Fig. 3

Multivalency of galectins is crucial for the attraction of glycosylated FGF4-Fc to the cell surface and for the modulation of FGF4-Fc trafficking and signaling. A Effect of endogenous galectins on FGF4-Fc signaling. Serum-starved NIH3T3 cells were washed with 50 mM lactose prior cell supplementation with various concentrations of FGF4-Fc (2–20 ng/mL). Cells were lysed and analyzed with WB using the indicated antibodies. CBB served as a loading control (left panel). Densitometric analyses of the effect of lactose washes on the activation of FGF-Fc-dependent signaling pathways by 20 ng/mL FGF4-Fc (right panel). Mean values from at least three independent experiments +/- SEM are shown. Statistical analyses were performed with Student’s t-test (*p < 0.05; **p < 0.005 and ***p < 0.001). B and C Effects of the wild type galectins (B) and their monovalent variants (C) on FGF4-Fc signaling. Serum-starved NIH3T3 cells were treated with FGF1 (100 ng/mL, control), different concentrations of FGF4-Fc (2 and 20 ng/mL) in the presence or absence of recombinant galectins (1 µg/mL) or their monovalent variants (5 µg/mL). Cells were lysed and analyzed with WB using the indicated antibodies (left panel). CBB and tubulin served as loading controls. Densitometric analyses of the effect of galectins on FGF4-Fc (20 ng/mL) signaling (right panel). Mean values from at least three independent experiments +/- SEM are shown. Statistical analyses were performed with Student’s t-test (*p < 0.05; **p < 0.005 and ***p < 0.001). The schemes of the wild type galectins and their engineered variants with altered valency are shown (left panel). D Effects of the wild type galectins and their engineered variants with altered valency on the cell binding and endocytosis of FGF4-Fc. U2OS-R1 cells were incubated for 30 min with FGF4-Fc (20 µg/mL) either at 4 °C (for cell binding analysis) or at 37 °C (for growth factor endocytosis analysis) in the presence of the studied galectins and their variants (20 µg/mL). Cells were either fixed (4 °C samples) or fixed and permeabilized (37 °C samples), nuclei were labelled with NucBlue and FGF4-Fc was detected with Zenon-AF-488 using fluorescence microscopy. Representative images from at least three independent experiments are shown. Scale bars represent 20 μm. To quantify cell binding by FGF4-Fc in the presence of galectin variants, the total fluorescence of at least 20 cells from three fields of view/condition was measured in three independent experiments using Zeiss ZEN 2.3 software. Statistical analyses were performed with Student’s t-test (*p < 0.05; **p < 0.005 and ***p < 0.001). The schemes of the wild type galectins and their engineered variants with altered valency are shown (left panel)