Galectin-9 depletion alters actomyosin cytoskeleton. (A) Total lysates from WT, gal9 KD, and gal9 KD + rGal9 moDCs were subjected to western blot and total PAK1, PAK_Thr423, and galectin-9 protein expression analyzed. Tubulin was used as a loading control. Immunoblot is representative of three independent experiments. (B) Quantification of mean ± SEM of PAK1_Thr423 and total PAK1 content normalized to corresponding tubulin for three independent donors. One-way ANOVA was performed. (C) Total lysates from WT, gal9 KD, and gal9 KD +rGal9 moDCs seeded onto 6-well plates were subjected to western blot and total mDia, pMLC (Ser19), and galectin-9 expression analyzed. Tubulin was used as a loading control. Immunoblot is representative of five independent experiments. (D) Quantification of normalized mDia and pMLC content shown in C. A one-sample t test was performed. n.s., P > 0.05; *P < 0.05. (E) WT or gal9 KD mature DCs were seeded onto coverslips and stained for pMLC and nucleus (DAPI). ROI in the left panel (yellow square) is depicted in the zoomed right panels. Scale bar = 10 µm; insets = 10 µm. (F and G) Quantification of data shown in (E). Box-and-whisker plots show mean (as +) pMLC intensity ratio in the whole cell (F) or at the perinuclear region (G) for two independent donors (10–15 cells analyzed per donor). Whiskers are generated using the Tukey method. Significance was determined by an unpaired t test. (H) Pairwise comparison of actin intensity at the cell front (red) and rear (blue) of 30 WT, gal9 KD, or gal9 KD + rGal9 moDCs from three independent donors, corresponding to rear/front ratio analysis shown in Fig. 6 D. (I) Distance of the nucleus to the front of the cell in WT, gal9 KD, and gal9 KD + rGal9 moDCs. The 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. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. n.s > 0.05. In H, two-tailed paired Student’s parametric t test was used. Source data are available for this figure: SourceData FS4.
Figure S4.

Galectin-9 depletion alters actomyosin cytoskeleton. (A) Total lysates from WT, gal9 KD, and gal9 KD + rGal9 moDCs were subjected to western blot and total PAK1, PAK_Thr423, and galectin-9 protein expression analyzed. Tubulin was used as a loading control. Immunoblot is representative of three independent experiments. (B) Quantification of mean ± SEM of PAK1_Thr423 and total PAK1 content normalized to corresponding tubulin for three independent donors. One-way ANOVA was performed. (C) Total lysates from WT, gal9 KD, and gal9 KD +rGal9 moDCs seeded onto 6-well plates were subjected to western blot and total mDia, pMLC (Ser19), and galectin-9 expression analyzed. Tubulin was used as a loading control. Immunoblot is representative of five independent experiments. (D) Quantification of normalized mDia and pMLC content shown in C. A one-sample t test was performed. n.s., P > 0.05; *P < 0.05. (E) WT or gal9 KD mature DCs were seeded onto coverslips and stained for pMLC and nucleus (DAPI). ROI in the left panel (yellow square) is depicted in the zoomed right panels. Scale bar = 10 µm; insets = 10 µm. (F and G) Quantification of data shown in (E). Box-and-whisker plots show mean (as +) pMLC intensity ratio in the whole cell (F) or at the perinuclear region (G) for two independent donors (10–15 cells analyzed per donor). Whiskers are generated using the Tukey method. Significance was determined by an unpaired t test. (H) Pairwise comparison of actin intensity at the cell front (red) and rear (blue) of 30 WT, gal9 KD, or gal9 KD + rGal9 moDCs from three independent donors, corresponding to rear/front ratio analysis shown in Fig. 6 D. (I) Distance of the nucleus to the front of the cell in WT, gal9 KD, and gal9 KD + rGal9 moDCs. The 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. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. n.s > 0.05. In H, two-tailed paired Student’s parametric t test was used. Source data are available for this figure: SourceData FS4.

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