Finally, we stringently evaluated RX1's TrxR1-dependency using TrxR1-knockout (ko) cells (MEF: mouse embryonic fibroblasts) , which we compared against their corresponding wildtype control (fl/fl). This knockout upregulates compensatory thiol-based reductive pathways to survive (e.g. the GSH system), with Trx being mainly reduced instead by Grx2. Therefore, we expected that the only circumstances under which we would see zero signal for a hitherto cellularly-active probe dependent on Se-containing TrxR1, would be if that probe was exclusively activated by TrxR1 in the cellular context. The TrxR1 ko MEFs showed zero RX1 activation, while the corresponding wildtype gave strong signal (Fig. ). As the mitochondrial TrxR2 is still functional in the knockout, this strongly suggests that cellular RX1 signal reports essentially on cytosolic TrxR1 at least in this cell line. Signal from mechanistic control G1 was suppressed by ca. 60% though not abolished in the knockout cells (Fig. ), again matching expectations that G1 is not fully TrxR1-selective in cells. To obtain still stronger proof of mechanism, we tested TrxR1-knock-in (ki) to the MEF TrxR1-knockout background . This fully restored the strong, dose-dependent RX1 signal generation (Fig. , Fig. ).