Figure 3.

(a) Structures of CyBam-γ-Glu and CyBam-N.C. (b) Increase in fluorescent intensity of CyBam-γ-Glu (20 μM) after incubation with increasing concentration of GGT (0–400 U/L). (c) Activation of CyBam-γ-Glu after incubation with GGT (100 U/L), leucine aminopeptidase (LAP; 800 U/L), and pig-liver esterase (PLE; 800 U/L) at 37 °C for 30 min in PBS pH 7.4 followed by pH adjustment to pH 5.2. Cathepsin B (CatB; 2.5 μg) was used in acetate buffer pH 5.2 for 30 min at 37 °C. (d) Confocal images of activation of CyBam-γ-Glu (20 μM) and CyBam-N.C. (20 μM) in SHIN-3 cells. The fluorescent signal from the probe and nucleus (Hoechst) is shown in red and blue, respectively. Images were taken using a 63× oil immersed lens (numerical aperture, N.A. 1.4). (e) Quantification of fluorescent signal after incubation of CyBam-γ-Glu (20 μM) in the presence of GGT inhibitors (DON, GGsTop) and CyBam-N.C. in SHIN-3 cells using flow cytometry. The geometric mean fluorescent intensity (±SD) of the fluorescent signal in the cells is shown (n = 6 independent experiments). (f) In vivo imaging of the SHIN-3-ZsGreen metastatic tumor model at 3 h. Green and red pseudocolors are used to represent the signal from the GFP and Cy7 channels, respectively. Fluorescent line graph showing a correlation between the fluorescent signal from GFP and Cy7 channel across the metastatic tumor in two different regions (A and B). Data points are displayed as mean ± SD, and the p-values were evaluated by the Student’s t-test (*** p-value ≤0.001).