Figure 3
Figure 3. SK-1 regulates EC permeability changes. (A) HUVECs were untreated (Nil) or treated with 5 μM DMS for 15 minutes (DMS), Ang-1 (0.2 μg/mL) for 30 minutes (Ang-1), or Ang-1 and DMS (Ang-1 + DMS). Permeability is given as the FITC-dextran passage in 30 minutes. Pooled data from 3 experiments are shown (*P < .01 vs untreated cells). (B) HUVECs were transfected with control siRNA or siRNA against hSK-1. After 48 hours, cells were lysed and SK-1 activity measured. Pooled data from 3 experiments are shown and are expressed as the fold change in relation to control cells where the SK-1 activity was set to 1.0 (*P < .001 vs control siRNA cells). (C) HUVECs were transfected with control siRNA or siRNA against hSK-1. Permeability was measured 48 hours later. Permeability is given as the FITC-dextran passage in 30 minutes. Pooled data from 3 experiments are shown (*P < .01 vs control siRNA cells). (D) HUVECs were infected with adenoviral carrying EV or human SK-1. After 48 hours, SK-1 activity was measured. Pooled data from 3 experiments are shown and are expressed as the fold change in relation to EV cells, which was normalized to 1.0 (*P < .001 vs EV cells). (E) HUVECs were infected with EV or SK-1 in adenovirus. Permeability was measured 72 hours later. Permeability is given as the FITC-dextran passage in 30 minutes. Shown are pooled data from 3 experiments (*P < .01 vs EV cells). (F) HUVECs were infected with EV or SK-1 in adenovirus. After 48 hours, cell lysates were immunoprecipitated with an anti-PECAM-1 antibody. Western blots for phosphotyrosine (top panel) and PECAM-1 (bottom panel) are shown. (G) HUVECs were transfected with control siRNA or siRNA against hSK-1. After 48 hours, cells were lysed and immunoprecipitated with an anti-PECAM-1 antibody. Western blots for phosphotyrosine (top panel) and PECAM-1 (bottom panel) are shown. (H) HUVECs were infected with EV (EV) or SK-1 (SK-1) in adenovirus. Forty-eight hours after infection, the cells were replated onto LabTek slides and washed and fixed 45 minutes after plating. Cells were stained with anti–VE-cadherin antibody. For imaging information, see “VE-cadherin staining” section in “Methods.” All data are mean plus or minus SEM.

SK-1 regulates EC permeability changes. (A) HUVECs were untreated (Nil) or treated with 5 μM DMS for 15 minutes (DMS), Ang-1 (0.2 μg/mL) for 30 minutes (Ang-1), or Ang-1 and DMS (Ang-1 + DMS). Permeability is given as the FITC-dextran passage in 30 minutes. Pooled data from 3 experiments are shown (*P < .01 vs untreated cells). (B) HUVECs were transfected with control siRNA or siRNA against hSK-1. After 48 hours, cells were lysed and SK-1 activity measured. Pooled data from 3 experiments are shown and are expressed as the fold change in relation to control cells where the SK-1 activity was set to 1.0 (*P < .001 vs control siRNA cells). (C) HUVECs were transfected with control siRNA or siRNA against hSK-1. Permeability was measured 48 hours later. Permeability is given as the FITC-dextran passage in 30 minutes. Pooled data from 3 experiments are shown (*P < .01 vs control siRNA cells). (D) HUVECs were infected with adenoviral carrying EV or human SK-1. After 48 hours, SK-1 activity was measured. Pooled data from 3 experiments are shown and are expressed as the fold change in relation to EV cells, which was normalized to 1.0 (*P < .001 vs EV cells). (E) HUVECs were infected with EV or SK-1 in adenovirus. Permeability was measured 72 hours later. Permeability is given as the FITC-dextran passage in 30 minutes. Shown are pooled data from 3 experiments (*P < .01 vs EV cells). (F) HUVECs were infected with EV or SK-1 in adenovirus. After 48 hours, cell lysates were immunoprecipitated with an anti-PECAM-1 antibody. Western blots for phosphotyrosine (top panel) and PECAM-1 (bottom panel) are shown. (G) HUVECs were transfected with control siRNA or siRNA against hSK-1. After 48 hours, cells were lysed and immunoprecipitated with an anti-PECAM-1 antibody. Western blots for phosphotyrosine (top panel) and PECAM-1 (bottom panel) are shown. (H) HUVECs were infected with EV (EV) or SK-1 (SK-1) in adenovirus. Forty-eight hours after infection, the cells were replated onto LabTek slides and washed and fixed 45 minutes after plating. Cells were stained with anti–VE-cadherin antibody. For imaging information, see “VE-cadherin staining” section in “Methods.” All data are mean plus or minus SEM.

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