Fig. 2: Preventing O-GlcNAcylation decrease through OGA silencing does not impact the entry of HCT116 cells into SN38-induced senescence.

HCT116 cells were transfected with a siRNA targeting OGA (si OGA) or a non-target control siRNA (siCTRL). 24 h later, the cells were treated with 1 nM SN38 for 72 hours. A Western blot analysis was performed to assess the expression of the three senescence markers p21, cyclin D1, and EZH2. The efficiency of siRNA was also confirmed by evaluating OGA los of expression and O-GlcNAcylation levels upregulation. GAPDH was used as a loading control. Results shown are representative of three independent experiments. B Quantitative fluorimetric determination of the SA-β-Galactosidase activity in the different experimental conditions. Results represent the individual values and mean +/- SEM of four independent experiments (n = 4) (ns: non-significant, *P < 0.05, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests). Quantification of relative protein expression analyzed by Western Blot (optical density measurement relative to GAPDH) of p21 (C), cyclin D1 (D) and EZH2 (E) from three independent experiments (n = 3) (individual values and mean +/- SEM, ns: non-significant, *P < 0.05, ***P < 0.001, ****P < 0.0001, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests).