Figure 2. Glycated PON1 triggers oxidative stress to induce ER stress and intracellular Ca²⁺ overload in HUVECs.

(A–C) HUVECs were incubated with glycated PON1 as indicated concentrations (2.5, 5, 10, 20 μg/ml) for 6 hours. The 3-NT level was assayed in total cell lysates by western blot in (A). NADPH oxidase activity in (B) and ROS production in (C) were also assessed. N = 3 per group. *P < 0.05 vs. Control (PON1 alone). (D,E) HUVECs were transfected with p67 siRNA in (D) or p22 siRNA in (E) for 48 hours followed by co-incubation with glycated PON1 (10 μg/ml, 6 hours). The levels of p-PERK, p-eIF2α, CHOP and ATF6 were assayed in total cell lysates by western blot. The blot was a representative picture from 3 independent experiments. (F) HUVECs were treated with 10 μg/ml gly-PON1 with or without BAPTA (0.5 mM) for 6 hours. Ratiometric measurement of intracellular Ca²⁺ was done. N = 3 per group. (G) HUVECs were treated with 10 μg/ml gly-PON1 and tunicamycin (10 μM) in presence or absence of BAPTA (0.5 mM) from 6 hours. The levels of p-PERK, p-eIF2α, CHOP, ATF6, STIM1 and ORAI1 were assayed in total cell lysates by western blot. These experiments were repeated for 3 times. The presented blots were cropped from the full blots shown in Supplementary Figures S4–8.