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Data Supplement for Arteriosclerosis, Thrombosis, and Vascular Biology: Volume 20, Issue 4 -- Page 989
This item has the following additional materials available:
Data Files
- Figure I. A, Antiapoptotic effect of EF-1 is mediated via ETA receptor in rat VSMCs. Subconfluent cells were deprived of serum for 6 hours with or without ET-1 (10-8; mol/L) or ETB; receptor agonist (BQ3020: 10-6 mol/L) in the absence or presence of ET receptor antagonists (BQ123, BQ788, TAK044: 10-7 mol/L). Both floating and adherent cells were analyzed as desccribed in print Figure 1A. B, In situ detection of serum deprivation-induced apoptosis in rat VSMCs. Subconfluent cells were serum deprived for 6 hours with or without ET-1 (10-8 mol/L) in the presence or absence of ETA receptor antagonist (BQ123) and ETB receptor antagonist (BQ788) at 10-7 mol/L. Adherent cells were stained in situ by TUNEL method.
- Figure II. A, DNA fragmentation by NO donor and its suppression by ET-1. Rat VSMCs in DMEM containing 1% FBS were incubated with FK409 (10-4 mol/L) or SNAP (10-4 mol/L) in the presence or absence of ET-1 (10-7 to 10-6 mol/L) for 6 hours. Fragmented DNAs extracted by NP-40 lysis method was fractionated on agarose gel electrophoresis as described in Figure 1A. B, Induction of apoptosis by NO donors and effect of ET-1. Subconfluent cells in DMEM containing 1% FBS were incubated with FK409 (10-4 mol/L) or SNAP (10-4 mol/L) in the presence and absence of ET-1 (10-7 mol/L) for 24 hours and subjected to flow cytometric analysis as described in print Figure 1B.
- Figure III. In situ detection of NO-induced apoptosis of rat VSMCs. Subconfluent cells in DMEM containing 1% FBS were incubated with FK409 (10-4 mol/L) with or without ET-1 (10-7 mol/L) in the presence or absence of ET receptor antagonists (BQ123, BQ788: 10-6 mol/L) for 6 hours. Adherent cells were stained in situ by TUNEL method, as described in Figure IB. B, ET-1 suppresses NO-induced apoptosis via ETA receptor. Rat VSMCs in DMEM containing 1% FBS were incubated with FK409 (10-4 mol/L) and/or ET-1 (10-7 mol/L) with or without ET receptor antagonists (BQ123, BQ788, TAK044: 10-6 mol/L). After 24 hours, both floating and adherent cells were separately collected and cell number was counted. Each column represents mean±SEM (n=6); values were calculated as the percentage of the floating dead cell number to total cell population. *P<0.005 vs control.
- Figure IV. ET-1 stimulates and NO inhibits MAP kinase activity in rat VSMCs. A, Cells were cotransfected with pFR-Luc, pFA-Elk1 (GAL4-Elk1), and pRL-TK (Renilla luciferase), deprived of serum for 24 hours, and stimulated with the indicated doses of ET-1 in the presence or absence of PD98059 or FK409. B, Cells were cotransfected with pFR-Luc, pFA-Elk1, and pRL-TK, together with pFC-MEK1, with an empty vector (control vector) or with the mutant form of MEK-1 (s218D/S222D, constitutively active form; S22a, dominant negative form). Transcriptional activity of GAL4-Elk1 after incubation with or without ET-1 (10-7 mol/L) was analyzed using Firefly luciferase as a reporter gene normalized with Renilla luciferase as an internal control. Each column represents mean±SEM (n=8). *P<0.01; **P<0.005; ***P0.001 vs control. [star]P<0.01 vs ET-1 (10-7 mol/L). #P<0.01 vs S218D/S222D.
Prepared by: the Data Supplement Manager
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