Fig. 1
Effects of S1PC on cell permeability, junctional/cytoskeletal proteins, and MLC2 phosphorylation in TNF-α-stimulated HUVECs. a Chemical structure of S1PC. b The concentration-dependent effect of S1PC on TNF-α-induced hyperpermeability of HUVECs. Vascular permeability assay was conducted after the stimulation with TNF-α (50 ng/mL) in the presence or absence of S1PC (75, 150 or 300 μM) for 24 h. Data are shown as mean ± SD, n = 4. Significant difference compared to the control group (##p < 0.01) or TNF-α-treated group (**p < 0.01) was determined by Dunnett’s multiple comparison test. c Effect of S1PC on TNF-α-induced disruption of junctional proteins in HUVECs. The membrane and cytoplasmic proteins were extracted after the stimulation with TNF-α (50 ng/mL) in the presence or absence of S1PC (300 μM) for 24 h and analyzed by western blotting with indicated antibodies. Quantitative data are shown as mean ± SD, n = 3–6. Significant difference compared to the control group (#p < 0.05) or TNF-α-treated group (*p < 0.05) was determined by Bonferroni’s multiple comparison test. d, e Effect of S1PC on TNF-α-induced disturbance of localization of junctional proteins, VE-cadherin and Claudin-5, and actin cytoskeleton in HUVECs. HUVECs were immunostained with indicated antibodies after the stimulation with TNF-α (50 ng/mL) in the presence or absence of S1PC (300 μM) for 24 h. Specific fluorescence: red for VE-cadherin (d) and Claudin-5 (e), green for F-actin and blue for nuclei stained with DAPI. Scale bar, 25 μm. f Effect of S1PC on TNF-α-induced MLC2 phosphorylation in HUVECs. Cell lysates were obtained after the stimulation with TNF-α (50 ng/mL) in the presence or absence of S1PC (300 μM) for 24 h and analyzed by western blotting with indicated antibodies. Quantitative data are shown as mean ± SD, n = 3. Significant difference compared to the control group (##p < 0.01) or TNF-α-treated group (**p < 0.01) was determined by Bonferroni’s comparison test