Figure 2. High glucose (HG) promotes phosphatase-dependent HIPK2 protein degradation.

A. Western blot analysis (lower panel) and relative quantification (upper panel) of endogenous HIPK2 protein levels in HCT116 cells cultured in HG with or without MG132, Calyculin A (CL-A) or Okadaic acid (OA). Anti-β-actin was used as protein loading control. A representative image is shown. Data of relative quantification of HIPK2 levels from three different experiments (upper panel) are presented as mean± S.E.M. (n=3) and quantified with respect to control set to 1.0. *P < 0.001. (HG versus ctr, MG132 versus HG, CL-A versus HG, OA versus HG). B. HEK-293 cells were transfected with HIPK2-GFP vector and 24 h after transfection switched in HG condition for 24 h with or without MG132, CL-A, or OA. Analysis of GFP-positive cells was performed by visualizing at least 200 DAPI-positive cells/group and quantified with respect to control (HIPK2-GFP/LG condition) set to 1.0. *P < 0.001. C. Western blot analysis (lower panel) and relative quantification (upper panel) of GFP levels in RKO cells transfected with HIPK2-GFP vector and 24 h after transfection switched in HG condition for 24 h with or without CL-A. Anti-β-actin was used as protein loading control. *P < 0.001. (HG versus HIPK2-GFP and HG/CL-A versus HG/HIPK2-GFP). D. Western blot analysis of endogenous HIPK2 protein in HCT116 cells cultured for 24 h in the presence of serum coming from different patients, evidenced as numbers at the height: one normo-glycemic sera (glycemia ≤ 90) (LG¹) and two hyperglycemic sera (glycemia ≥ 300) derived from patients with type 2 diabetes (DM2) (HG¹ and HG²), with or without CL-A. Anti-β-actin was used as protein loading control.