Cellular TrxR1-selectivity: By now we had shown that, in line with our pathway hypothesis, RX1 was monothiol resistant and could therefore be a protein-selective probe, while its regioisomeric control G1 had some lability to physiological GSH concentrations. Under cell-free conditions, both RX1 and G1 were rapidly activated by TrxR1, dependent upon its selenolthiol active site, and had minor sensitivity to TrxR2; both were sensitive to fully reduced Trx but almost entirely nonresponsive to the alternative vicinal dithiol thioredoxin-related protein TRP14; and were inert to GR/Grx. We now entered cell-based assays, aiming to test the probes' TrxR-dependency indicated in the cell-free assays as well as their potential for functional TrxR1-selectivity in the cellular context, by elucidating the cellular mechanism of activation of these compounds (see Supporting Information for assay conditions, processing methods, full data for all probes and accompanying discussions; and Supporting Note 5 for discussion of fluorescence normalisation).