Globotriaosylceramide gb3

C8 lactosyl(β)-ceramide (LacCer), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Atto 647N 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from Sigma-Aldrich (Darmstadt, Germany). Globotriaosylceramide (Gb3) was obtained from Matreya (State College, PA, USA). Shiga toxin B-subunit (StxB) was kindly provided by enGenes Biotech (Vienna, Austria). UDP-Glo Glycosyltransferase assay kit and ultra-pure UDP-Galactose (UDP-Gal) were from Promega (Charbonnières-les-Bain, France). UDP-Gal for QCM-D analysis was purchased from Carbosynth (Compton, UK). HisTrap FF column was purchased from Cytiva (Marlborough, MA, USA) and Superdex 200 Increase 10/300 GL from GE Healthcare (Chicago, MA, USA). All other chemical reagents were of analytical or liquid chromatography grade.

Cell viability was assessed using the CellTiter‐Glo luminescent cell viability assay kit (Promega, Madison, WI), as described previously.²², ²³ Briefly, cells (4000 cells/well; 2500 cells/well for MCF‐12A) were grown in 96‐well plates overnight and then switched to 5% FBS medium containing drugs for 72 hours treatments. For combination treatment, cells were cultured in 5% FBS medium containing respective agents for 48 hours in advance and then cocultured with drugs for an additional 72 hours. Cell viability was assessed in a Synergy HT microplate reader (BioTek, Winnooski, VT, USA), following incubation with CellTiter‐Glo reagent. A new GCS inhibitor, Genz‐161 (GENZ 667161, (S)‐quinuclidin‐3‐yl(2‐(2‐(4‐fluorophenyl)thiazol‐4‐yl)propan‐2‐yl carbamate) was kindly provided by Sanofi Genzyme (Framingham, MA).³⁶, ³⁷ Doxorubicin hydrochloride (Dox) was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Monosialoganglioside (NH₄⁺ salt, GM2), disialoganglioside (NH₄⁺ salt; GD3), and globotriaosylceramide (Gb3) were purchased from Matreya (State College, PA, USA).