Mab1154

The paraffin blocks were cut into sections of 4 μm, mounted on polylysine-coated microslides, dewaxed, and rehydrated. Then, tissue sections were incubated in 3 % hydrogen peroxide at room temperature. For antigen retrieval, tissue slides were immersed in citric acid and boiled in a microwave oven. The sections were washed in distilled water and phosphate buffered saline (PBS, Gibco), and then subjected to bovine serum to block unspecific binding agents. This step was followed by overnight exposure (4 °C) to the mouse anti-human Gal-3 antibody (mab1154, R&D Systems Inc., Minneapolis, MN, USA) in a humidified chamber. After being rinsed in PBS, the sections were incubated with the bridging rabbit anti-mouse immunoglobulins conjugated with horseradish peroxidase (HRP)-labelled dextran polymer for 1 h. After washing in PBS, diaminobenizidine tetrahydrochloride (DAB) solution was applied, followed by running tap water as well as nuclear counterstaining with haematoxylin (Sigma, USA). The stained slides were viewed under microscopy (OLYMPUS OPTICAL CO., Ltd.) under 400× magnification. Positive cells were characterized by the brown staining of Gal-3 antibody. The intensity and distribution of the staining reaction were evaluated by two blinded, independent observers.

Monoclonal antibodies against CD146/MCAM (mab 932) and galectin-3 (mab 1154), biotinylated goat anti-galectin-3 polyclonal antibody (BAF1154) were from R&D Systems (Abingdon, UK). Antibodies against AKT (9272S) and Phospho-AKT (Phospho T308) were purchased from Cell Signalling (Hitchin, UK). Antibody against CD146/MCAM rabbit polyclonal antibody was obtained from Proteintech (Rosemont, IL, USA). Protein A/G PLUS-Agarose beads were from Santa Cruz Biotechnology (Heidelberg, Germany). Calcein AM cell labelling solution was obtained from Invitrogen. O-glycanase (Endo-O-Glycosidase, GK80090) was purchased from Agilent (Santa Clara, CA, USA). ShRNA plasmid DNA for CD146/MCAM (TRCN0000151337), control shRNA plasmid (SHC002v), and N-glycanase PNGaseF (F8435) were from Sigma Aldrich (Dorset, UK). Matrigel matrix was obtained from Corning Life Sciences (Deeside, UK).