Legends for supplementary figures

Figure S1: Galectin-3 expression in Gal-3 KD and control Sc cells by confocal microscopy.

Sh1 and Sc cells were fixed, permeabilized, immunostained and analyzed by confocal microscopy. (A) Galectin-3 expression (FITC, green) was evaluated in both Sc cells and in Sh1 cells at low magnification. Phalloidin was used to stain the actin cytoskeleton (red, Texas Red). Images were merged to study co-localization. (B) Galectin-3 expression in Sc cells nucleus (high magnification). Nuclei are colored in blue by DAPI whereas MUC1 appeared in Red (texas-red). Co-localization of galectin-3 – DAPI in the nucleus appeared in white.

Figure S2: MUC1 expression in Gal-3 KD and control Sc cells by flow cytometry.

Cells were detached with trypsin-EDTA, rinsed, resuspended at 106 cells/ml in PBS with 1% Bovine Serum Albumin (BSA), and incubated with a phycoerythrin (PE)-labelled anti MUC1 antibody (sc-7313PE, Santa Cruz Biotechnologies, 5mg/ml) for 60 min at room temperature before being analyzed with a Beckman-Coulter Epics XL-MCL4 cytometer. For the evaluation of intracellular and membrane labelling, cells were fixed with ParaFormAldehyde (PFA) 3% for 20 min, permeabilized with 0.2% saponin in D-PBS with Ca2+ and Mg2+ and 1% BSA for 20 min at room temperature before being incubated with the anti-MUC1 antibody. Figures are representative of 2 independent experiments run in duplicate.

(A) Cell surface staining with phycoerythrin (PE)-labelled anti MUC1 antibody followed by analysis with flow cytometry shows similar cell surface MUC1 expression in Sh1 and in Sc controls cells. (B) Cell surface and intracellular staining with PE labelled anti MUC1 antidody was performed on permeabilized cells. The mean MUC1 intensity increased by 2.1 fold in permeabilized Sc cells by comparison with non permeabilized cells whereas the increase was weaker (1.1 fold, mean of 2 independent experiments) in Sh1 cells (permeabilized vs non permeabilized).

Figure S3: MUC1 and EGFR associate to EEA1 in galectin-3 expressing cells.

Cells were processed for immunofluorescence microscopy using anti-EEA1 (FITC, green), anti MUC1 or anti EGFR (both Texas Red) antibodies, and then incubated with secondary fluorophore labelled antibodies (e.g. DyLight 594 or 488). Images were merged to study (A) EEA1 and MUC1 co-localization (in yellow color) or (B) EEA1 and EGFR co-localization (in yellow color) in un-treated or treated Sc (panels a & b) or Sh1 cells (panels c & d), respectively.

Figure S4: MUC1 and EEA1 co-localization in galectin-3 treated Sh1 cells.

Cells were processed for immunofluorescence microscopy using anti-EEA1 (FITC, green), and anti-MUC1 (Texas Red, red) antibodies, and merge images were performed to study co-localization (in yellow color). Zooms of interesting sub-cellular areas are depicted.

1