Figure 7.
Galectin-9 interaction with CD44 regulates DC migration. (A) IP of galectin-9 or isotype negative control in whole-cell lysates obtained from day 7 matured moDCs. Co-IP complexes were resolved and probed against galectin-9, Vamp-3, and CD44. Graph shows quantification of the CD44 signal. Data shown are representative of three independent experiments. (B) CD44 expression in WT, gal9 KD, and gal9 KD + rGal9 moDC lysates. Tubulin was used as a loading control. Graph shows mean CD44 expression of four independent donors (black symbols), and immunoblot is representative of four independent experiments. (C) Super-resolution dSTORM images of the basal membranes of day 7 matured WT, gal9 KD, and gal9 KD + rGal9 moDCs stained for CD44. ROI (red square) in the upper panel is depicted in the zoomed bottom panels (5,000 × 5,000 nm) to visualize CD44 nanoscale organization. (D) Mean ± SEM CD44 cluster diameter in WT, gal9 KD, and gal9 KD + rGal9 was calculated per cell (gray dots) based on pair correlation analysis for 4 independent donors. One-way ANOVA with Tukey’s multiple comparisons was performed. (E) RhoA_GFP was incubated with either whole DC lysates from three independent donors pulled together (+) or nothing (−), and immunocomplexes were resolved by western blot and probed with specific antibodies against GEF-H1 and RhoA. Western blot is representative of two independent experiments. (F) Confocal microscopy of PLA assessing the proximity of CD44 to RhoA and GEF-H1 in mature moDCs. RhoA-GEF-H1 and isotypes were used as a positive and negative control, respectively. Scale bar: 5 µm. (G) Quantification of the number of PLA foci per cell area (black symbols) from images shown in F. Data represent the mean ± SEM from four independent donors (15–20 cells were analyzed/condition). One-way ANOVA with Dunn’s multiple comparison correction was performed. (H) Quantification of the number of PLA foci (CD44-RhoA) per cell (black symbols) in WT or gal9 KD moDCs untreated or treated with 5 µg/ml of RhoA activator II. Data represent the mean ± SEM from two independent donors. 25–30 cells were analyzed per condition. One-way ANOVA was performed. (I) Quantification of the number of PLA foci (RhoA-GEF-H1) per cell (black symbols) in WT or gal9 KD moDCs. Data represent the mean ± SEM from three independent donors (20 cells were analyzed per condition). An unpaired t test was used to assess significance. n.s, P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001. IP, immunoprecipitation. Source data are available for this figure: SourceData F7.

Galectin-9 interaction with CD44 regulates DC migration. (A) IP of galectin-9 or isotype negative control in whole-cell lysates obtained from day 7 matured moDCs. Co-IP complexes were resolved and probed against galectin-9, Vamp-3, and CD44. Graph shows quantification of the CD44 signal. Data shown are representative of three independent experiments. (B) CD44 expression in WT, gal9 KD, and gal9 KD + rGal9 moDC lysates. Tubulin was used as a loading control. Graph shows mean CD44 expression of four independent donors (black symbols), and immunoblot is representative of four independent experiments. (C) Super-resolution dSTORM images of the basal membranes of day 7 matured WT, gal9 KD, and gal9 KD + rGal9 moDCs stained for CD44. ROI (red square) in the upper panel is depicted in the zoomed bottom panels (5,000 × 5,000 nm) to visualize CD44 nanoscale organization. (D) Mean ± SEM CD44 cluster diameter in WT, gal9 KD, and gal9 KD + rGal9 was calculated per cell (gray dots) based on pair correlation analysis for 4 independent donors. One-way ANOVA with Tukey’s multiple comparisons was performed. (E) RhoA_GFP was incubated with either whole DC lysates from three independent donors pulled together (+) or nothing (−), and immunocomplexes were resolved by western blot and probed with specific antibodies against GEF-H1 and RhoA. Western blot is representative of two independent experiments. (F) Confocal microscopy of PLA assessing the proximity of CD44 to RhoA and GEF-H1 in mature moDCs. RhoA-GEF-H1 and isotypes were used as a positive and negative control, respectively. Scale bar: 5 µm. (G) Quantification of the number of PLA foci per cell area (black symbols) from images shown in F. Data represent the mean ± SEM from four independent donors (15–20 cells were analyzed/condition). One-way ANOVA with Dunn’s multiple comparison correction was performed. (H) Quantification of the number of PLA foci (CD44-RhoA) per cell (black symbols) in WT or gal9 KD moDCs untreated or treated with 5 µg/ml of RhoA activator II. Data represent the mean ± SEM from two independent donors. 25–30 cells were analyzed per condition. One-way ANOVA was performed. (I) Quantification of the number of PLA foci (RhoA-GEF-H1) per cell (black symbols) in WT or gal9 KD moDCs. Data represent the mean ± SEM from three independent donors (20 cells were analyzed per condition). An unpaired t test was used to assess significance. n.s, P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001. IP, immunoprecipitation. Source data are available for this figure: SourceData F7.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal