Figure 3. Global protein glutathionylation increases upon the addition of palmitate during repletion of glucose and oxygen after OGD (OGD/R).

(A-C) In-gel fluorescence detection of global levels of glutathionylation during repletion of glucose or oxygen after OGD (A), repletion of palmitate after OGD (B), or co-addition of fatty acid oxidation inhibitors during OGD/R (C). HL-1/GS M4 cells incubated with azido-Ala were subjected to the indicated conditions after OGD (1% O₂ and no glucose) in a hypoxic chamber, or co-treated with Etomoxir (Et), Ranolazine (Ra), or 4-pentenoic acid (PA) in the presence of palmitate (1 mM) and BSA (170 μM). Cells were then lysed and analyzed by in-gel fluorescence (glutathionylation level) or Coomassie stains (protein loading control) after click reaction with Cy5-alkyne. Data are representative of at least 3 independent experiments. (D-G) Analysis of redox environment during OGD/R. HL-1 cells were analyzed for ROS (D), a ratio of NADP⁺ over NADPH (E), a ratio of oxidized glutathione (GSSG) over reduced glutathione (GSH) (F), and viability (G) after repletion of glucose, oxygen, or palmitate for 2 h, following OGD for 8 h. Data represent the mean ± SD, n = 3 independent experiments. Difference is significant by one-way ANOVA followed by Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001.