Fig. 1: Drug repurposing screen to identify modulators of O-GlcNAc.

a Schematic of O-GlcNAc regulation by enzymes OGT and OGA with commonly used OGT inhibitor OSMI-4a/b and OGA inhibitor TMG. b Canonical splicing pathways of OGT and OGA transcripts. High O-GlcNAc favors nonproductive OGT transcripts and the productive OGA transcript. Low O-GlcNAc favors the productive OGT transcript and the nonproductive OGA transcript. c Western blot of O-GlcNAc, OGT, and OGA levels in HCT116 cells after indicated treatment for 24 h (n = 3). d Schematic of GFP-β-OGT reporter for OGT splicing. e Schematic of GFP-β-OGT reporter screen. Lower O-GlcNAc correlates with increased GFP signal, while elevated O-GlcNAc correlates with decreased GFP signal. f Sample images of GFP-β-OGT reporter cells following indicated treatment for 24 h. Scale bar 100 μm. g Dose-response change in mean nuclei GFP per well with indicated compound for 24 h (n = 5 biological replicates). Data are represented as mean ± s.d. h Screening results with the Drug Repurposing Library in GFP-β-OGT reporter cells. i Average Δ nuclei mean GFP versus average viability in GFP-β-OGT reporter across n = 2 replicates. Viability determined by nuclei counts. j Schematic of filter for autofluorescent compounds in wild-type HCT116 cells. k Average Δ nuclei mean GFP in wild-type HCT116 cells versus average Δ nuclei mean GFP in GFP-β-OGT reporter cells from n = 3 replicates to filter autofluorescent compounds. l Activity of selective and nonselective ROCK inhibitors in GFP-β-OGT reporter. Source data are provided as a Source Data file.