Fig. 1

UDP-HexNAc and protein O-GlcNAcylation are increased in pmm2 zebrafish mutants (A?C) (A) Schematic of sugar phosphate pathway. Arrows indicate whether metabolite abundance is increased or decreased in PMM2-CDG. Green, increased. Red, decreased. (B) Graph shows the level of glutamine trends higher in 6-dpf pmm2m/m larvae, while (C) UDP-HexNAc is significantly increased. n = 5 biological replicates per genotype consisting of 10 embryos each. (D) Western blot of protein O-GlcNAcylation comparing wild-type (pmm2+/+) and mutant (pmm2m/m) larvae 4?8 dpf shows increased O-GlcNAc modification present in mutants. n = 4 biological replicates of 10 embryos each per time point. (E) Graphs quantitating total O-GlcNAc levels across each lane show 2-fold higher levels present in mutants. Data are presented as fold change relative to wild-type samples and as values normalized only to total protein loaded, as indicated by Ponceau stains. (F) RNA-seq analysis of transcript abundances in WT and mutant larvae 5 and 8 dpf identify that several genes involved in the hexosamine biosynthetic and O-GlcNAc pathways are differentially expressed. Heatmaps show relative transcript abundances. n = 5 biological replicates of 10 embryos each per time point. p values are shown within the heatmap. (G) qRT-PCR analyses confirm gfpt abundance is nearly 9-fold higher in pmm2m/m mutants. n = 4 biological replicates of 10 embryos each per time point. (H) Western blot analyses comparing Gfpt, Ogt, and Oga protein abundances in 4- to 8-dpf WT and mutant larvae. (I?K) Graphs show quantitation of protein abundance values normalized only to total protein loaded, as indicated by Ponceau stains. (I) Gfpt, (J) Ogt, and (K) Oga. Error = SEM, Student?s t test, ?p < 0.05, ??p < 0.01. See also Figure S1.