(A) Western blot of NAGK, GFAT1, and ACTIN loading control from TU8988T, MiaPaCa2, and HPAC parental (wildtype, WT) and NAGK knockout (KO) populations. NAGK was knocked out using two independent sgRNAs (sg1, sg2). (B) Representative wells from a colony-forming assay for parental and NAGK knockout lines. (C) Quantitation of colony-forming assay data in B (n = 3). (D) Western blot for NAGK, GFAT1, and loading control ACTIN in parental pancreatic ductal adenocarcinoma (PDA) cell lines. Band density was quantitated, normalized to control, and presented below the blot. (E) Schematic overview of the Leloir pathway of galactose catabolism integrated with the hexosamine biosynthetic pathway (HBP) and GlcNAc salvage pathway. (F) Quantitated data from colony formation assays in parental and GFAT1 knockout clonal TU8988T cell lines in base media (DMEM), positive control GlcNAc, and N-acetyl-galactosamine (GalNAc) (n = 3) (G) Western blot for GFAT1, NAGK, and loading control VINCULIN (VNC) in parental TU8988T and HPAC, GFAT1 knockout clones, and GFAT/NAGK double targeted lines. (H–K) Proliferation time course of (H,I) parental TU8988T and GFAT1 knockout line B9 in base media, GalNAc positive control, 60 kDa HA, or 5 kDa HA; (J,K) GFAT1/NAGK double targeted clones in base media, GalNAc positive control, 60 kDa HA, or 5 kDa hyaluronic acid (HA) (n = 3). (L) Western blot for proteome O-GlcNAcylation (O-GlcNAc), GFAT1, NAGK, and VCN in parental (WT), GFAT1 knockout (B9), and GFAT1/NAGK double knockout (B9–sg1) TU8988T cells treated with 10 mM GalNAc, DMEM, CM, or o-HA. (M) Liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis of UDP-GlcNAc from the samples in L (n = 3). (N) Schematic overview of the HA metabolism through the GlcNAc salvage pathway to fuel glycosylation in GFAT1 knockout PDA. Error bars represent mean ± SD. n.s., non-significant; *p < 0.05; **p < 0.01.