RhoA downstream signaling is impaired in galectin-9–depleted DCs. (A) Heat map of proteins involved in cytoskeletal rearrangements analyzed by RPPA. Whole-cell lysates obtained from mature WT, gal9 KD, or gal-9 KD +rGal9 DCs were denatured, arrayed on nitrocellulose-coated slides, and probed with antibodies against the specified protein targets. Data shown represent mean protein expression normalized against the loading control. (B) Metascape pathway enrichment analysis from all proteins found to be differentially regulated by RPPA between WT and gal9 KD moDCs. (C and D) WT, gal9 KD, or gal9 KD + rGal9 mature DCs were embedded in 3D collagen gels and stained for pMLC, actin (phalloidin), and nucleus (Hoechst). Box-and-whisker plots show mean (as +) pMLC (C) or actin (D) intensity ratio at the rear over the front of the cell (based on the nucleus localization) for 4 independent donors (10–15 cells analyzed per donor). Whiskers are generated using the Tukey method. Representative high-resolution Airyscan images of WT, gal9 KD, or gal9 KD + rGal9 moDCs are shown. Scale bar = 10 µm. Two-way ANOVA followed by Dunnett’s test for multiple comparisons was performed in all panels. (E) Average actin intensity for the entire width of WT (black), gal9 KD (red), and gal9 KD + rGal9 (blue) moDCs at each position forward from the rearmost point of the cell (left) and backward from the frontmost point of the cell (right) for the rear and front 25 μm. 15 cells were analyzed across three independent donors with bar representing SEM; all values were normalized to the maximum actin intensity of each cell. (F) Distance of the nucleus to the rear of the cell in WT, gal9 KD, and gal9 KD + rGal9 moDCs. Box-and-whisker plot shows mean (as +) nuclear distance for four independent donors (10–15 cells analyzed per donor). Whiskers are generated using the Tukey method. Representative high-resolution Airyscan images of WT, gal9 KD, or gal9 KD + rGal9 moDCs stained for actin (phalloidin) and nucleus (Hoechst) are shown. Scale bar = 10 µm. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. n.s., P > 0.05; ***P < 0.001; ****P < 0.0001. n.s., P > 0.05; *P < 0.05; ***P < 0.01; ****P < 0.001.
Figure 6.

RhoA downstream signaling is impaired in galectin-9–depleted DCs. (A) Heat map of proteins involved in cytoskeletal rearrangements analyzed by RPPA. Whole-cell lysates obtained from mature WT, gal9 KD, or gal-9 KD +rGal9 DCs were denatured, arrayed on nitrocellulose-coated slides, and probed with antibodies against the specified protein targets. Data shown represent mean protein expression normalized against the loading control. (B) Metascape pathway enrichment analysis from all proteins found to be differentially regulated by RPPA between WT and gal9 KD moDCs. (C and D) WT, gal9 KD, or gal9 KD + rGal9 mature DCs were embedded in 3D collagen gels and stained for pMLC, actin (phalloidin), and nucleus (Hoechst). Box-and-whisker plots show mean (as +) pMLC (C) or actin (D) intensity ratio at the rear over the front of the cell (based on the nucleus localization) for 4 independent donors (10–15 cells analyzed per donor). Whiskers are generated using the Tukey method. Representative high-resolution Airyscan images of WT, gal9 KD, or gal9 KD + rGal9 moDCs are shown. Scale bar = 10 µm. Two-way ANOVA followed by Dunnett’s test for multiple comparisons was performed in all panels. (E) Average actin intensity for the entire width of WT (black), gal9 KD (red), and gal9 KD + rGal9 (blue) moDCs at each position forward from the rearmost point of the cell (left) and backward from the frontmost point of the cell (right) for the rear and front 25 μm. 15 cells were analyzed across three independent donors with bar representing SEM; all values were normalized to the maximum actin intensity of each cell. (F) Distance of the nucleus to the rear of the cell in WT, gal9 KD, and gal9 KD + rGal9 moDCs. Box-and-whisker plot shows mean (as +) nuclear distance for four independent donors (10–15 cells analyzed per donor). Whiskers are generated using the Tukey method. Representative high-resolution Airyscan images of WT, gal9 KD, or gal9 KD + rGal9 moDCs stained for actin (phalloidin) and nucleus (Hoechst) are shown. Scale bar = 10 µm. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. n.s., P > 0.05; ***P < 0.001; ****P < 0.0001. n.s., P > 0.05; *P < 0.05; ***P < 0.01; ****P < 0.001.

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