Figure 5. STIM1-mediated NFAT activity is necessary for LPS-induced proinflammatory gene expression in ECs.

(A) Ex vivo imaging of pulmonary vascular Ca²⁺ levels in freshly prepared lung slices from wild-type mice challenged with LPS (1 mg/kg) for 20 hours. AcLDL was used as an endothelial marker. Scale bar: 50 μm. (B) Quantification of Fluo-4 fluorescence was measured from multiple regions of the lung slices. ECs from wild-type and Stim1^ΔEC mice were challenged with LPS (1 μg/ml) for 16 hours. ECs were loaded for 30 minutes with Fluo-4/AM. (C) Representative traces from wild-type and Stim1^ΔEC ECs. (D) Quantification of oscillation frequency. ECs from wild-type and Stim1^ΔEC mice were transduced with adenovirus encoding NFATc3-GFP for 36 hours. Following adenoviral transduction, ECs were treated with LPS for 16 hours. (E) Representative images showing NFAT nuclear translocation. Scale bar: 20 μm. Arrowheads indicate the nuclear translocated NFAT. (F) Quantification of nuclear NFAT-positive cells. (G) NFAT-dependent luciferase activity was measured in wild-type and Stim1 KD ECs 16 hours after LPS (1 μg/ml) treatment. (H) Quantification of cytokine protein expression in wild-type and Stim1^ΔEC ECs treated with LPS for 16 hours. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.