Purging the ER of its H₂O₂ content selectively retards PRDX4-mediated ER oxidation. (A) Bar diagram showing specific activities of catalase in extracts of untransfected MEF^DKO cells and cells expressing catalase variants targeted to the indicated compartments. (B–D) Plot of the relationship between H₂O₂ concentration in the media and the excitation ratio of cytoHyper, mitochondrially located HyPer (mitoHyPer), or ERHyPer, expressed alone or alongside catalase in MEF^DKO cells (with insets of fixed anti-catalase immunostained cells showing the localization of the protein). The titration measuring the ER-localized probe was performed in presence of 2 mM DTT. The plots are presented along their corresponding ratiometric photomicrographs (B–D, bottom panels). The catalase-expressing cells are marked by the coexpression of mCherry (encoded on the same plasmid, denoted by white arrows). Note, the ratio indicates a more reduced state (color coded in blue-green) at the intermediate H₂O₂ concentration in catalase-positive cells compared with the mostly oxidized state (color coded yellow-red) in catalase-negative cells, as the latter are desensitized to H₂O₂ by catalase overexpression. (E) Traces of oxidation recovery of ERroGFP2 after a DTT pulse (2 mM, 1 min) in MEF^DKO cells with catalase expressed in the indicated compartments. (F) Bar diagram of the t_1/2 to recovery of the oxidized form of ERroGFP2 after the reductive DTT pulse, calculated from fitting the data in E. Shown are mean t_1/2 values ± SEM (n > 10; **, P < 0.01). (G) Traces of oxidation recovery of ERroGFP2 after a DTT (2 mM, 1 min) pulse in parental TKO cells [Cat(−)] and TKO cells expressing catalase in their ER [ER Cat], identified by the presence of the coexpressed mCherry marker, as in E–G]. Inset shows a bar diagram of the corresponding ERroGFP2 reoxidation t_1/2 values. Shown are means ± SEM (n > 10).