Fig. 6.

Reduction of Fast-TRFS (TRFS9) by TCEP and Selective activation of Fast-TRFS by TrxR. a Reduction of Fast-TRFS by TCEP. Fast-TRFS (100 μM) was incubated with TCEP (1 mM) for 0 min (a0), 1 min (a1), 30 min (a30) and 240 min (a240), and the mixture was analyzed by HPLC-MS. b Time-dependent emission spectra of Fast-TRFS toward TrxR. Fast-TRFS was incubated with TrxR/NADPH (50 nM/200 μM), and the emission spectra were recorded every 1 min for 15 min. c Time course of the fluorescence increase of Fast-TRFS with TrxR/NADPH and U498C TrxR/NADPH. d Response of Fast-TRFS to various relevant biological species. The fluorescence increase at 460 nm was determined after they were incubated with Fast-TRFS for 15 min. The Sec (10 μM) was generated in situ by mixing Cys (1 mM) and selenocystine (5 μM). e Inhibition of the cell lysate-mediated Fast-TRFS reduction by TrxR inhibitor AF. The NADPH-pretreated HeLa cell lysate (0.5 mg mL⁻¹) was treated with AF for 30 min, and further incubated with Fast-TRFS and NADPH (200 μM) for additional 20 min. The fluorescence increase was determined. f Reduction of Fast-TRFS by lysates (0.5 mg mL⁻¹) from the genetically manipulated HeLa cells in the presence of NADPH (200 μM). g Time course of the fluorescence increase of Fast-TRFS, TRFS-red and TRFS-green with TrxR/NADPH (50 nM/200 μM). All reactions were performed in TE buffer at 37 °C. The excitation/emission wavelengths for Fast-TRFS, TRFS-green and TRFS-red are 345/460 nm, 438/538 nm and 615/661 nm, respectively. The concentration of the probes in (b–g) was 10 μM. Source data are provided as a Source Data file