Fig. 3

Epalrestat-mediated improvement of cartilage phenotypes is associated with a reduction in UDP-HexNAc and O-GlcNAcylation (A) Confocal images of WT and pmm2 mutant cartilages 7 dpf show treating with epalrestat 5 dpf significantly improves multiple phenotypes. Yellow arrows highlight vacuoles present in mutant cells. n = 20 embryos per genotype per condition. Scale bar, 10 ?m. (B) Graphs show chondrocyte shape, the level of cellular intercalation (organization), and cellular vacuolation are all improved in pmm2 mutant cartilages following epalrestat treatment. Shape was assigned as the degree of ?roundness? according to the ratio of the short and long axis, plotted here as average cell shape per animal. See Figure S5 for individual cellular measurements. n = 10 larvae scored across three independent experiments per genotype per condition. Error = SEM, Student?s t test, ?p < 0.05, ????p < 0.0001. (C) Graphs of UDP-HexNAc and glutamine abundance in DMSO and epalrestat-treated WT (pmm2+/+) and mutant (pmm2m/m) larvae 6 dpf. n = 12?20 biological replicates per genotype per condition, each containing 10 embryo per sample. Error = SD, two-way ANOVA multivariant analyses, p values shown in graphs. (D) Western blots show protein O-GlcNAcylation in WT and mutant larvae treated with either DMSO (?) or epalrestat (+) with reduced levels in treated mutants. ROI, region of interest used for quantitation. (E) Graphs show the level of protein O-GlcNAc in the three ROIs denoted on western blot. n = 3 biological replicates per genotype per condition with 10 larvae per sample. Error = SEM, Student?s t test, ??p < 0.01. (F) Western blot show epalrestat treatment reduces Dpagt1 and NgBR abundance in mutant larvae, but has less effect on Gfpt and Alg9. This is quantitated in graphs of G) Dpagt1, H) NgBR, and I) Gfpt where data represent three biological replicates per genotype per condition of 10 embryo samples. Error = SEM, Student?s t test, ??p < 0.01. (J?M) Immunohistochemical stains of 7dpf WT and mutant fli1a:EGFP positive cartilage sections show Dpagt1(J, red) and NgBR (K, red) abundances are locally reduced following epalrestat treatment. (L and M) Graphs show pixel intensity of Dpagt1 (L) and NgBR (M) relative to EGFP. Each data point represents a single cell. n = 10?20 cells in 6?10 embryos per genotype per condition. Error = SEM, Student?s t test, ?p < 0.05 and ????p < 0.0001. See also Figure S3.