Both patient-derived GNE mutations severely affect de novo sialic acid biosynthesis. (A) Establishment of Gne-knockout mouse endothelial cell line MS1 validated by western blotting. Hprt, housekeeping protein. (B) Altered glycans on the cell surface of Gne-KO MS1 cells examined by flow cytometry using SiaFind Lectenz (pan-sialic acid probe) and RCA1 lectin (asialylated glycan lectin). The unstained control was used as a negative control. (C) Gne-KO MS1 cells were lentivirally infected with empty control or each GNE-Flag3 variant. (D) Expression analysis of each GNE protein by western blotting. Exogenously transduced GNE (exo) and endogenous Gne (endo) are distinguished by size based on Flag3 tag on transduced GNE. WT, wild type. (E) Infected cells were analyzed for SiaFind Lectenz binding by flow cytometry. (F) Expression of cytoplasmic GNE was reduced in MS1 cells transduced with mutant GNE imaged by confocal microscopy. Flag3, Flag3-tagged GNE; DAPI, nuclear marker. (G) Two pathways of sialic acid biosynthesis. (H) ManNAz-derived NeuAz on cell surface glycans of cells that were metabolically labeled with ManNAz for 3 days. ∗∗P < .01; ∗∗∗P < .001.