We next explored how chemical inhibition of TrxR affects signal generation. We relied on the metal-free compounds TRi-1 and TRi-3 which are inhibitors with good cellular TrxR-selectivity, rather than the common thiophilic gold complex auranofin which has potential pitfalls (see Supporting Note 1 and Fig. ). Cellular RX1 signal was strongly inhibited by acute TRi treatment, with just 2 µM TRi-1 reducing signal by 66%; whereas G1 signal was only reduced by 15% and SS00 signal was unaffected by this treatment (Fig. , Fig. ). TRi-3 dose-dependently reproduced the same inhibitory effects. This supports that cellular RX1 activation is highly dependent on the activity of the TRi-1/3 target TrxR, whereas activation of its regioisomer G1 is largely TrxR-independent.