Nucblue live dye

Serum-starved U2OS-R1 cells were incubated in DMEM supplemented with heparin (40U/mL) with FGF4-Fc (20 µg/mL) in the presence or absence of galectins (20 µg/mL) for 30 min on ice to assess cell binding and at 37 °C for 30 min to study FGF4-Fc endocytosis. Cells were subsequently washed with ice cold PBS, fixed in 4% paraformaldehyde solution and permeabilized with 0.1% Triton in PBS (for cells incubated at 37 °C). Nuclei were stained with NucBlue Live dye (Thermo Fisher Scientific), Zenon AF-488 (Thermo Fisher Scientific) was used for detection of FGF4-Fc. Early endosomes were detected with rabbit anti-human polyclonal early endosome antigen 1 (EEA1) antibody (#ab2900, Abcam) and anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (#A11037, Thermo Fisher Scientific). Wide-field fluorescence microscopy was carried out using a Zeiss Axio Observer Z1 fluorescence microscope (Zeiss, Oberkochen, Germany) as described in [33 ]. Images were processed with Zeiss ZEN 2.3 software (Zeiss, Oberkochen, Germany), and Adobe Photoshop CS6 (Adobe, San Jose, CA, USA). For quantification of the 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.

U2OS-R1 cells were incubated with 10 µg/mL of DyLight550-labeled wild type FGF1 alone or in combination with non-labeled FGF1 R35E mutant in 1:1 and 1:10 molar ratio, for 1 h on ice. The cells were subsequently washed with PBS and fixed with 4% paraformaldehyde (20 min, at 4 °C). Next, the cells were washed with PBS and nuclei were stained for 10 min with NucBlue Live dye (Thermo Fisher Scientific). Wide-field fluorescence microscopy was carried out using a Zeiss Axio Observer Z1 microscope (Zeiss, Oberkochen, Germany). Images were taken using LD-Plan-Neofluar 40×/0.6 Korr M27 objective and Axiocam 503 camera. FGF1-DyLight550 signal was visualized with 540/552-nm bandpass excitation filter and 575/640-nm bandpass emission filter. NucBlue Live signal was visualized with 335/383-nm bandpass excitation filter and 420/470-nm emission filter. Image analysis was carried out using ZEN 2.3 (Zeiss, Oberkochen, Germany) and ImageJ (NIH, Bethesda, MD, USA).