ERHyPer responds to exogenous H₂O₂ in a chemically imposed reducing environment. (A) Plot of the rate of in vitro oxidation of HyPer (1 µM) as a function of H₂O₂ or oxidized PDI (PDI_ox) concentration, calculated from the linear phase of the initial oxidation reaction traced ratiometrically (Fig. S2). (B) Schema representing response modes of ERroGFP2 and ERHyper. Both probes undergo oxidation by PDI. Unlike roGFP2, reduced HyPer also undergoes rapid oxidation by H₂O₂. Thus, HyPer’s responsiveness to H₂O₂ can be unmasked in settings with a low concentration of oxidized PDI. (C) Traces of time-dependent changes to the redox-sensitive excitation ratio of HyPer exposed to various concentrations of H₂O₂ in vitro, in the continued presence of DTT. (D) Fluorescent photomicrographs of MEFs transiently transfected with ERHyPer encoding vector, immunostained for calreticulin, as an ER marker. (E) Trace of time-dependent changes to the oxidation state of ERHyPer (top plot) or ERroGFP2 (bottom plot) expressed in MEF cells challenged repeatedly with H₂O₂ (arrows) in the continuous presence of DTT. The general oxidant, diamide, was added in excess, to reveal the responsiveness of both probes at the end of the experiment. (F) Trace of time-dependent changes to the redox state of ERHyPer expressed in MEF cells, first briefly exposed to DTT, followed by wash out, and then exposed to DTT, followed by introduction of H₂O₂ to the reducing medium.