Figure 4

PM-activated TRPM2 promotes ZO-1 degradation by calpain. (A) Human lung microvascular ECs grown in 6-well dishes to approximately 95% confluence were treated with TRPM2 antibody or control IgG (5 μg/ml) for 4 hr, and then challenged with PM (100 μg/ml) for 6 hr. Cell lysates were analyzed by Western blotting with ZO-1 antibody. 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 control. **p < 0.05 compared to PM challenge. (B) ECs grown in 6-well dishes to approximately 80% confluence were treated with TRPM2 siRNA or control siRNA (100 ng/ml) for 48 hr, and cell lysates were analyzed by Western blotting with ZO-1 antibody. (C) ECs grown in 6-well dishes to approximately 95% confluence were treated with TRPM2 siRNA or control siRNA (100 ng/ml) for 48 hr, and then challenged with PM (100 μg/ml) for 6 hr. Cell lysates were analyzed by Western blotting with ZO-1 antibody. 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 control. **p < 0.05 compared to PM challenge. (D) ECs grown on ECIS gold electrodes were treated with TRPM2 antibody or control IgG (5 μg/ml) for 4 hr, and then challenged with PM (100 μg/ml). Changes in TER were measured with ECIS. *p < 0.05 compared to PM-challenged group. (E) ECs grown on 100 mm dishes were treated with TRPM2 siRNA or control siRNA (100 ng/ml) for 48 hr, and then plated onto gold electrodes for ECIS measurement. 24 hours after replating, the ECs were challenged with PM (100 μg/ml) and changes in TER were measured with ECIS. *p < 0.05 compared to PM-challenged group.