Fig 1: Effects of ATRA and O-GlcNAc cycle inhibitors on the intracellular levels of galectins. HL-60 cells were grown in IMDM medium supplemented with either FBS or ITS and treated with O-GlcNAc-modulating drugs as specified in Figure 1. Representative Western blots and the ensuing quantification of galectin-1, galectin-3, galectin-9, and galectin-10 levels in HL-60 cells are shown. The fold changes were calculated as a ratio of galectin band intensities to ß-actin as a loading control with the subsequent normalization to the average of untreated cells (CTRL). Significant differences (p < 0.05) between treatments are labeled by different letters as per one-way ANOVA followed by a Tukey’s multiple comparisons test for each media. The results are presented as means ± SD, n = 3–5.
Fig 2: Proposed model of O-GlcNAc-mediated expression and secretion of galectins. The model implies that neutrophilic differentiation of HL-60 cells is associated with a decrease of O-GlcNAcylated proteins in cells including galectins (resting galectins are in blue and O-GlcNAcylated galectins are in red), which results in their secretion. Shape represents the type of galectins: prototype (gal-1, gal-10), chimera type (gal-3), and tandem-repeat type (gal-9). Whereas the secretion of all these galectins is stimulated by a drop in O-GlcNAc, changes in intracellular concentration can vary.
Fig 3: Sensitivity of galectin gene expression to disruptions in O-GlcNAc homeostasis: (a) Changes in the expression of six galectin genes (LGALS1, LGALS3, LGALS8, LGALS9, LGALS10, and LGALS12) in HL-60 cells grown in IMDM medium supplemented with either FBS or ITS and treated with O-GlcNAc-modulating drugs as specified in Figure 1. Significant differences (p < 0.05) between treatments are labeled by different letters as per one-way ANOVA followed by a Tukey’s multiple comparisons test for each media. The results are presented as means ± SD, n = 3–5; (b) Pairwise correlation analysis of gene expression in HL-60 cells: left, heatmap showing pairwise correlation patterns between expression of genes (log2-transformed values) encoding galectins and cell differentiation marker NCF1. Significant (p < 0.05) Pearson’s correlation coefficients are reported for each pairwise comparison while blanks cells indicate that the correlation is not significant; right, comparison of pairwise Pearson’s correlations between IMDM-FBS and IMDM-ITS samples using the ‘cocor’ R package, significant p values are shown, ns—not significant, n = 15 for each gene (3 biological replicates for each treatment including control).
Fig 4: Effects of ATRA and O-GlcNAc cycle inhibitors on the secretion of galectins from HL-60 cells. The cells were grown for 72 h in IMDM-ITS, treated with O-GlcNAc-modulating drugs as specified in Figure 1, and cell supernatants were collected for analysis of galectin levels as described in Materials and Methods: (a) Immunodot blots (inserts; 1, 2, 3, 4, 5 labels correspond to CTRL, ATRA, TG, DON, and AC samples, respectively) for galectin-1, galectin-3, galectin-9, and galectin-10 in serum-free cell supernatants and their quantification by densitometric analysis with normalization to control samples; (b) Accumulation of secreted galectin-1, galectin-3, and galectin-9 in serum-free culture medium at 72 h, as detected by ELISA. Significant differences (p < 0.05) between treatments are labeled by different letters as per one-way ANOVA followed by a Tukey’s multiple comparisons test. The results are presented as means ± SD, n = 3–5.
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