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. 2019 May;60(6):2369–2379. doi: 10.1167/iovs.19-26606

Figure 3.

Figure 3

REDD1 deletion is sufficient to prevent hyperglycemia-induced ROS. R28 retinal cells were maintained in DMEM containing 5 mmol/L glucose and supplemented with 10% FBS. (A) Cells were exposed to medium containing 5 mmol/L glucose, 30 mmol/L glucose, or 5 mmol/L glucose plus 25 mmol/L mannitol as an osmotic control for 24 hours. ROS were visualized with DCFDA. DCF fluorescence and bright-field (BF) images are shown. (B) DCF fluorescent intensity per well was quantified using a plate reader. (C, D) REDD1, caspase-3 cleavage, and tubulin protein expression were assessed by Western blotting. Protein molecular mass (kDa) is indicated at the right of blots. (E) Relative cell death was assessed by ELISA of cytoplasmic nucleosomes. R28 WT and REDD1 KO (sgREDD1) cells were maintained in DMEM containing 5 mmol/L glucose supplemented with 10% FBS (F–H, J–L). Cells were exposed to medium containing 5 mmol/L or 30 mmol/L glucose for 24 hours. (F) REDD1 mRNA expression was assessed by qPCR. (G) ROS were visualized with DCFDA, and DCF fluorescent intensity was quantified (H). (I) ROS levels were similarly evaluated in REDD1+/+ and REDD1−/− MEF. REDD1 and tubulin protein expression were assessed by Western blotting. Protein loading was evaluated by reversible protein stain. (J) Mitochondrial superoxide was assessed by MitoSOX stain. (K, L) Mitochondrial membrane potential was evaluated with JC-1 dye. JC-1 aggregates and monomers were quantified. Results are representative for three experiments. Within each experiment, 3 to 10 independent samples were analyzed. Values are means + SEM. *P < 0.05 versus Veh; #P < 0.05 versus WT. nd, not detected.