Neuraminic acid

Cell surface α-2,6-linked sialic acid content was determined by using the FITC-conjugated Sambucus Nigra Agglutinin lectin (SNA). COS-7 cells were grown in DMEM supplemented with 10% FBS (HyClone, Thermo Scientific, Waltham, MA, USA). One day after plating, cells were detached and transfected using electroporation with 2.5 μg of each plasmid cDNA according to the supplier's protocol (Nucleofector™, Lonza, Basel, Switzerland). 24 h post-transfection, cells were transferred to a 96-well Ultra™ plate (PerkinElmer Inc., USA), allowed to attach, and cultivated for 24 h at both normoxic and hypoxic conditions in the presence of neuraminic acid (10 mM, Sigma-Aldrich). Cells were then fixed with 2% p-Formaldehyde, quenched with 1% BSA-PBS before staining with FITC-conjugated SNA-lectin (1 μg/ml, Vector Laboratories Ltd, UK) for 1 h. Before final washing with PBS and imaging using the high content imaging system (Operetta, PerkinElmer Inc.). Cell nuclei were counter-stained with the Hoechst dye to allow selection of individual cells for quantification. For normalization, identical cell samples were subjected to SDS-PAGE and western blotting with the anti-CD75 antibody (#AP21150PU-N, Acris Antibodies GmbH, Germany) to determine the level of ST6Gal-I protein in each sample (Suppl. Fig. S5).

Lysogeny broth (LB), isopropyl β-d-thiogalactopyranoside (IPTG), NAD⁺, and NADH were
purchased from Research Products International. The protease inhibitor
cocktail, lysozyme, DNase I, UDP-GlcNAc, ManNAc, neuraminic acid,
uridine 5′-diphosphate (UDP), cytidine 5′-triphosphate
(CTP), cytidine 3′,5′-cyclic monophosphate, phosphoenolpyruvate
(PEP), pyruvate kinase, lactate dehydrogenase, sialic acid aldolase,
pyrophosphatase, kanamycin, dithiothreitol (DTT), imidazole, and HEPES
were obtained from Sigma-Aldrich. Ammonium bicarbonate, 2-mercaptoethanol,
KCl, MnCl₂, and MgCl₂ were acquired from Sigma-Aldrich.
Vivaspin 20 spin filters and HisTrap and HiTrap Q HP columns were
obtained from Cytiva. The 10 kDa Nanosep spin filters were purchased
from Pall Corp. (Port Washington, NY). Deuterium oxide was acquired
from Cambridge Isotope Laboratories Inc., and ¹⁸O-labeled
water (98%) was obtained from Medical Isotopes Inc.