Fig. 3.

Sec-TRs but not Cys-TRs accelerate roGFP reduction after oxidative insult. (A) Schematic of the dynamic response of the roGFP probe to changes in intracellular thiol status. In the presence of oxidants a disulfide bond is formed in roGFP that increases fluorescence intensity after excitation of the probe at 405 nm (Ox). Under reduced conditions, fluorescence intensity is increased after excitation at 488 nm (Red). The ratio of oxidized to reduced roGFP reflects cellular redox status. (B) Basal cytosolic redox state in cells co-transfected with Cyto-roGFP, TRX1, and the indicated TR1 expression vectors (n=20 cells). (C and D) Basal mitochondrial oxidation state in cells transfected with Mito-roGFP, TRX2, and the indicated amount of TR2 expression vectors (n=20, p value determined by students t-test). (E) The basal cellular redox state in C10 cells expressing Cyto-roGFP was assessed by ratiometric imaging prior to adding 40 uM menadione (MD) for 20 min. After washing out MD by 5 exchanges with fresh imaging media the same cells were re-examined over time to quantify change in oxidation (ΔOx, see Materials and methods) of the cyto-roGFP probe. (F) The experiment in panel E was repeated in cells co-expressing the indicated TR1 expression vector and Cyto-roGFP, and the ΔOx was calculated post menadione washout after 10 min (n=10, *p<0.05). (G) The experiment in E was repeated using cells co-transfected with Mito-roGFP and the indicated TR2 expression vectors and the ΔOx was calculated post menadione washout after 10 min (n=10, *p<0.05).