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Figure 3 | Particle and Fibre Toxicology

Figure 3

From: Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation

Figure 3

PM induces calcium leakage via ROS-activated TRPM2. (A) Human lung microvascular ECs were plated on glass cover slips and loaded with 5 μM Fura-2 AM in 1 ml of basic medium. ECs were rinsed twice and treated with PM (100 μg/ml). Fura-2 fluorescence was measured at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm (0–60 min). Fura-2 has excitation wavelengths of 380 nm in its free form and 340 nm when it is bound to calcium; therefore, relative calcium concentrations are represented by the ratio of 340/380. (B) ECs plated on glass cover slips were treated with TRPM2 antibody or control IgG (5 μg/ml, 4 hr pretreatment). ECs were then rinsed twice and subjected to the same calcium measurement procedures with PM treatment (100 μg/ml, 0–60 min). *p < 0.05 compared to control IgG group at the same time point. (C) ECs were transfected with TRPM2 siRNA or control siRNA for 48 hrs. ECs were then rinsed twice and subjected to the same calcium measurement procedures with PM treatment (100 μg/ml, 0–60 min). EC cell lysates were analyzed by Western blot with antibodies to TRPM2 and β-actin to confirm silencing. *p < 0.05 compared to control siRNA group at the same time point. (D-E) ECs plated on glass cover slips (95% confluent) were treated with NAC (5 mM, 1 hr pretreatment) or 3-AB (1 mM, 1 hr pretreatment). ECs were then rinsed twice and subjected to the same calcium measurement procedures with PM treatment (100 μg/ml, 0–60 min). *p < 0.05 compared to PM-only group at the same time point.

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