Fig. 1: SN38-induced senescence of HCT116 cells is accompanied by a decrease in GFAT2, OGT, OGA, and O-GlcNAcylation levels.

A Scheme depicting the Hexosamine Biosynthetic Pathway and O-GlcNAcylation processes. GFAT1/2: Glutamine Fructose-6-Phosphate Amido Transferase 1/2 (rate limiting enzyme of the HBP), OGT: O-GlcNAc Transferase, OGA: O-GlcNAcase. B HCT116 cells were treated with increasing concentrations of SN38 ranging from 1 to 25 nM for 96 hours. Cell apoptosis was investigated by Western Blot analyses of cleaved-caspase 7 and cleaved-PARP1. GAPDH was used as a loading control. Data shown are representative of three independent experiments. C–E HCT116 cells were treated with 1 nM SN38 for 96 or with DMSO as a negative control. C Top: Representative images of the microscopic analysis of cell morphology showing an increase in the size of senescent cells. Pictures were taken at the same magnification (x200). Bottom: SA-β-Galactosidase activity assay demonstrating blue staining of senescent cells. D Left: Western blot analysis of O-GlcNAcylation levels, OGT, OGA, and both isoforms of GFAT (GFAT1 and GFAT2), as well as the expression of p21, cyclin D1, and EZH2, three senescence markers. Right: Quantification of relative protein expression from four independent experiments (optical density measurement relative to GAPDH) (n = 4) (individual values and mean +/- SEM, ns: non-significant, *P < 0.05, **P < 0.01, multiple unpaired t-tests). E qRT-PCR analysis of p21, cyclin D1, EZH2, GFAT1, GFAT2, OGT, and OGA transcript expression. Results represent the individual values and mean +/- SEM of five independent experiments (n = 5) (ns: non-significant, **P < 0.01, ***P < 0.001, ****P < 0.0001, multiple unpaired t-tests).