Sluggish transit of H₂O₂ into the ER lumen. (A) Plot showing the dependence of the redox state of cytoHyPer-expressed in RINm5F cells (measured by excitation ratio) on concentration of H₂O₂, introduced to the culture medium in the continued presence or absence of DTT (2 mM). (B) Plot of the relationship between HyPer oxidation state (linearly normalized, by setting the maximal observed value to 1 and the minimal observed value to 0) in the different compartments and concentration of H₂O₂ in the media in RINm5F cells exposed continuously to DTT (as in A). (C) Bar diagram of the mean H₂O₂ concentration in the culture media required to effect 50% of maximal oxidation of cytosolic, mitochondrial, or ER-localized HyPer (the midpoint in the traces shown in B; mean ± SEM, n = 4). **, P < 0.01. (D) Titration of H₂O₂ as described in C, performed in the presence of DTT in RINm5F or MEF cells. Note the enhanced sensitivity of RINm5F cells compared with the MEFs.