Figure 2.

Hydrolysis of S223-AM. (A) S223-AM was exposed to PBS (left), U2OS cell culture supernatant (middle), and lysates from INS cells (right). The hydrolysis of S223-AM was followed over time by HPLC and the peak integrals of S223-AM and the products S223 and OXO were plotted. The decay of S223-AM and the appearance of the products were fitted to single exponential kinetics shown as solid lines. (B) The fraction of S223 in the total product (S223 and OXO) after exposure of S223-AM to different media. The thin grey bars represent the expected fraction of S223 if OXO had been formed with the same rate constant as that observed in PBS. (C) Summary of the kinetic parameters determined for the hydrolysis of S223-AM in different media. Full kinetics in PBS, culture supernatant and lysates from INS cells that allowed the accurate determination of the rate constants of the decay of S223-AM (k_decay) and the formation of S223 (k_S223) and OXO (k_OXO) was recorded as shown in (A). Limited kinetics that allowed an estimation of k_decay were recorded for the remaining conditions. The half-life of S223-AM τ_1/2 were calculated from k_decay. PBS, phosphate buffer saline; Acetylesterase, 1 unit orange skin acetylesterase in PBS; RPMI, RPMI medium supplemented with foetal calf serum; supernatant, RPMI medium supplemented with serum taken from a culture dish with U2OS cells after 3 day of culture; U2OS, HUVEC, HEK293T, and INS, lysates of respective cells.