Figure 2

Activated calpain is required for PM-mediated ZO-1 degradation and EC barrier disruption. (A) Human lung microvascular ECs grown in 60-mm dishes to approximately 95% confluence were treated with ALLN (30 μM), calpeptin (CALP, 10 μM), NAC (5 mM), or BAPTA-AM (50 μM) for 1 hr, and then challenged with PM (100 μg/ml) for 1 hr. Cell lysates were subjected to calpain activity assay by using the calpain activity kit (Calbiochem). Calpain activity was normalized to protein concentration and expressed as fold changed compared to control. *p < 0.05 compared to control. **p < 0.05 compared to PM only group. (B) ECs grown on ECIS gold electrodes were treated with ALLN (30 μM), calpeptin (CALP, 10 μM) for 1 hr, and then challenged with PM (100 μg/ml). Changes in TER were measured with ECIS. *p < 0.05 compared to PM only group. (C) ECs grown on 6-well plates were treated with ALLN (30 μM), calpeptin (CALP, 10 μM), or BAPTA-AM (50 μM) for 1 hr, and challenged with PM (100 μg/ml) for 1–6 hr. Cell lysates were analyzed by Western blotting with antibody to ZO-1. Changes in levels of ZO-1 are expressed as fold changes and normalized to β-actin. Shown are representative blots from three independent experiments. *p < 0.05 compared to PM-1 hr group. **p < 0.05 compared to PM-6 hr group