of three analyzed, and numbers in the inset indicate gMFI. (D and E) WT or gal9 KD moDCs were matured and levels of HLA-DR, CD80, CD86, and <t>CD83</t> analyzed by flow cytometry. The graph depicts the mean ± SEM surface expression (gMFI) of four independent donors. (F) WT or gal9 KD moDCs were placed in the upper chamber of a transwell system and subjected to a CCL21 chemokine gradient. Migrated cells were collected at the bottom chamber 1, 2, and 4 h after seeding and measured by flow cytometry. Graphs show a representative donor of four independent donors analyzed. (G) Quantification of data shown in F. Graph shows mean percentage ± SEM of migratory DCs relative to the total number of seeded cells for each time point and genotype. An unpaired t test between WT and gal9 KD DCs was performed. (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 graph corresponds to one independent donor (labeled donor 1–4; 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). n.s., P > 0.05; *P < 0.05. gMFI, geometrical mean fluorescence intensity. Source data are available for this figure: .
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Galectin-9 depletion impairs chemokine-driven DC migration independently of cell maturation. (A and B) Galectin-9 expression was assessed by western blot (A) or flow cytometry (B) 48 h in WT or gal9 KD moDCs. Immunoblot is representative of four independent experiments. In B, numbers in the inset indicate gMFI. Cells were gated based on their forward-side scatter after which cell clusters were gated out and only single cells were analyzed. WT, light gray population; gal9 KD moDCs, orange population. A black dotted line represents isotype control values. (C) CCR7 surface expression in moDCs treated as per (B). Graph is a representative donor out of three analyzed, and numbers in the inset indicate gMFI. (D and E) WT or gal9 KD moDCs were matured and levels of HLA-DR, CD80, CD86, and <t>CD83</t> analyzed by flow cytometry. The graph depicts the mean ± SEM surface expression (gMFI) of four independent donors. (F) WT or gal9 KD moDCs were placed in the upper chamber of a transwell system and subjected to a CCL21 chemokine gradient. Migrated cells were collected at the bottom chamber 1, 2, and 4 h after seeding and measured by flow cytometry. Graphs show a representative donor of four independent donors analyzed. (G) Quantification of data shown in F. Graph shows mean percentage ± SEM of migratory DCs relative to the total number of seeded cells for each time point and genotype. An unpaired t test between WT and gal9 KD DCs was performed. (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 graph corresponds to one independent donor (labeled donor 1–4; 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). n.s., P > 0.05; *P < 0.05. gMFI, geometrical mean fluorescence intensity. Source data are available for this figure: .
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Galectin-9 depletion impairs chemokine-driven DC migration independently of cell maturation. (A and B) Galectin-9 expression was assessed by western blot (A) or flow cytometry (B) 48 h in WT or gal9 KD moDCs. Immunoblot is representative of four independent experiments. In B, numbers in the inset indicate gMFI. Cells were gated based on their forward-side scatter after which cell clusters were gated out and only single cells were analyzed. WT, light gray population; gal9 KD moDCs, orange population. A black dotted line represents isotype control values. (C) CCR7 surface expression in moDCs treated as per (B). Graph is a representative donor out of three analyzed, and numbers in the inset indicate gMFI. (D and E) WT or gal9 KD moDCs were matured and levels of HLA-DR, CD80, CD86, and CD83 analyzed by flow cytometry. The graph depicts the mean ± SEM surface expression (gMFI) of four independent donors. (F) WT or gal9 KD moDCs were placed in the upper chamber of a transwell system and subjected to a CCL21 chemokine gradient. Migrated cells were collected at the bottom chamber 1, 2, and 4 h after seeding and measured by flow cytometry. Graphs show a representative donor of four independent donors analyzed. (G) Quantification of data shown in F. Graph shows mean percentage ± SEM of migratory DCs relative to the total number of seeded cells for each time point and genotype. An unpaired t test between WT and gal9 KD DCs was performed. (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 graph corresponds to one independent donor (labeled donor 1–4; 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). n.s., P > 0.05; *P < 0.05. gMFI, geometrical mean fluorescence intensity. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Galectin-9 regulates dendritic cell polarity and uropod contraction by modulating RhoA activity

doi: 10.1083/jcb.202404079

Figure Lengend Snippet: Galectin-9 depletion impairs chemokine-driven DC migration independently of cell maturation. (A and B) Galectin-9 expression was assessed by western blot (A) or flow cytometry (B) 48 h in WT or gal9 KD moDCs. Immunoblot is representative of four independent experiments. In B, numbers in the inset indicate gMFI. Cells were gated based on their forward-side scatter after which cell clusters were gated out and only single cells were analyzed. WT, light gray population; gal9 KD moDCs, orange population. A black dotted line represents isotype control values. (C) CCR7 surface expression in moDCs treated as per (B). Graph is a representative donor out of three analyzed, and numbers in the inset indicate gMFI. (D and E) WT or gal9 KD moDCs were matured and levels of HLA-DR, CD80, CD86, and CD83 analyzed by flow cytometry. The graph depicts the mean ± SEM surface expression (gMFI) of four independent donors. (F) WT or gal9 KD moDCs were placed in the upper chamber of a transwell system and subjected to a CCL21 chemokine gradient. Migrated cells were collected at the bottom chamber 1, 2, and 4 h after seeding and measured by flow cytometry. Graphs show a representative donor of four independent donors analyzed. (G) Quantification of data shown in F. Graph shows mean percentage ± SEM of migratory DCs relative to the total number of seeded cells for each time point and genotype. An unpaired t test between WT and gal9 KD DCs was performed. (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 graph corresponds to one independent donor (labeled donor 1–4; 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). n.s., P > 0.05; *P < 0.05. gMFI, geometrical mean fluorescence intensity. Source data are available for this figure: .

Article Snippet: A donkey anti-goat secondary antibody conjugated to Alexa Fluor 488 was used (#A-11055, 1:400; Invitrogen [vol/vol]). moDCs were incubated for 30 min on ice with antibodies against HLA-DR (#555811, clone G46-6, FITC-labeled; BD BioSciences), CD80 (#557227, clone C3H, PE-labeled; BD BioSciences), CD86 (#555658, clone 2331, PE-labeled; BD BioSciences), CD83 (#130-094-186, clone HB15, APC-labeled; Miltenyi), and CCR7 (#130-094-286; Miltenyi Biotec).

Techniques: Migration, Expressing, Western Blot, Flow Cytometry, Control, Plasmid Preparation, Negative Control, Labeling, Fluorescence