Figure 2.

Mast cell induced endothelial ER stress is accompanied by mitochondrial dysfunction, reactive oxygen species (ROS) production, and increased permeability. mBECs were treated for 24 h with MCCM from cultures of mast cells isolated from the skin of sickle or control mice. mBECs were pre-treated with Salubrinal (5 μM) or vehicle for 30 min before addition of MCCM, where applicable. (A) Sickle MCCM significantly decreased mitochondrial membrane potential detected by JC-1. An increased green to red fluorescence ratio is indicative of decreased membrane potential. *p < 0.05. (B) Sickle MCCM significantly increases the production of ROS, which is ameliorated by pre-treatment with Salubrinal. ROS in mBEC is shown as fluorescence units of oxidized 2′,7′-dichlorodihydrofluorescein. (C) Sickle MCCM significantly increases the endothelial permeability, which is ameliorated by pre-treatment with Salubrinal. Evans blue leakage through mBEC monolayer following incubation with control or sickle MCCM, measured at 650 nm is shown. *p < 0.05, ****p < 0.0001.