Figure 2.

Hyperglycemic conditions suppress proteasome-independent REDD1 degradation by promoting oxidative stress. A: HA-REDD1 was expressed in REDD1-KO MIO-M1 cells by transient transfection. Cells were exposed to medium containing 30 mmol/L glucose (HG) or 5 mmol/L glucose plus 25 mmol/L mannitol as an osmotic control (OC) for 4 h. Cycloheximide (CHX) was used to inhibit protein synthesis. REDD1 and tubulin protein expression were evaluated by Western blotting (n = 6). Representative blots are shown. Protein molecular mass in kDa is indicated at right of blots. B: ROS were visualized in WT MIO-M1 cells with DCFDA. DCF fluorescent intensity was quantified after exposure to HG or OC in the presence and absence of the antioxidant NAC. C: HA-REDD1 protein degradation was assessed in REDD1-KO MIO-M1 cells by CHX-chase assay. D: HEK293 Tet-On HA-REDD1 cells were exposed to culture medium containing H₂O₂ (HP) for 2 h, followed by CHX-chase in the presence/absence the proteasome inhibitor MG-132 (n = 4). E: HA-REDD1 was expressed in REDD1-KO MIO-M1 cells, and REDD1 protein degradation was evaluated by CHX-chase following exposure to HP and/or MG-132. F: HA-REDD1 was expressed in REDD1-KO MIO-M1 cells, and REDD1 protein degradation was evaluated by CHX chase in the presence of MG-132 following exposure to either HG or OC (n = 4). Data are represented as mean ± SD. *P < 0.05 vs. OC or Veh; #P < 0.05 vs. control.