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Blood, Vol. 113, Issue 2, 488-497, January 8, 2009
Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis
Blood Carmona et al.
113: 488
Supplemental materials for: Carmona et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 2.91 MB)
- Figure S1. Activation of Rap1 by angiogenic growth factors (JPG, 47.8 KB)
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(A) Expression of Rap1a and Rap1b in HUVEC as assessed by RT-PCR. Primers directed against GAPDH were used as loading control. (B) Comparison of the relative expression of Rap1a and Rap1b mRNA in HUVEC and HMVEC by using a gene microarray analysis (n=3) C/D, Rap1 activity assay, HUVEC were stimulated with VEGF (B) or bFGF (C). The level of GTP-bound (active) Rap1 was assessed.
- Figure S2. Silencing of Rap1 decrease Rap1 protein level (JPG, 40.6 KB)
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48 hours after transfection with siRNAs targeted against Rap1a, Rap1b, Rap1a/Rap1b, HUVEC were lysed. Cell lysates were subjected to Western blot analysis using antibody against Rap1 (recognizes both Rap1a and Rap1b) and tubulin.
- Figure S3. Silencing of Rap1 inhibits endothelial migration on collagen and adhesion on collagen and vitronectin (JPG, 58.4 KB)
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48 hours after transfection with siRNAs targeting Rap1a, Rap1b, and both Rap1a and Rap1b, or scrambled siRNA, cells were allowed to migrate (modified boyden chambers) or to adhere on collagen. (A–C) Migration (A) (n=6) and adhesion (B) (n=5) assays on collagen and adhesion (C) (n=3) on vitronectin with siRNA-transfected endothelial cells (*P<0.05 vs. scr, #P<0.05 vs. scr+bFGF). The data are presented as mean ± SEM. (D) HUVEC were transfected with RapGAP1-FLAG or mock control. 24 h after transfection, surface expression of β1-integrin subunit (CD29) was quantified using FACS. Data are represented as mean % of gated positive cells for CD29 ± SEM (n=3). (D) HUVEC were transfected with RapGAP1-FLAG or mock control. 24 h after transfection, surface expression of β1 integrin subunit (CD29) was quantified using FACS. Data are represented as mean % of gated positive cells for CD29 ± SEM (n=3).
- Figure S4. Silencing of RAPL inhibits sprout formation (JPG, 32.6 KB)
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(A) Endothelial cells transfected with siRNAs targeted against RAPL or scrambled control. Statistical summary of spheroids assay with RAPL siRNA I (5′ CTGGAAGACTGCTTCTTCA 3′) and II (5′ CTTCAGAACTTCCTAACAA 3′) or scrambled transfected endothelial cells. Spheroids were stimulated with or without bFGF 50ng/mL. Cumulative length of all sprouts originating from an individual spheroid was quantified after 24 hours. Statistical summary represents the mean ± SEM (*P<0.05 vs. scrambled; # P<0.05 vs bFGF+scrambled). (B) Spheroid angiogenic sprouting assay in presence of blocking monoclonal β1-integrin antibodies or murine isotype control antibodies. Data are given as mean ± SEM (n=3, *P<0.05 vs. IgG, **P<0.05 vs. bFGF+IgG).
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